CN108368522A - Method and system for high-throughput unicellular genetic manipulation - Google Patents

Abstract

Provided herein are methods and systems for introducing nucleic acid manipulation agents into single cells. This high-throughput delivery of nucleic acid manipulation reagents into single cells and subsequent genetic manipulation of such cells allows for large-scale genetic analysis that can be used, for example, to study biological pathways and drug target discovery.

Description

Methods and systems for high-throughput single-cell genetic manipulation

Cross References to Related Applications

This application claims the benefit of US Provisional Application No. 62/243,917, filed October 20, 2015, which is hereby incorporated by reference in its entirety for all purposes.

Background of the invention

Advances in the development of nucleic acid manipulation reagents have allowed simple and efficient manipulation of nucleic acids (eg, DNA and RNA) in target cells. RNA interference (RNAi) agents such as single interfering RNA (siRNA) and short hairpin RNA (shRNA) allow cleavage, degradation and/or translational repression of a target RNA with appropriate complementary sequences. The development of CRISPR reagents provides DNA-encoded, RNA-mediated DNA- or RNA-targeted sequence-specific targeting. The CRISPR system can be used to generate small insertions or deletions that cause efficient and inactivating mutations in a target nucleic acid. In addition, CRISPR reagents are also used to precisely insert donor DNA into the target cell genome. This nucleic acid manipulation reagent enables researchers to precisely manipulate specific genomic elements and facilitates the functional elucidation of target nucleic acids in biology and disease.

Nucleic acid manipulation reagents have great potential for use in high-throughput applications such as genome-wide mutation screening, drug target discovery, and large-scale production of transgenic cells and organisms for research and commercial purposes. However, with the development of novel nucleic acid manipulation reagents for high-throughput purposes, there is also a need to develop systems and methods for high-throughput introduction of these reagents into cells. Standard array screening methods require arraying of cell and nucleic acid manipulation reagents into multi-well plates as a single reagent per well. Such screening often requires special facilities that handle many plates using automation. Therefore, large-scale application of these nucleic acid manipulation reagents can be an expensive and time-consuming process. Accordingly, new systems and methods for high-throughput delivery of nucleic acid manipulation reagents are needed.

Summary of the invention

Provided herein are compositions, systems and methods for delivering agents into individual cells.

In a first aspect, provided herein is a method of delivering an agent into an individual cell. Such methods include, but are not limited to, the steps of: (a) providing a plurality of capsules, wherein the capsules contain an agent for altering expression of at least one gene product in a cell; (b) delivering the capsules to discrete compartments wherein the discrete partitions further include individual cells; and (c) causing the capsule to release the contents of the capsule into the discrete partitions under conditions that enable the individual cells to take up the agent, thereby The agent is delivered into the individual cells.

In some embodiments and in accordance with the above, the agent for altering the expression of at least one gene product comprises: (i) a first regulatory element operably linked to at least one encoding CRISPR system a nucleotide sequence of a guide RNA that hybridizes to a target sequence in a DNA molecule within the individual cell; and (ii) a second regulatory element operably linked to a Nucleotide sequence of RNA-guided nuclease or RNA-guided nuclease fusion protein. In some embodiments, the components (i) and (ii) are on the same or different vectors, and the RNA-guiding nuclease and the guide RNA do not naturally occur together.

In an exemplary embodiment, the second regulatory element is operably linked to a nucleotide sequence encoding an RNA-guiding nuclease. In such embodiments, the guide RNA targets the target sequence and the RNA-guided nuclease cleaves the DNA molecule, thereby altering the expression of the at least one gene product. In some embodiments, the agent for altering gene expression further comprises a donor nucleic acid inserted into the DNA molecule after cleavage of the DNA molecule by the RNA-guided nuclease.

In another exemplary embodiment, the second regulatory element is operably linked to a nucleotide sequence encoding an inactivated RNA-guided nuclease, whereby the guide RNA targets the target sequence and the The deactivated RNA-guided nuclease interferes with transcription of the nucleic acid encoding the at least one gene product, thereby altering expression of the at least one gene product.

In yet another exemplary embodiment, the second regulatory element is operably linked to a nucleotide sequence encoding an RNA-guided nuclease fusion protein, whereby the guide RNA targets the target sequence and the RNA The directed nuclease fusion protein interferes with the expression of the at least one gene product, thereby altering the expression of the at least one gene product. In some cases, the RNA-guided nuclease fusion protein comprises an inactivated RNA-guided nuclease and a transcriptional activator or transcriptional repressor. In some cases, the nuclease fusion protein comprises an inactivated RNA-guided nuclease and an epigenetic modifier.

In certain embodiments and according to the above, the RNA-guided nuclease is a Cas9 protein or a Cpf1 protein.

In certain embodiments and in accordance with the above, the capsule is configured to release its contents upon application of a stimulus. In some embodiments, the stimulus is selected from chemical stimulation, electrical stimulation, thermal stimulation, magnetic stimulation, change in pH, change in ion concentration, reduction of disulfide bonds, photostimulation, and combinations thereof. In some embodiments, the stimulus is a thermal stimulus.

In other embodiments and according to any of the above, the plurality of capsules comprises about 100 to 100,000 different agents for altering the expression of at least one gene product such that different individual cells receive different agents .

In other embodiments and according to any of the above, said capsule further comprises one or more additives for compatibility of said capsule or its contents with said individual cells. In some embodiments, the one or more additives include a transfection agent.

In certain embodiments and according to any one of the above, the agent for altering the expression of at least one gene product further comprises an oligonucleotide comprising a nucleic acid barcode sequence. In some of these embodiments, different individual cells receive different nucleic acid barcode sequences.

In some embodiments, the reagent for altering expression of at least one of the gene products described above further comprises a pair of Cas9 nickases or a Cas9 fusion protein, compared to when an RNA-guided nuclease is used, the The Cas9 nickase or Cas9 fusion protein improves the specificity of the CRISPR system.

In certain embodiments and according to any of the above, said target sequence has little or no relatedness within the genome of said cell.

In other embodiments and according to any one of the above, the agent for altering the expression of at least one gene product further comprises increasing the concentration of A reagent for the frequency of homologous recombination. In some embodiments, these reagents comprise a Cas9 nuclease or a nuclease-free Cas9 protein encoded with at least one nucleotide sequence encoding the CRISPR system guide RNA.

In other embodiments and according to any one of the above, said guide RNA further comprises a spacer identical to the sequence of a targeting protospacer within the genome of said cell.

In another exemplary embodiment and according to any of the above, one or both of said first and second regulatory elements are inducible promoters. In some embodiments, the inducible promoter is selected from the group consisting of a light-inducible promoter, a heat-inducible promoter, and a chemical-inducible promoter.

In a second aspect, provided herein is a method for delivering an agent to a cell. Such methods include, but are not limited to, the steps of: (a) providing said agent releasably coupled to microcapsules; (b) isolating said microcapsules into discrete partitions, wherein said discrete partitions also include individual cells; and (c) releasing said agent under conditions that enable uptake of said agent into said cell.

In some embodiments of this second aspect, the reagent comprises one or more of a nuclease encoding an RNA guide, a clustered regularly interspaced short palindromic repeat (CRISPR), or a DNA molecule capable of interacting with the cell. A vector of at least one of the CRISPR guide RNAs to which the target sequence hybridizes, and one or more conditionally inducible promoters. In certain embodiments, the reagent comprises: (i) a first regulatory element operable in a eukaryotic cell operably linked to at least one nucleotide encoding a CRISPR system guide RNA sequence, the CRISPR system guide RNA hybridizes to the target sequence in the cell; and (ii) a second regulatory element operable in eukaryotic cells, the second regulatory element is operably linked to the coding RNA guide The nucleotide sequence of a nuclease. In some embodiments, components (i) and (ii) are on the same or different vectors, and the RNA-guiding nuclease and the guide RNA do not naturally occur together.

In an exemplary embodiment of this second aspect, said second regulatory element is operably linked to a nucleotide sequence encoding an RNA-guiding nuclease, whereby said guide RNA targets said target sequence and said The RNA-guided nuclease cleaves the DNA molecule, thereby altering expression of the at least one gene product. In some embodiments, the agent for altering gene expression further comprises a donor nucleic acid inserted into the DNA molecule after cleavage of the DNA molecule by the RNA-guided nuclease.

In another exemplary embodiment of this second aspect, said second regulatory element is operably linked to a nucleotide sequence encoding an inactivated RNA-guided nuclease, whereby said guide RNA targets said The target sequence and said inactivated RNA-guided nuclease interferes with transcription of nucleic acid encoding said at least one gene product, thereby altering expression of said at least one gene product.

In yet another exemplary embodiment of this second aspect, said second regulatory element is operably linked to a nucleotide sequence encoding an RNA-guiding nuclease fusion protein, whereby said guide RNA targets said target sequence and the RNA-guided nuclease fusion protein interferes with the expression of the at least one gene product, thereby altering the expression of the at least one gene product. In some embodiments, the RNA-guided nuclease fusion protein comprises an inactivated RNA-guided nuclease and a transcriptional activator or transcriptional repressor. In certain embodiments, the nuclease fusion protein comprises an inactivated RNA-guided nuclease and an epigenetic modifier.

In some embodiments of this second aspect, the RNA-guided nuclease is a Cas9 protein. In other embodiments, the RNA-guided nuclease is a Cpf1 protein. In some embodiments, the vector is capable of stably integrating into the genome of the cell.

In other embodiments, the releasing step comprises applying a stimulus to the microcapsules to release the agent. In some embodiments, the stimulus is selected from chemical stimulation, electrical stimulation, thermal stimulation, magnetic stimulation, change in pH, change in ion concentration, reduction of disulfide bonds, photostimulation, and combinations thereof.

In some embodiments, uptake of the agent into the cell is facilitated by electroporation.

In other embodiments, the microcapsules further comprise one or more additives to improve the compatibility of the agent uptake into the cells. In some embodiments, the one or more additives include a transfection agent.

In some embodiments, the microcapsules comprise members selected from droplets in emulsions and cross-linked polymers.

In other embodiments, the microcapsules comprise beads. In certain embodiments, the beads are gel beads.

In other embodiments, the microcapsules further comprise a population of nucleic acid barcode sequences releasably coupled thereto, wherein substantially all of the barcode sequences comprise the same barcode sequence. In some embodiments, the barcode sequence further comprises a hairpin sequence.

In other embodiments, the reagent further comprises a pair of Cas9 nickases or Cas9 fusion proteins that improve the specificity of the CRISPR system compared to when RNA-guided nucleases are used .

In a third aspect, provided herein is a method for altering gene expression in a plurality of cells. The method includes, but is not limited to, the steps of: (a) providing a plurality of capsules, wherein the capsules contain an agent for altering the expression of at least one gene product, the agent comprising an engineered, non-naturally occurring cluster of regularly spaced short palindromic repeat (CRISPR) system; (b) delivering the capsule into discrete compartments containing individual cells; (c) providing a stimulus to cause the capsule to act in such a way that the agent is delivered to the individual cell releases its contents under certain conditions. In some embodiments, the CRISPR system comprises one or more vectors comprising: (i) a first regulatory element operably linked to at least one coding The nucleotide sequence of the CRISPR-Cas system guide RNA, the CRISPR-Cas system guide RNA can hybridize with the target sequence in the DNA molecule of the cell; and (ii) a second regulatory element, the second regulatory element can be Operably linked to a nucleotide sequence encoding an RNA-guiding nuclease, wherein components (i) and (ii) are located on the same or different vectors of the system. In this aspect, following application of said stimulus, said guide RNA hybridizes to said target sequence, and said RNA-guided nuclease cleaves said DNA molecule containing said target sequence, thereby altering said at least one Expression of the gene product. In some embodiments of this aspect, the agent for altering gene expression further comprises a donor nucleic acid inserted into the DNA molecule following cleavage of the DNA molecule by the RNA-guided nuclease.

In a fourth aspect, provided herein is a method for altering gene expression in a plurality of cells. The method includes, but is not limited to, the steps of: (a) providing a plurality of capsules, wherein the capsules contain an agent for altering the expression of at least one gene product, the agent comprising an engineered, non-naturally occurring cluster of regularly spaced short palindromic repeat (CRISPR) system; (b) delivering the capsule into discrete compartments containing individual cells; (c) providing a stimulus to cause the capsule to act in such a way that the agent is delivered to the individual cell releases its contents under certain conditions. In some embodiments, the CRISPR system comprises one or more vectors comprising: (i) a first regulatory element operably linked to at least one coding The nucleotide sequence of the CRISPR-Cas system guide RNA, the CRISPR-Cas system guide RNA can hybridize with the target sequence in the DNA molecule of the cell; and (ii) a second regulatory element, the second regulatory element can be Operably linked to a nucleotide sequence encoding an inactivated RNA-guiding nuclease, wherein components (i) and (ii) are located on the same or different vectors of the system. In this aspect, following application of said stimulus, said guide RNA hybridizes to said target sequence, and said deactivated RNA-guided nuclease interferes with transcription of nucleic acid encoding said at least one gene product, thereby altering Expression of the at least one gene product.

In a fifth aspect, provided herein is a method for altering gene expression in a plurality of cells. The method includes, but is not limited to, the steps of: (a) providing a plurality of capsules, wherein the capsules contain an agent for altering the expression of at least one gene product, the agent comprising an engineered, non-naturally occurring cluster of regularly spaced short palindromic repeat (CRISPR) system; (b) delivering the capsule into discrete compartments containing individual cells; (c) providing a stimulus to cause the capsule to act in such a way that the agent is delivered to the individual cell releases its contents under certain conditions. In some embodiments, the CRISPR system comprises one or more vectors comprising: (i) a first regulatory element operably linked to at least one coding The nucleotide sequence of the CRISPR-Cas system guide RNA, the CRISPR-Cas system guide RNA can hybridize with the target sequence in the DNA molecule of the cell; and (ii) a second regulatory element, the second regulatory element can be Operably linked to a nucleotide sequence encoding an RNA-guided nuclease fusion protein, wherein components (i) and (ii) are on the same or different vectors of the system. In this aspect, following application of said stimulus, said guide RNA hybridizes to said target sequence, and said RNA-guided nuclease fusion protein interferes with expression of said at least one gene product, thereby altering said at least one expression of a gene product. In some embodiments, the RNA-guided nuclease fusion protein comprises an inactivated RNA-guided nuclease and a transcriptional activator or transcriptional repressor. In certain embodiments, the nuclease fusion protein comprises an inactivated RNA-guided nuclease and an epigenetic modifier.

In some embodiments of the third, fourth and fifth aspects, the RNA-guided nuclease is a Cas9 protein or a Cpf1 protein.

In some embodiments of the third, fourth and fifth aspects, said different capsules comprise guide RNAs capable of hybridizing to different target sequences within said individual cells such that expression of different gene products is altered in different cells .

In other embodiments, the plurality of capsules comprises from about 500 to about 100,000 capsules. In some embodiments, the plurality of capsules comprises about 10,000 to about 50,000 capsules. In other embodiments, the plurality of capsules comprises from about 15,000 to about 30,000 capsules, wherein only a single capsule is delivered into each discrete partition.

In other embodiments, the capsules comprise droplets in an emulsion. In other embodiments, the capsule comprises a polymer gel. In some embodiments, the polymer gel is polyacrylamide. In other embodiments, the capsules comprise gel beads.

Brief description of the drawings

Figure 1 provides a schematic representation of a microfluidic device for delivery of nucleic acid manipulation reagents into compartments containing single cells, as described herein.

I. Overview

The present disclosure provides methods, compositions and systems suitable for delivering agents into single cells. In particular, the methods, compositions, and systems provided herein allow high-throughput delivery of reagents for manipulating one or more target nucleic acids in individual cells. In some instances, such nucleic acid manipulation reagents alter the expression of a gene product encoded by a target nucleic acid. This high-throughput delivery of nucleic acid manipulation reagents into single cells and subsequent genetic manipulation of such cells allows for large-scale genetic analysis that can be used, for example, to study biological pathways and drug target discovery. Furthermore, such high-throughput gene editing could facilitate the production of genetic plants and animals and the development of cell-based therapeutics.

In general, provided herein is a method for delivering nucleic acid manipulation reagents into individual cells. The method includes the step of providing a plurality of capsules, each capsule carrying reagents for nucleic acid manipulation in an individual cell. Provided capsules are delivered into discrete compartments comprising one or more cells. After delivery of the capsule into the compartment comprising the cells, the capsule is caused to release its contents into the discrete compartment, typically through the use of a stimulus. Nucleic acid manipulation reagents are taken up by cells in the presence of uptake reagents (eg, transfection reagents or electroporation buffer).

After release from the capsule, the nucleic acid manipulation reagents can be taken up by the single cells using any suitable method. For example, cells can be electroporated, wherein the electroporated cells are capable of taking up an agent for altering expression of a gene product in the presence of an electroporation buffer. In another instance, a transfection reagent can be used to allow transfection of an agent for altering expression of a gene product. Virus-based systems can also be used to introduce nucleic acid manipulation reagents into single cells.

The capsules described herein serve as vehicles for delivering suitable reagents for nucleic acid manipulation of target nucleic acids to cells (eg, single cells) in compartments. Such agents are useful, for example, in altering the expression of gene products. Agents that alter expression of a gene product may alter expression by acting on the coding region of the gene of interest or by acting on a non-coding regulatory region (eg, an enhancer or promoter) of the gene of interest. Such agents can alter the expression of a gene product by increasing or decreasing the expression of the gene product.

The reagents used in the subject methods can allow high-throughput manipulation of one specific target nucleic acid (ie, DNA or RNA) or allow high-throughput alteration of the expression of multiple different target nucleic acids in a single cell. Where alteration of multiple different target nucleic acids is desired, multiple different target nucleic acids may be altered within a single cell, or a single target nucleic acid may be altered within a single cell, depending on the reagents contained in each capsule. For example, a plurality of capsules used with the subject methods may contain about 100 to 10,000 different agents for altering the expression of at least one gene product such that different individual cells receive different agents.

Any suitable reagent for manipulating target nucleic acids can be used with the systems and methods provided herein. Exemplary reagents include, but are not limited to, zinc finger nucleases; transcription activator-like effector nucleases (TALENs); reengineered homing nucleases; RNA interference (RNAi) agents and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas nuclease system.

In some cases, nucleic acid manipulation reagents for use with the subject methods and systems include CRISPR system reagents. Such reagents include, for example, nucleic acids encoding nucleases, such as RNA-guided nucleases (eg, Cas9 nuclease or Cpf1 nuclease); and nucleic acids encoding guide RNAs (gRNAs). Exemplary CRISPR system reagents and methods for use in the subject methods and systems are described in further detail herein, and are also known in the art, for example in Shalem et al., Nature Reviews Genetics 16:299-311 (2013); Zhang et al., Human Molecular Genetics 23(R1):R40-6 (2014); and Zhu et al. Cell 157:1262-1278 (2014), for all purposes and in particular with respect to reagents related to the CRISPR system All teachings are hereby incorporated by reference in their entirety.

In an exemplary CRISPR system, the gRNA/RNA-guided nuclease complex is recruited to the genomic target sequence by base pairing between the gRNA sequence and the complement of the genomic target sequence. For successful binding of an RNA-guided nuclease, the genomic target sequence must generally contain the correct protospacer adjacent motif (PAM) sequence immediately following the target sequence (learn more about PAM sequences). The binding of the gRNA/RNA-guided nuclease complex localizes the RNA-guided nuclease to the genomic target sequence such that the RNA-guided nuclease can cleave both DNA strands at the target sequence, resulting in a double-strand break (DSB). RNA-guided nucleases that can be used with the methods and systems provided herein include, but are not limited to, Cas9 nuclease and Cpf1 nuclease.

This DSB can then be repaired by (1) the non-homologous end joining (NHEJ) DNA repair pathway or (2) the homology directed repair (HDR) pathway. The NHEJ repair pathway typically produces insertions/deletions (indels) at DSB sites that result in frameshifts and/or premature stop codons, effectively disrupting the open reading frame (ORF) of the target gene. Genomic alterations of this type are useful, for example, for loss-of-function gene function studies. The HDR path requires the presence of repair templates, which are used to repair DSBs. Specific nucleotide changes can be introduced into target genes by using HDR with repair nucleic acid templates. For example, the HDR approach can be used to introduce gain-of-function mutations or to modify regulatory elements.

The RNA-guided nucleases used with the subject systems and methods depend on the particular type of genetic alteration desired. For example, the RNA-guided nuclease can be an inducible RNA-guided nuclease (eg, Cas9 or Cpf1) that is optimized for expression in a time- or cell-type-dependent manner. Mutant Cas9 nucleases exhibiting improved specificity can also be used (see, e.g., Ann Ran et al. Cell 154(6) 1380-89 (2013), for all purposes and in particular with regard to interactions with mutant Cas9 nucleases. All teachings relating to nucleases are incorporated herein by reference in their entirety). In addition, deactivated RNA-guided nucleases (ie, nuclease-free) can serve as sinks for other proteins that affect gene expression at the target site (eg, transcriptional repressors or activators).

Guide RNAs (gRNAs) used with the subject systems and methods can target coding regions or regulatory non-coding regions (eg, enhancers and promoters). The number and type of gRNA used will depend on the application of the systems and methods described herein. For example, the systems and methods can be used for large-scale mutagenesis using guide RNA libraries containing multiple guide RNAs targeting multiple different target sequences. The systems and methods can also be used to introduce a specific change in a large number of a specific cell type or many different types of cells using a specific gRNA. For example, specific gRNAs can be used to correct a disease loss-of-function gene or to inactivate a disease gene associated with a dominant-negative condition.

In applications requiring the introduction of specific alleles or mutations, nucleic acid manipulation reagents also include homologous repair template nucleic acids comprising said specific allelic mutations. Homologous repair template nucleic acids introduce allelic-specific mutations into the genome of cells when Cas-induced DSBs are repaired through the HDR pathway. In some cases, homologous repair templates are used to introduce specific mutations into wild-type cells. In other cases, a homologous repair template is used to introduce a wild-type allele into a mutant cell (eg, a cell containing a mutation associated with a particular disease). Homologous repair templates may also include markers for identifying and sorting cells containing specific mutations, such as nucleic acid or fluorescent barcode markers as described herein. In such applications requiring the introduction of specific mutations via homologous repair template nucleic acid, the reagents may also include one or more reagents that promote the HDR pathway over HNEJ to repair DSBs. Such agents include, but are not limited to, agents that inhibit genes involved in HNEJ repair, such as DNA ligase IV (see, e.g., Maruyana et al. Nat Biotechnol. 33(5):538-42 (2015), which is for all purposes And in particular all teachings related to agents that inhibit genes involved in HNEJ repair are hereby incorporated by reference in their entirety.

The subject capsules can serve as carriers for nucleic acid manipulation reagents in a variety of different ways. For example, the agent can be encapsulated. Such capsules may have an outer barrier surrounding an inner fluid center or core, such as a droplet in an emulsion. In other cases, capsules may comprise cross-linked polymers or porous matrices capable of entraining and/or retaining materials within their matrices. Capsules for use with the subject systems and methods may also comprise beads (eg, gel beads) to which agents described herein are attached.

Capsules for use with the methods and systems provided herein are configured to release their contents (eg, agents) upon application of a stimulus after the capsule has been delivered or separated into discrete compartments containing individual cells. Individual capsules can contain reagents for altering the expression of one gene product (eg, one guide RNA) or more than one gene product (eg, more than one guide RNA). In addition, the subject capsules may also contain other reagents that facilitate the delivery of nucleic acid manipulation reagents into cells, such as transfection reagents.

Each capsule may also include markings that allow identification and/or classification of the capsules. For example, in applications where multiple different reagents and/or cell types are used, such labels are useful, eg, for dispensing or introducing capsules containing reagents for editing a specific target sequence with a specific cell type. Suitable labels include, for example, fluorescent labels and unique nucleic acid barcodes as described herein.

According to the subject methods, capsules containing the agents described herein are "delivered" or "isolated" into discrete compartments containing individual cells. As used herein, "deliver" and "isolate into" are used interchangeably to describe the process of introducing a capsule containing an agent into a compartment containing cells whose gene expression is desired to be altered.

Any suitable cell can be used with the subject methods and systems described herein. Exemplary cells include, but are not limited to, bacterial, plant, yeast, and mammalian cells, including human cells. Depending on the application of the subject method, single or multiple cell types can be used. In certain instances, cells (eg, stem cells) are used to make cell-based therapies. In other cases, fertilized embryos at the one-cell stage can be used to generate transgenic animals.

In some aspects, the compartments or partitions containing cells for nucleic acid manipulation include partitions that are flowable within a fluid stream. These compartments may comprise, for example, microcapsules or microvesicles with an outer barrier surrounding an inner fluidic center or core, or they may be porous matrices capable of entraining and/or retaining material within their matrix. In some aspects, however, these partitions include droplets of aqueous fluid within a non-aqueous continuous phase (eg, an oil phase). Various containers are described, for example, in U.S. Patent Publication No. 2014/0155295, the entire disclosure of which is incorporated by reference for all purposes and in particular with respect to all teachings relating to partitions and droplets used in accordance with the present invention. method is incorporated into this article as a whole. Likewise, emulsion systems for forming stable droplets in non-aqueous or oily continuous phases are described in detail, for example, in US Patent Publication No. 2010/0105112.

In the case of droplets in emulsions, the assignment of individual cells to discrete partitions can generally be achieved by introducing a flow of cells in an aqueous fluid into a flow of a non-aqueous fluid such that Droplets are produced at the junction. By providing an aqueous cell-containing flow at a certain level of cell concentration, the occupancy level of the resulting partition in terms of cell number can be controlled. In some cases, where single-cell partitions are desired, it may be desirable to control the relative flow rates of the fluids so that the partitions contain on average less than one cell per partition, in order to ensure that those partitions that are occupied are primarily mono-occupied. Likewise, it may be desirable to control the flow rate so that a higher percentage of partitions is occupied, eg allowing only a smaller percentage of unoccupied partitions. In some aspects, flow and channel structure are controlled to ensure a desired number of single occupied partitions, unoccupied partitions below a certain level, and multiple occupied partitions below a certain level. In many cases, the systems and methods are used to ensure that the vast majority of occupied partitions (partitions containing one or more capsules) contain no more than 1 cell per occupied partition. In some cases, the allocation process is controlled so that less than 25% of occupied partitions contain more than one cell, and in many cases less than 20% of occupied partitions have more than one cell, and in some cases , less than 10% or even less than 5% of the occupied partitions comprise more than one cell per partition.

In some cases, microfluidic channel networks are particularly suitable for creating partitions as described herein. Examples of such microfluidic devices include those described in detail in Provisional U.S. Patent Application No. 61/977,804, filed April 4, 2014, the entire disclosure of which is for all purposes and with particular reference to microfluidic devices. All teachings relating to the device are hereby incorporated by reference in their entirety. Alternative mechanisms may also be employed in dispensing individual cells, including porous membranes through which the aqueous mixture of cells is extruded into the non-aqueous fluid. Such systems are generally available from, for example, Nanomi, Inc.

In the case of droplets in emulsions, the assignment of individual cells to discrete partitions can generally be achieved by introducing a flow of cells in an aqueous fluid into a flow of a non-aqueous fluid such that Droplets are produced at the junction. By providing an aqueous cell-containing flow at a certain level of cell concentration, the occupancy level of the resulting partition in terms of cell number can be controlled. In some cases, where single-cell partitions are desired, it may be desirable to control the relative flow rates of the fluids so that the partitions contain on average less than one cell per partition, in order to ensure that those partitions that are occupied are primarily mono-occupied. Likewise, it may be desirable to control the flow rate so that a higher percentage of partitions is occupied, eg allowing only a smaller percentage of unoccupied partitions. In some aspects, flow and channel structure are controlled to ensure a desired number of single occupied partitions, unoccupied partitions below a certain level, and multiple occupied partitions below a certain level.

Each cell-containing compartment may also include identification markers which advantageously allow identification, tracking and sorting of specific cells and/or reagents. For example, in systems and methods employing CRISPR systems and multiple cell types, such identification markers may allow certain guide RNAs to be sorted and distributed with specific cell types among multiple different cell types. Suitable labels may include, for example, fluorescent labels and nucleic acid barcode labels as described herein. For example, in mutagenesis screens, such barcodes can also help identify specific mutations associated with specific phenotypes.

After isolation or delivery of a capsule containing an agent into a compartment containing an individual cell, the capsule is allowed to release its contents into the compartment under conditions that enable the cell to take up the agent. In some instances, a stimulus delivered to the capsule is used to cause the capsule to release its contents. Any suitable stimulus may be used to cause the capsule to release its contents. Exemplary stimuli include, but are not limited to, chemical stimuli, electrical stimuli, thermal stimuli, magnetic stimuli, changes in pH, changes in ion concentrations, reduction of disulfide bonds, and photostimulation.

Once the reagents are released from the capsule, the cells take up the nucleic acid manipulation reagents. Uptake by cells can be facilitated by including uptake reagents in the partitions, such as transfection reagents or electroporation buffer.

Individual capsules containing cells that have undergone nucleic acid manipulation events can then be further sorted and analyzed depending on the application. For example, in phenotypic screening, such cells can be placed under selection conditions for a particular phenotype, and any suitable technique, including, for example, fluorescence, luminescence, and high-content imaging techniques, can be used to characterize cells with a particular phenotype . See, eg, Hasson et al., Nature 504:291-295 (2013); Neumann et al., Nature Methods 3:385-390 (2006); and Moffat et al., Cell 124:1283-1298 (2006). Nucleic acid manipulations in cells that have been selected for a particular phenotype can also be analyzed using appropriate sequencing techniques, including, for example, next-generation sequencing techniques.

In some cases, individual cells that have been selected for a particular phenotype are lysed. This lysis can occur within compartments containing individual cells of a particular phenotype, or cells containing the same phenotype can be pooled prior to lysis. Following lysis, reverse transcription of mRNA from selected cells can be performed in the compartments described herein to generate single-cell transcriptome profiles. In cases where individual cells are lysed within a partition, reagents for reverse transcription can then be introduced into each partition. Following reverse transcription, the cDNA transcripts are sequenced to identify specific transcripts that are differentially expressed in specific cells over time or after exposure to specific conditions compared to cells that do not exhibit the desired phenotype. This differential expression hints at genes that contribute to specific phenotypes. See, e.g., U.S. Patent Application Publication No. 2014/0227684, the entire disclosure of which is incorporated by reference in its entirety for all purposes and in particular with respect to the entire teachings relating to methods of reverse transcription in assigned individual cells This article.

The subject methods and systems provided herein are useful in a variety of applications requiring high-throughput alteration of at least one gene product in a cell. For example, the subject methods and systems can be used for large-scale mutagenesis. For example, large-scale mutagenesis is useful for drug development, biological pathway studies, and gene function studies. The subject methods and systems can be used to mass produce transgenic plants and animals. The subject methods and systems are also useful for large-scale production of cell-based therapeutics. For example, the subject methods can be used to generate T cells with modified chimeric antigen T cell receptors useful in the treatment of cancer.

Also provided herein are microfluidic devices for delivering an agent as described above (eg, an agent for altering the expression of at least one gene product). Such microfluidic devices may include a network of channels for carrying out delivery processes such as those set forth in FIG. 1 .

II. Workflow Overview

In an exemplary aspect, the methods and systems described herein provide for high-throughput delivery of agents in target cells. In particular, the methods and systems provided herein are used to deliver nucleic acid manipulation reagents. Such nucleic acid manipulation reagents are useful, for example, to alter the expression of a gene product encoded by a target nucleic acid. Nucleic acid manipulation reagents can also be used to introduce specific mutations in a particular target nucleic acid, thereby producing a mutated gene product that functions differently than its wild-type counterpart.

In a first step, a plurality of capsules comprising reagents for editing a target nucleic acid in a target cell are provided. Any suitable reagent for editing a target nucleic acid can be used with the systems and methods provided herein.

The systems and methods provided herein allow for high-throughput alteration of at least one target nucleic acid in target cells. The target nucleic acid selected for modification may in some cases be a sequence with little or no relatedness within the genome of the target cell. Different target nucleic acids can be located within the same gene or on different genes. Additionally, a target nucleic acid can encompass an entire gene or a portion of a gene. In some cases, the subject methods provided herein are used for high-throughput nucleic acid manipulation of multiple target cells, wherein one target nucleic acid is manipulated in each cell. In certain instances, the subject methods provided herein are used to manipulate multiple target cells, wherein each single cell manipulates the expression of one gene product. In such embodiments, where the subject methods are used to manipulate the expression of one gene product per single cell, more than one target nucleic acid in the gene encoding the gene product can be manipulated in each single cell. For example, each single cell can be associated with 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleic acid manipulation reagents for different target nucleic acids are dispensed together.

In other cases, the methods described herein are used in single cells2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 ,20,25,30,35,40,45,50,55,60,65,70,75,80,85,90,95,100,200,300,400,500,600,700,800,900 Or high-throughput alterations of 1,000 or more different target nucleic acids. In some cases, the methods described herein are used in single cells from 2 to 10, 15 to 25, 20 to 30, 35 to 40, 45 to 55, 50 to 60, 65 to 75, 70 to 80, 75 to 85, High-throughput alterations of 80 to 90, 85 to 95, or 90 to 100 different target nucleic acids.

In certain instances, the methods described herein are used in at least 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000、10,000、20,000、30,000、40,000、50,000、60,000、70,000、80,000、90,000、1000,000、2000,000、300,000、400,000、500,000、600,000、700,000、800,000、900,000、100万、150百万、200 High-throughput alterations of 10,000, 3, 4, 5, 6, 7, 8, 9, or 10 million or more single cells. In some embodiments, the method is for 50 to 1,000, 1,000 to 5,000, 5,000 to 10,000, 10,000 to 50,000, 50,000 to 100,000, 100,00 to 200,000, 200,000 to 300,000, 300,000 to 400,000, 400,000 500,000 to 1 million, 1 million to 2 million, 2 million to 3 million, 3 million to 4 million, 4 million to 5 million, 5 million to 6 million, 6 million to 7 million, 7 million to 8 million, 8 million to High-throughput alterations of 9 million or 9 million to 10 million single cells or more.

Nucleic acid manipulation reagents can act on DNA (eg, CRISPR system reagents) and/or RNA (eg, RNAi reagents) nucleic acid targets. The nucleic acid manipulation reagents described herein can be used to alter a target nucleic acid in such a way that the expression of one or more gene products encoded by the target nucleic acid is altered. For example, in some instances, a nucleic acid manipulation agent reduces the expression and/or function of one or more gene products. In such cases, nucleic acid manipulation reagents may be targeted to regions encoding gene products or regulatory regions that control transcription of the nucleic acid. In some instances, nucleic acid manipulation agents increase the expression of one or more gene products. Nucleic acid manipulation agents that can be used to increase the expression of a gene product include those that target regulatory regions that affect the transcription of a target nucleic acid. In some instances, nucleic acid manipulation agents act by recruiting transcriptional repressors, activators, and/or recruitment domains that affect gene expression at the target site without No irreversible mutations are introduced into the target nucleic acid. In other cases, nucleic acid manipulation reagents are used to introduce new mutations into a target gene of interest such that the mutation confers a new function compared to the wild-type gene product (ie, a gain-of-function mutation). The nucleic acid manipulation reagents described herein can also be used to introduce mutations that are antagonistic to the wild-type form of a gene product (ie, dominant negative mutations).

Suitable reagents for use with the provided subject systems and methods Nucleic acid manipulation reagents include, but are not limited to, zinc finger nucleases; transcription activator-like effector nucleases (TALENs); reengineered homing nucleases; RNAi reagents, Such as small interfering RNA (siRNA) and small hairpin RNA (shRNA); and clustered regularly interspaced short palindromic repeat (CRISPR)/RNA guide nuclease system. Nucleic acid manipulation reagents can be delivered to compartments of single cells, for example, in the form of expression plasmids encoding the reagents, mRNA encoding the reagents, or viral vectors encoding the reagents.

In some cases, target nucleic acid manipulation reagents used with the subject methods and systems include CRISPR system reagents. Such reagents include, for example, nucleic acids encoding RNA-guided nucleases (e.g., Cas9 nuclease or Cpf1 nuclease) and nucleic acids encoding guide RNAs (gRNAs), including CRISPR RNA (crRNA) and transactivating CRISPR Combination of RNA (tracrRNA). The nucleic acid encoding the RNA-guiding nuclease and guide RNA can each be operably linked to a regulatory element and can be contained on a single vector or on different vectors. The selected vector may be capable of stably integrating into the cellular genome. In some instances, the RNA-guiding nuclease (eg, Cas9 nuclease or Cpf1 nuclease) and guide RNA do not occur together in nature. Exemplary CRISPR system reagents and methods for use in the present invention are described in further detail herein, for example, in Shalem et al., Nature Reviews Genetics 16:299-311 (2013); Zhang et al., Human Molecular Genetics 23(R1) : R40-6 (2014); Zetche et al., http://dx.doi.org/10.1016/j.cell.2015.09.038 and Zhu et al. Cell 157: 1262-1278 (2014), the literature published It is hereby incorporated by reference in its entirety for all purposes and in particular with respect to all teachings relating to the reagents of the CRISPR system.

In CRISPR systems, the gRNA/RNA-guided nuclease complex is recruited to the genomic target sequence by base pairing between the gRNA sequence and the complementary sequence of the target nucleic acid. The binding of the gRNA/RNA-guided nuclease complex localizes the RNA-guided nuclease (e.g., Cas9 nuclease or Cpf1 nuclease) to the genomic target sequence such that the wild-type nuclease can cleave both DNA strands, resulting in double Strand breaks (DSBs).

The DSB can be repaired by (1) the non-homologous end joining (NHEJ) DNA repair pathway or (2) the homology directed repair (HDR) pathway. NHEJ repair pathways typically generate insertions/deletions (indels) at DSB sites that result in frameshifts and/or premature stop codons, effectively disrupting the open reading frame (ORF) of the target nucleic acid, thereby reducing Expression of the gene product encoded by the target nucleic acid. Such genetic alterations are useful, for example, for gene function studies. The HDR path requires the presence of repair templates, which are used to repair DSBs. Specific nucleotide changes can be introduced into target genes by using HDR with repair templates. For example, the HDR approach can be used to introduce gain-of-function mutations or specific point mutations into target single cells. RNA-guided nucleases for use with the subject methods provided herein can include any suitable nuclease compatible with the CRIPSR system. Suitable nucleases include, but are not limited to, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Cbf1, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Cpf1, Csb1, Csb2, Csb3, Csx17, Csx14, Csx1O, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3 , Csf4, C2c1, C2c2, C2c3, homologues thereof and modified forms thereof.

The RNA-guided nucleases used with the subject systems and methods depend on the particular type of genetic manipulation desired. For example, the RNA-guided nuclease can be an inducible RNA-guided nuclease optimized for expression in a time- or cell-type-dependent manner. Suitable inducible promoters that can be linked to RNA-guided nucleases include, but are not limited to, light (e.g., green or blue light-inducible promoters), heat-inducible promoters (e.g., HSP promoters), and chemical-inducible promoters. promoters (eg, antibiotic, copper, alcohol, and steroid-inducible promoters). See, eg, Papatriantafyllou et al., Nature Reviews Molecular Cell Biology 13, 210 (2012); Yu et al., Protist 163(2):284-95 (2012); and Lee et al., Appl Environ Microbiol 76(10):3089 - 3096 (2010), which is hereby incorporated by reference in its entirety for all purposes and in particular with respect to all teachings relating to inducible promoters. Exemplary promoters include, for example, tetracycline-inducible promoters, metallothionein promoters, tetracycline-inducible promoters, methionine-inducible promoters (e.g., MET25, MET3 promoters); and galactose-inducible promoters ( GAL1, GAL7 and GAL 10 promoters). Other suitable promoters include ADH1 and ADH2 alcohol dehydrogenase promoters (repressed in glucose, induced when glucose is depleted and ethanol is produced), CUP1 metallothionein promoter (in the presence of Cu2 ⁺ , Zn2 ⁺ induction), PHO5 promoter, CYC1 promoter, HIS3 promoter, PGK promoter, GAPDH promoter, ADC1 promoter, TRP1 promoter, URA3 promoter, LEU2 promoter, ENO promoter, TP1 promoter and AOX1 promoter .

Mutant RNA-guided nucleases exhibiting improved specificity can also be used (see, e.g., Ann Ran et al. Cell 154(6) 1380-89 (2013), for all purposes and in particular with respect to All teachings relating to mutant RNA-guiding nucleases with increased specificity for target nucleic acids are hereby incorporated by reference in their entirety). Nucleic acid manipulation reagents may also include deactivated RNA-guided nucleases (eg, such as Cas9 (dCas9)). The deactivated RNA-guided nucleases provided herein are useful in applications where cleavage at a particular target nucleic acid is undesirable. Binding of deactivated Cas9 to individual nucleic acid elements can inhibit transcription by sterically hindering the RNA polymerase machinery and preventing transcriptional elongation. Furthermore, deactivated Cas can serve as a homing device (e.g., transcriptional repressors, activators, and recruitment domains) for other proteins that affect gene expression at target sites without introducing irreversible mutations. target nucleic acid. For example, dCas9 can be fused to transcriptional repressor domains such as KRAB or SID effectors to promote epigenetic silencing at target sites. Cas9 can also be converted into a synthetic transcriptional activator by fusion with VP16/VP64 or p64 activation domains.

Such deactivated RNA-guided nucleases (eg, dCas9) can also be used as homing devices for epigenetic modification tools. Inactivated RNA-guided nucleases fused to epigenetic modification tools can be used to modify histone tails and DNA molecules, such as histone methylation and demethylation, histone acetylation, cytosine methylation, and hydroxylation Methylation. For example, an inactivated RNA-guided nuclease can be fused to a functional domain of a DNA methyltransferase for targeting CpG promoter site methylation. Inactivating nucleases can be fused with epigenetic modification tools to remove methylation from key promoter CpGs (eg, the hydrogenase catalytic domain of TET1). See, eg, Falahi et al., Mol. Cancer Res. 11: 1029-1039 (2013); Mendenhall et al., Nat. Biotechnol. 31: 1133-1136 (2013); and Hilton et al., Nat. Biotechnol. 33: 510 - 517 (2015), which is hereby incorporated by reference in its entirety for all purposes and in particular with respect to all teachings relating to epigenetic modification tools.

In some cases using CRISPR system reagents, the guide RNA is attached to a bead capsule (eg, a gel bead), and the nucleic acid encoding the RNA-guided nuclease is carried as a droplet. In this case, the guide RNA and nuclease can be dispensed together prior to distribution with the target cells. Alternatively, each of the guide RNA and RNA-guided nuclease can be dispensed directly with the target cells. In some embodiments, the guide RNA and the RNA-guided nuclease are dispensed together prior to being dispensed with the target cells. In certain embodiments, the guide RNA is dispensed with the target cell prior to the RNA-guided nuclease and the target cell are dispensed. In other embodiments, the RNA-guided nuclease is dispensed with the target cells prior to the dispense of the guide RNA with the target cells. In some instances of the subject methods, the guide RNA and the RNA-guided nuclease are each delivered to the target cell using bead capsules.

Guide RNAs (gRNAs) used with the subject systems and methods can target nucleic acids to coding regions or regulatory non-coding regions (eg, enhancers and promoters). The number and type of gRNA used will depend on the application of the systems and methods described herein. For example, the systems and methods can be used for large-scale mutagenesis using guide RNA libraries containing multiple gRNAs targeting multiple different target sequences. The systems and methods can also be used to introduce a specific change in a large number of a specific cell type or many different types of cells using specific gRNAs. For example, specific gRNAs can be used to correct a disease loss-of-function gene or to inactivate a disease gene associated with a dominant-negative condition. In some cases, only one specific guide RNA is used. In some cases, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55 ,60,65,70,75,80,85,90,95,100,150,200,250,300,350,400,450,500,550,600,650,700,750,800,850,900 , 950 or 1,000 different guide RNAs, each different guide RNA corresponding to a different target nucleic acid for alteration. In some cases, 2 to 50, 50 to 100, 100 to 150, 150 to 200, 200 to 250, 250 to 300, 300 to 350, 350 to 400, 450 to 500, 500 to 550, 550 to 600, 600 to 650, 650 to 700, 700 to 750, 750 to 800, 850 to 900, 900 to 950, and 950 to 1,000 different guide RNAs. In other cases, at least 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000 or 10,000 different guide RNAs are used. In some cases, 2,000 to 3,000, 2,500 to 3,000, 3,000 to 4,000, 3,500 to 4,500, 4,000 to 5,000, 4,500 to 5,500, 5,000 to 6,000, 5,500 to 6,500, 6,000 to 7,000, 6,500 to 7,0000, 7 7,500 to 8,500, 8,000 to 9,000, 8,500 to 9,500, or 9,000 to 10,000 different guide RNAs. Where more than one guide RNA is used, each different guide RNA can be associated with a different barcode tag that allows identification and classification of the guide RNAs as described below. For example, fluorescent labeling can allow the assignment of specific gRNAs to specific single cells by fluorescent cell sorting techniques. Such barcodes can be included as part of the guide RNA or as part of a capsule carrying the guide RNA as described below. In some cases, where cells are assigned with more than one guide RNA, all guide RNAs to be assigned with a particular cell contain the same barcode. In such cases, guide RNAs distributed with different cells contain different barcodes. Such configurations advantageously allow the sorting and assignment of guide RNAs to specific cells. Furthermore, such configurations may also advantageously allow tracking and identification of cells containing a particular nucleic acid manipulation following a nucleic acid manipulation event.

Repair of double-strand breaks generated by RNA-guided nucleases can be repaired by error-prone non-homologous end joining (NHEJ) or homology-directed repair (HDR). NHEJ typically produces small insertions or deletions (indels) that are unpredictable in nature, but frequently cause efficient and inactivating mutations in the target nucleic acid. In contrast, the HDR approach is adapted for precise insertion of donor DNA into target nucleic acids.

In applications requiring the introduction of specific alleles using the CRISPR system (e.g., replacing a mutant allele with a wild-type allele or replacing a wild-type allele with a mutant allele), nucleic acid manipulation reagents may also include A homology repair template nucleic acid comprising said specific allele. Homologous repair template nucleic acids introduce specific alleles into the genome of target cells when repairing RNA-guided nuclease-induced DSBs through the HDR pathway. Homologous repair templates may also include markers for identifying and sorting cells containing specific mutations or alleles. In such applications requiring the introduction of specific alleles via a homologous repair template nucleic acid, the reagents may also include one or more reagents that promote the HDR pathway over HNEJ to repair DSBs. Such agents include, but are not limited to, agents that inhibit genes involved in HNEJ repair, such as DNA ligase IV. See, e.g., Maruyana et al. Nat Biotechnol.33(5):538-42 (2015), which is cited for all purposes and in particular for all teachings related to agents that inhibit genes involved in HNEJ repair of DSBs The method is incorporated into this article as a whole.

Other exemplary nucleic acid manipulation reagents that can be used with the systems and methods provided herein include reagents that silence the expression of one or more target genes through the RNA interference (RNAi) pathway, including but not limited to small hairpin RNA (shRNA) , double-stranded RNA (dsRNA), small interfering RNA (siRNA), and shRNA (shRNAmir) embedded in microRNA (miRNA) precursors.

In some cases, nucleic acid manipulation reagents are used to introduce a detectable label in a target nucleic acid. The amount of detectable label involved depends on the application of the method. As noted above, a detectable label may, for example, be included in a homology repair template nucleic acid. In some cases, detectable labels are used to monitor specific nucleic acid changes introduced into the genome of target cells. In such cases, different detectable labels can be used to distinguish between different alterations (eg, different fluorophores or nucleic acid sequences). In some cases, there may be more than one agent targeting a particular target nucleic acid (eg, multiple "chunked" gRNAs targeting overlapping regions of a particular gene). In this case, the same detectable label can be used for all gRNAs targeting the same gene. Detectable labels include labels that allow for the non-invasive detection of specific nucleic acid alterations in cells. For example, when used as a large-scale screen, such detectable labels may advantageously allow the identification of nucleic acid manipulations associated with a particular phenotype of interest.

Detection of a detectable label can be by any suitable method, including fluorescence spectroscopy or by other optical means. In some cases, the detectable label is a fluorophore that, upon absorption of energy, emits radiation at a defined wavelength. Fluorescently detectable labels include, for example, dansyl-functionalized fluorescent moieties (see, e.g., Welch et al., Chem. Eur. J. 5(3):951-960 (1999)); fluorescent labels Cy3 and Cy5 (see, e.g., Zhu et al., Cytometry 28:206-211 (1997)). Suitable detectable labels are also disclosed in Prober et al., Science 238:336-341 (1987); Connell et al., BioTechniques 5(4):342-384 (1987); Ansorge et al., Nucl.AcidsRes.15(11) : 4593-4602 (1987) and Smith et al., Nature 321: 674 (1986). Other commercially available fluorescent labels include, but are not limited to, fluorescein, rhodamine (including TMR, Texas Red, and Rox), alexa, bodipy, acridine, coumarin, pyrene, benzanthracene, and cyanine.

The nucleic acid manipulation reagents described herein are carried by capsules (eg, microcapsules) to compartments containing target cells. As used herein, a capsule includes any suitable container or solid matrix for carrying one or more nucleic acid manipulation reagents. Capsules include, but are not limited to, wells, microwells, holes, droplets (eg, in emulsions), spots, and beads. In some cases, a capsule includes an outer barrier surrounding an inner fluid center or core, such as a droplet in an emulsion. In other cases, capsules may comprise cross-linked polymers or porous matrices capable of entraining and/or retaining materials within their matrices. In some instances, the capsules are beads. Suitable beads include, for example, gel beads, paraffin beads and wax beads. In some instances, the capsules are gel beads. In some cases where the capsule is a bead, the nucleic acid manipulation reagent is releasably coupled to the capsule. For example, in some cases, nucleic acid manipulation reagents such as shRNA, siRNA, or guide RNA oligonucleotides are attached to the beads. In some cases, the capsule is a droplet, wherein nucleic acid manipulation reagents are encapsulated within the droplet.

Nucleic acid manipulation reagents can be coupled or immobilized on bead capsules using any suitable method. For example, coupling/immobilization can be via any form of chemical bonding (eg, covalent bonds, ionic bonds) or physical phenomena (eg, van der Waals forces, dipole-dipole interactions, etc.). In some cases, the coupling/immobilization of nucleic acid manipulation reagents to gel beads or any other capsules described herein may be reversible, such as, for example, via labile moieties (e.g., via chemical cross-linking agents, including those described herein). chemical cross-linking agent). Upon application of a stimulus, the unstable moiety can be cleaved and the immobilized reagent released. In some cases, the labile moiety is a disulfide bond. For example, where nucleic acid manipulation reagents (eg, guide RNAs) are immobilized to gel beads via disulfide bonds, exposing the disulfide bonds to a reducing agent can cleave the disulfide bonds and release the nucleic acid manipulation reagents from the beads.

In some examples, all nucleic acid manipulation reagents used to alter one or more particular target nucleic acids (ie, a "set" of nucleic acid manipulation reagents) are carried in the same capsule. For example, where a CRISPR system is used in conjunction with a bead capsule, an oligonucleotide encoding a CRISPR guide RNA specific for a particular target nucleic acid and a tube nucleotide encoding an RNA-guided nuclease can be releasably linked to The same grain. In some cases, more than one nucleic acid manipulation reagent can be used to alter the expression of a product encoded by a target nucleic acid. For example, a "chunking" approach can be used that includes multiple agents targeting overlapping regions along the length of the target nucleic acid (eg, multiple gRNAs targeting different sequences in the target nucleic acid). In such examples, all of the different reagents targeting different regions of a particular target nucleic acid can be carried by the same capsule.

In some cases, each component of a set of nucleic acid manipulation reagents (eg, CRISPR guide RNA and RNA-guided nuclease oligonucleotides) is carried by a different capsule. In such methods and systems, individual capsules carrying different components are each introduced or segregated into the same compartment containing the target cells, thereby distributing the target and target cells with a complete set of "nucleic acid manipulation reagents" to allow manipulation of a specific target nucleic acid . In certain embodiments, the guide RNA is carried by beads (eg, gel beads), and the RNA-guided nuclease is carried using microdroplet capsules.

The capsules of the subject systems and methods provided herein may also include markers to allow identification, isolation, and separation of the capsules during one or more steps of the methods. Labels include, but are not limited to, fluorescent labels and oligonucleotide "barcodes." Where oligonucleotide barcodes are used, the barcode may be comprised of the same oligonucleotide as the oligonucleotide encoding one or more agents for altering gene product expression (e.g., shRNA, siRNA, or gRNA). on or on a different oligonucleotide. In the case of bead capsules, the barcode can be attached directly to the capsule. A barcode (eg, bead) attached to the capsule can be releasably linked. Each bead can typically have a large number of oligonucleotide molecules attached. Specifically, the number of barcode molecules on a single bead can be at least about 10,000 barcode molecules, at least 100,000 barcode molecules, at least 1,000,000 barcode molecules, at least 100,000,000 barcode molecules, and in some cases at least 1 billion barcode molecules molecular.

Reagents and labels can be released from capsules (eg, beads) upon application of specific stimuli to the capsules. In some cases, the stimulus can be photostimulation, eg, by cleavage of a photolabile bond of a releasable oligonucleotide. In some cases, thermal stimulation may be used, where an increase in the temperature of the bead environment may result in cleavage of linkages or other release of oligonucleotides from the bead. In some cases, a chemical stimulus can be used, thereby cleaving the linkage of the oligonucleotide to the bead, or can otherwise cause the release of the oligonucleotide from the bead. Examples of this type of system are described in U.S. Patent Publication No. 2014/0155295, and U.S. Provisional Application Nos. 61/940,318, filed February 7, 2014, U.S. Provisional Application Nos. 61/991,018, filed May 9, 2014, and U.S. Patent Publication No. 2014/0378345, the entire disclosure of which is incorporated by reference in its entirety for all purposes and in particular with respect to the entire teachings relating to methods of releasably attaching oligonucleotides to beads This article. In one instance, such compositions comprise a polyacrylamide matrix as described above for cell encapsulation, and can be degraded by exposure to a reducing agent, such as DTT, to release the attached oligonucleotides. In some cases, the agent is released from the capsule by applying the stimulus in such a manner and under conditions that the capsule dissolves.

According to the methods and systems described herein, capsules containing nucleic acid manipulation reagents are delivered or isolated into discrete compartments containing individual cells. As used, "delivered into" and "isolated into" are used interchangeably to describe the process of producing a compartment comprising at least one cell and at least one set of nucleic acid manipulation reagents . As used herein, a "set" of nucleic acid manipulation reagents refers to the nucleic acid manipulation reagents necessary to perform the editing of one or more specific target nucleic acids in a cell. For example, a set of nucleic acid manipulation reagents in a CRISPR system includes at least one nucleic acid encoding an RNA-guiding nuclease and a guide RNA for localizing the RNA-guiding nuclease to a desired target nucleic acid. Reagents in a set of nucleic acid manipulation reagents may be contained in the same capsule or in different capsules. In some cases, nucleic acid manipulation reagents (eg, shRNA, siRNA, or gRNA) are attached to bead capsules, wherein the beads are delivered into compartments such that a single bead and a single cell are contained within separate compartments. When single-cell/single-group nucleic acid manipulation reagent occupancy is the most desired state, it should be understood that there will often be multiple occupied partitions (in terms of cells, beads, or both), or unoccupied partitions (in terms of cells, beads, or for both). In some cases, the ratio of nucleic acid reagent-carrying cells to capsules in a single partition is 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10.

In some cases where the CRISPR system nucleic acid manipulation reagents are used, the guide RNA and the nuclease components of the RNA guide can be contained on separate beads. In some cases of this configuration, the ratio of beads carrying guide RNA to beads carrying RNA-guided nuclease in a particular partition is 10:1, 9:1, 8:1, 7:1, 6: 1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10. In some cases of this configuration, cells, beads carrying guide RNA, and beads carrying RNA-guided nuclease are present in a partition in a 1:1:1 ratio.

As used herein, a partition refers to a vessel or container, which may comprise a variety of different forms, such as pores, tubes, micro- or nanopores, through-holes, and the like. However, in a preferred aspect, the partitions are flowable within the fluid flow. These containers may comprise, for example, microcapsules or microvesicles with an outer barrier surrounding an inner fluid center or core, or they may be porous matrices capable of entraining and/or retaining material within their matrix. In some aspects, these partitions can include droplets of aqueous fluid within a non-aqueous continuous phase (eg, an oil phase). A variety of different containers are described, for example, in US Patent Publication No. 2014/0155295. Likewise, emulsion systems for forming stable droplets in non-aqueous or oily continuous phases are described in detail, for example, in US Patent Publication No. 2010/0105112. In some cases, microfluidic channel networks are particularly suitable for creating partitions as described herein. Examples of such microfluidic devices include those described in detail in Provisional U.S. Patent Application No. 61/977,804, filed April 4, 2014, the entire disclosure of which is for all purposes and with particular reference to microfluidic devices. All teachings relating to the device are hereby incorporated by reference in their entirety. Alternative mechanisms may also be employed in distributing individual cells, including porous membranes through which the aqueous mixture of cells is extruded into the non-aqueous fluid. Such systems are commonly available, for example, from Nanomi, Inc.

In the case of droplets in emulsions, distributing cells and capsules carrying nucleic acid manipulation reagents into discrete compartments can typically be accomplished by flowing an aqueous sample-containing stream into the junction and also passing a dispensing fluid (e.g., fluorine A non-aqueous stream of carburetor flows into the junction such that aqueous droplets are formed within the flowing stream distribution fluid, wherein such droplets include sample material. The relative amounts of cells and capsules carrying nucleic acid manipulation reagents in any particular partition can be adjusted by controlling various parameters of the system, including, for example, the concentration of cells or capsules carrying nucleic acid manipulation reagents in the aqueous stream, the aqueous Flow rate of flow and/or non-aqueous flow, etc.

Microfluidic devices can be used to provide controlled distribution of cells and capsules containing nucleic acid reagents. In some cases, a microfluidic device comprising a network of microfluidic channel structures is used to deliver nucleic acid manipulation reagents and cells into the same compartment. Examples of such microfluidic devices include those described in detail in Provisional U.S. Patent Application No. 61/977,804, filed April 4, 2014, the entire disclosure of which is for all purposes and with particular reference to microfluidic devices. All teachings relating to the device are hereby incorporated by reference in their entirety.

An example of a microfluidic channel configuration for co-dispensing cells and beads containing an agent oligonucleotide that alters gene expression is schematically illustrated in FIG. 1 . As described herein, in some aspects a substantial percentage of all occupied partitions will include both beads and cells, and in some cases some of the resulting partitions will be unoccupied. In some cases, some of the partitions may have a distribution of beads and cells that is not 1:1. In some cases it may be desirable to provide multiple occupancy partitions, for example containing two, three, four or more cells and/or beads within a single partition. As shown, channel segments 102 , 104 , 106 , 108 , and 110 are disposed in fluid communication at channel junction 112 . An aqueous stream containing individual cells 114 is passed through channel segment 102 to channel junction 112 . These cells may be suspended in an aqueous fluid, as described above, or may have been pre-encapsulated prior to the dispensing process.

Referring to FIG. 1 , an aqueous flow of cells 114 flows through channel segment 102 to channel junction 112 . Simultaneously, an aqueous flow comprising beads 116 carrying nucleic acid manipulation reagents flows through channel segment 104 to channel junction 112 . Non-aqueous dispensing fluid 116 is introduced into channel junction 112 from each side channel 106 and 108 , and the combined stream flows into outlet channel 110 . Within channel junction 112 , the two combined aqueous streams from channel sections 102 and 104 are combined together and distributed into droplets 218 comprising co-distributed cells 114 and beads 116 . As previously mentioned, by controlling the flow characteristics of each of the combined fluids at channel junctions 112 as well as controlling the geometry of the channel junctions, the combination and distribution can be optimized to achieve the desired bead within the resulting partitions 118 Occupancy levels of granules, cells, or both.

In some cases, where single cell and/or bead partitions are required, it may be necessary to control the relative flow rates of the fluids so that the partitions contain on average less than one cell and/or bead per partition in order to ensure that those that are occupied The partitions are primarily single-occupied. Likewise, it may be desirable to control the flow rate so that a higher percentage of partitions is occupied, eg allowing only a smaller percentage of unoccupied partitions. In a preferred aspect, flow and channel configuration are controlled to ensure a desired number of single occupied zones, unoccupied zones below a certain level, and multiple occupied zones below a certain level.

The number of microfluidic channels can depend on the number of different reagents used in a particular application. In some cases where the method is performed with two or more nucleic acid manipulation reagents on separate capsules, each of the two or more nucleic acid manipulation reagents is carried on a separate microfluidic channel via a separate microfluidic channel. in an aqueous stream. This configuration allows control over the amount of each individual reagent that is dispensed with each cell. For example, in the case of using a CRISPR system, the guide RNA is introduced into the partition via a first channel in a first aqueous flow, and the RNA-guided nuclease (e.g., Cas9 nuclease or CpfI nuclease) is introduced in a second channel via a second channel. Two aqueous streams are introduced into the partitions. In another example, where a CRISPR system is used, the guide RNA and the RNA-guided nuclease are introduced into the compartments by the same aqueous flow through the same channel. In some cases, the streams carrying each of the two or more nucleic acid manipulation reagents are combined into a partition, and the stream carrying the full set of nucleic acid manipulation reagents is delivered to the partition containing the target cells. In other cases, streams carrying two or more nucleic acid manipulation reagents are each delivered separately to the compartments containing the target cells. Although certain aspects of the subject systems and methods are described herein in the context of a CRISPR system, those skilled in the art will recognize that other nucleic acid manipulation reagents, including those described herein, can also be used with the subject systems and methods In conjunction with.

A network of channels, eg as described herein, may be fluidly coupled to appropriate fluidic components. For example, inlet channel segments (eg, channel segments 102 , 104 , 106 , and 108 ) are fluidly coupled to a suitable source of material that they deliver to channel junction 112 . For example, channel section 102 is fluidly coupled to a source of an aqueous suspension of cells 114 to be analyzed, while channel section 104 is fluidly coupled to a source of aqueous suspension of beads 116 carrying nucleic acid manipulation reagents. Channel sections 106 and 108 then fluidly connect to one or more sources of non-aqueous fluid. These sources may include any of a variety of different fluidic components ranging from simple reservoirs defined in or connected to the body structure of the microfluidic device to fluidic conduits that deliver fluid from sources outside the device, manifolds, etc. A sort of. Likewise, outlet channel segment 110 may be fluidly coupled to a receptacle or conduit for dispensed cells. Again, this may be a reservoir defined in the body of the microfluidic device, or it may be a fluidic conduit for delivering the dispensed cells to a subsequent process operation, instrument or component.

In many cases, the systems and methods are used to ensure that the vast majority of occupied partitions (partitions containing one or more microcapsules) contain no more than 1 target cell per occupied partition. In some cases, the allocation process is controlled so that less than 25% of the occupied partitions contain more than one target cell, and in many cases, less than 20% of the occupied partitions have more than one target cell, and in some In cases, less than 10% or even less than 5% of the occupied partitions comprise more than one cell per partition.

Additionally or alternatively, in many cases it is desirable to avoid creating an excessive number of empty partitions. While this can be achieved by providing a sufficient number of target cells into the distribution region, a poissonian distribution is expected to increase the number of partitions that will include multiple cells. Thus, according to aspects described herein, the flow directed to one or more cells or other fluids in a distribution zone is controlled such that in many cases no more than 50% of the resulting partitions are unoccupied, i.e. including Less than 1 target cell, no more than 25% of the generated partitions, no more than 10% of the generated partitions may be unoccupied. Furthermore, in some aspects, these flows are controlled to exhibit a non-Poisson distribution of single occupied partitions while providing a lower level of unoccupied partitions. Stated again, in some aspects, the range of unoccupied partitions mentioned above can be achieved while still providing any of the single occupancy rates described above. For example, in many cases, using the systems and methods described herein creates multiple occupancy The resulting partitions at a rate of less than 50%, below 40%, below 30%, below 20%, below 10%, and in some cases below 5% of unoccupied partitions.

As will be appreciated, the occupancy rates described above also apply to partitions that include both target cells and capsules carrying nucleic acid manipulation reagents. Specifically, in some aspects, a substantial proportion of the total occupied partitions will include both capsules and cells. Specifically, it may be desirable to have at least 50% of the partitions occupied by at least one cell and at least one set of nucleic acid manipulation reagents, or at least 75% of the partitions may be so occupied, or even at least 80% or at least 90% of the partitions may be so occupied occupy. Furthermore, in those cases where it is desired to provide a single cell and a single set of nucleic acid manipulation reagents within a partition, at least 50% of the partition may be so occupied, at least 60%, at least 70%, at least 80%, or even at least 90% of the partition may be so occupied.

Although described above with respect to providing substantially single-occupancy partitions, in some cases it may be desirable to provide, for example, two, three, four or more cells and/or capsules within a single partition (e.g. , multiple occupancy partitions of beads), said capsules containing said set of nucleic acid manipulation reagents. Thus, as mentioned above, the flow characteristics of the fluid containing cells and/or capsules (eg, beads) and the dispensing fluid can be controlled to provide such multiple occupancy zones. Specifically, the flow parameters can be controlled to provide an occupancy of greater than 50%, greater than 75%, and in some cases greater than 80%, 90%, 95% or higher of the zone. In particular embodiments, the flow parameters are controlled to provide greater than 50%, greater than 75%, and in some cases greater than 80%, 90%, 95% or more of a partition for a single cell and a set of reagents for nucleic acid manipulation Multiple occupancy required.

Additionally, in many cases, capsules within a single partition may contain different nucleic acid manipulation reagents associated therewith. For example, in methods and systems employing CRISPR systems, a first capsule can contain a first guide RNA and a second capsule can contain an oligonucleotide encoding an RNA-guided nuclease. In some cases where two or more target nucleic acids are edited, the third capsule may comprise a second guide RNA that targets a different nucleic acid than the first guide RNA. In such cases, it may be advantageous to introduce different capsules from different capsule sources (i.e., containing different associated reagents) into a common channel or droplet generation junction through different channel inlets into such common channel or droplet generation junction. Droplets are produced in the junction. In such cases, the flow rate and frequency of the different capsules entering the channel or junction can be controlled to provide the desired ratio of microcapsules from each source while ensuring that the desired pairing or combination of such capsules enters the compartment with the desired number of cells . In an exemplary embodiment, capsules containing different nucleic acid manipulation reagents are delivered in a 1:1 ratio into the cell-containing compartments.

The compartments described herein are often characterized as having extremely small volumes, e.g., less than 10 μL, less than 5 μL, less than 1 μL, less than 900 picoliters (pL), less than 800 pL, less than 700 pL, less than 600 pL, less than 500 pL, less than 400 pL, less than 300 pL , less than 200 pL, less than 100 pL, less than 50 pL, less than 20 pL, less than 10 pL, less than 1 pL, less than 500 nanoliters (nL) or even less than 100 nL, 50 nL or even less.

For example, in the case of droplet-based partitioning, a droplet may have less than 1000 pL, less than 900 pL, less than 800 pL, less than 700 pL, less than 600 pL, less than 500 pL, less than 400 pL, less than 300 pL, less than 200 pL, less than 100 pL, less than 50 pL, less than A total volume of 20 pL, less than 10 pL or even less than 1 pL. In the case of co-distribution with beads, it will be appreciated that the volume of sample fluid within a partition, for example comprising co-distributed cells, may be less than 90%, less than 80%, less than 70%, less than the volumes described above. less than 60%, less than 50%, less than 40%, less than 30%, less than 20% or even less than 10% of the volume described above.

As described elsewhere herein, partitioning the species can result in partitioned populations. In such cases, any suitable number of partitions can be generated to create a partition population. For example, in the methods described herein, one can generate A partition population of about 1,000,000 partitions, at least about 5,000,000 partitions, at least about 10,000,000 partitions, at least about 50,000,000 partitions, at least about 100,000,000 partitions, at least about 500,000,000 partitions, or at least about 1,000,000,000 partitions. Furthermore, the partition population may include both unoccupied partitions (eg, empty partitions) and occupied partitions.

In addition to the capsules containing at least one nucleic acid manipulation reagent, one or more other reagents can also be dispensed with the cells undergoing nucleic acid editing. For example, one or more agents for facilitating the uptake of at least one nucleic acid manipulation agent into a cell. In some cases, one or more transfection reagents are dispensed with the cell and nucleic acid manipulation reagents. In the case of electroporation for uptake of nucleic acid manipulation reagents, an electroporation buffer may be included in the cell compartment undergoing nucleic acid editing. Such additional reagents can be dispensed to the cells together with the capsules containing the full set of nucleic acid manipulation reagents or can be delivered to the cells separately from the capsules containing the nucleic acid manipulation reagents.

After dispensing cells and capsules containing at least one nucleic acid manipulation agent, the capsules are caused to release the nucleic acid manipulation agent, thereby enabling individual cells to take up the agent. In some cases, the stimulus can be photostimulation, eg, by cleavage of a photolabile bond of a releasable oligonucleotide. In some cases, thermal stimulation may be used, where an increase in the temperature of the bead environment may result in cleavage of linkages or other release of oligonucleotides from the bead. In some cases, a chemical stimulus can be used, thereby cleaving the linkage of the oligonucleotide to the bead, or can otherwise cause the release of the oligonucleotide from the bead. In the case of light or thermal stimulation, the stimulus can be introduced by heat or a light source through openings in the microfluidic channel carrying the partitions into the compartments containing the cell and nucleic acid manipulation reagents. The chemical stimulus can be dispensed with the target cells prior to dispensing the target cells and the capsules carrying the nucleic acid manipulation reagents. In this example, nucleic acid manipulation reagents will only be released from the capsule in the presence of target cells and chemical stimuli.

Upon release of the nucleic acid manipulation reagent from the capsule, uptake of the nucleic acid manipulation reagent by the cells can be performed using any suitable method. As mentioned herein, one or more cellular uptake agents may be included to facilitate the uptake of nucleic acid manipulation agents into cells. In some cases, the one or more cellular uptake reagents are transfection reagents including, for example, polymer (eg, DEAE dextran)-based transfection reagents and cationic liposome-mediated transfection reagents. Electroporation of cells can also be used to facilitate the uptake of nucleic acid manipulation reagents. By applying an external field, an altered transmembrane potential is induced in the cell, and when the net transmembrane potential (sum of applied and resting potential differences) is greater than a threshold, a transient permeable structure is created in the membrane and electroporation is achieved. See, eg, Gehl et al., Acta Physiol. Scand. 177:437-447 (2003). Cells used with the subject systems and methods can be electroporated prior to delivery of the nucleic acid manipulation reagents or after dispensing the nucleic acid manipulation reagents with the cells. In some cases, an electroporation buffer can be delivered to the compartment containing the nucleic acid manipulation reagents and target cells to allow electroporation of the target cells and uptake of the nucleic acid reagents. Nucleic acid manipulation reagents can also be delivered to target cells by viral transduction. Suitable viral delivery systems include, but are not limited to, adeno-associated virus (AAV) retroviral and lentiviral delivery systems. Such viral delivery systems are particularly useful where cells are resistant to transfection. Where a viral delivery system is used, a virus (e.g., adeno-associated virus (AAV), retrovirus, or lentivirus) carrying the agent (e.g., nucleotides encoding the agent) can be encapsulated in a capsule, The capsules are then delivered to the cell-containing compartments. The virus in turn introduces the agent from the agent into the single cell upon release from the capsule. The method using a virus-mediated delivery system may also include the steps of preparing a viral vector encoding a nucleic acid manipulation agent and packaging the vector into viral particles. Other delivery methods for nucleic acid reagents include lipofection, nucleofection, microinjection, gene guns, virosomes, liposomes, immunoliposomes, polycations or lipid:nucleic acid conjugates, naked DNA, artificial viruses Reagents enhance uptake of particles as well as nucleic acids. See also Neiwoehner et al., Nucleic Acids Res. 42:1341-1353 (2014), which is hereby incorporated by reference in its entirety for all purposes and particularly for all teachings related to agent delivery systems.

Each partition of the subject method may comprise a particular cell type or different cell types, depending on the desired application. For example, the subject methods can be used in high-throughput genetic screens for one specific cell type or containing specific alleles (e.g., replacing a mutant allele in a specific gene with a wild-type allele or replacing a wild-type allele with a mutant allele). type allele) cell type mass production. In some cases, the subject methods were used to characterize gene function across different cell types. In some cases, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 , 85, 90, 95, or 100 or more different cell types are used with the subject method. In applications using more than one cell type, a single partition may contain a single cell, multiple cells of the same cell type, or multiple cells of different cell types.

The methods and systems provided herein can be used to alter eukaryotic or prokaryotic cells. A eukaryotic cell may be a cell of or derived from a particular organism such as a mammal (including but not limited to a human, mouse, rat, rabbit, dog, or non-human primate). Such cells may be isolated as a blood or tissue sample from an organism, or may be an established cell line. Examples of cell lines that can be used with the subject systems and methods include, but are not limited to, C8161, CCRF-CEM, MOLT, mIMCD-3, NHDF, HeLa-S3, Huh1, Huh4, Huh7, HUVEC, HASMC, HEKn, HEKa, MiaPaCell , Panel, PC-3, TF1, CTLL-2, C1R, Rat6, CV1, RPTE, A10, T24, J82, A375, ARH-77, Calu1, SW480, SW620, SKOV3, SK-UT, CaCo2, P388D1, SEM -K2, WEHI-231, HB56, TIB55, Jurkat, J45.01, LRMB, Bc1-1, BC-3, IC21, DLD2, Raw264.7, NRK, NRK-52E, MRC5, MEF, Hep G2, HeLa B , HeLa T4, COS, COS-1, COS-6, COS-M6A, BS-C-1 monkey kidney epithelial cells, BALB/3T3 mouse embryonic fibroblasts, 3T3Swiss, 3T3-L1, 132-d5 embryonic fibroblasts Cells; 10.1 Mouse fibroblasts, 293-T, 3T3, 721, 9L, A2780, A2780ADR, A2780cis, A172, A20, A253, A431, A-549, ALC, B16, B35, BCP-1 cells, BEAS- 2B, bEnd.3, BHK-21, BR 293, BxPC3, C3H-10T1/2, C6/36, Cal-27, CHO, CHO-7, CHO-IR, CHO-K1, CHO-K2, CHO-T , CHODhfr-/-, COR-L23, COR-L23/CPR, COR-L23/5010, COR-L23/R23, COS-7, COV-434, CML T1, CMT, CT26, D17, DH82, DU145, DuCaP , EL4, EM2, EM3, EMT6/AR1, EMT6/AR10.0, FM3, H1299, H69, HB54, HB55, HCA2, HEK-293, HeLa, Hepa1c1c7, HL-60, HMEC, HT-29, Jurkat, JY cells, K562cells, Ku812, KCL22, KG1, KYO1, LNCap, Ma-MeI 1-48, MC-38, MCF-7, MCF-10A, MDA-MB-231, MDA-MB-468, MDA-MB-435 , MDCK II, MDCK II, MOR/0.2R, MONO-MAC6, MTD-1A, MyEnd, NC I-H69/CPR, NCI-H69/LX10, NCI-H69/LX20, NCI-H69/LX4, NIH-3T3, NALM-1, NW-145, OPCN/OPCT cell line, Peer, PNT-1A/PNT2, RenCa, RIN-5F, RMA/RMAS, Saos-2 cells, Sf-9, SkBr3, T2, T-47D, T84, THP1 cell lines, U373, U87, U937, VCaP, Vero cells, WM39, WT-49, X63 , YAC-1, YAR and their transgenic varieties. Cell lines are available from a variety of sources known to those of skill in the art (see, eg, American Type Culture Collection (ATCC) (Manassus, Va.)).

Cells that have undergone nucleic acid manipulation events may be further processed, depending on the application of the method. In a phenotype-specific screen, cells can be subjected to phenotype-specific selection conditions and screened for the phenotype. For example, in a screen for growth mutants, cells can be transferred to microwells or suitable compartments that allow cell growth, and the presence or absence of growth determined after a defined period of time. Screening for phenotypes can be performed using any suitable technique including, for example, fluorescence, luminescence, and high-content imaging techniques. See, eg, Hasson et al., Nature 504:291-295 (2013); Neumann et al., Nature Methods 3:385-390 (2006); and Moffat et al., Cell 124:1283-1298 (2006). Where the phenotype is cell-autonomous, the phenotype can be selected for fluorescent or cell surface markers by cell sorting. Nucleic acid mutations attributable to a desired phenotype can be identified by any suitable method. In some cases, next-generation sequencing technologies can be used to determine nucleic acid manipulations. As discussed herein, detectable labels can also be used to track specific nucleic acid mutations. For example, fluorescent or oligonucleotide "barcode" tags can be used to track specific nucleic acid manipulations associated with specific target phenotypes.

III. Devices, Systems and Kits

Also provided herein are microfluidic devices for dispensing target cells with capsules carrying nucleic acid manipulation reagents as described above. Such microfluidic devices may include a network of channels for carrying out dispensing processes such as those set forth in FIG. 1 . Examples of particularly useful microfluidic devices are described in US Provisional Patent Application No. 61/977,804, filed April 4, 2014. Briefly, these microfluidic devices can include a network of channels (such as those described herein) for distributing cells into individual compartments and co-distributing such cells with, for example, nucleic acid manipulation reagents disposed on beads. These channel networks can be disposed within a solid body (such as a glass, semiconductor, or polymer body structure in which the channels are defined), wherein those channels are connected at their ends for receiving various input fluids and for output from the channel network. Reservoir communication for final deposition of dispensed cells etc. For example, and with reference to FIG. 1 , an aqueous suspension of cells 114 may be provided to a reservoir fluidly coupled to channel 102 while an aqueous suspension of beads 116 carrying nucleic acid manipulation reagents may be provided to a reservoir coupled to channel 104 . Channel sections 106 and 108 may be provided with a non-aqueous solution, such as oil, into which aqueous fluid is dispensed in droplets at channel junction 112 . Finally, an outlet reservoir can be fluidly coupled to channel 110 into which the dispensed cells and beads can be delivered and harvested. As will be appreciated, while described in the form of a reservoir, it should be appreciated that the channel segments may be coupled to any of a variety of different fluid source or sink components, including piping, manifolds, or other fluid components of the system.

Also provided are systems for controlling the flow of these fluids through a network of channels, eg, by applied pressure differentials, centrifugal force, electrokinetic pumping, capillary or gravity flow, and the like.

Also provided herein are kits for high-throughput alteration of nucleic acids in multiple target cells. The kit may include one, two, three, four, five or more up to all dispensing fluids, including aqueous buffers and non-aqueous dispensing fluids or oils; Capsule (e.g., bead) associated nucleic acid manipulation reagents; microfluidic devices; additional reagents for cellular uptake of nucleic acid manipulation reagents; and instructions for use of any of the foregoing in the methods described herein .

IV. Application

The subject systems and methods provided herein can be used to produce larger populations of individual cells each carrying the same mutation or different mutations. For example, the systems and methods can be used to perform high-throughput genome-scale screening. Such screens can be performed with libraries capable of interfering with a variety of target nucleic acids in the genome of the target cell. In some cases using a CRISPR system, the nucleic acid manipulation reagents comprise a library of guide RNAs that span the genome of the target cell. In some cases, the nucleic acid manipulation reagent comprises 10 ¹ , 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ , or 10 ¹⁰ or more different targeting cells in the genome The same number of guide RNAs for different target nucleic acids. Such methods can be used, for example, in positive screens to identify perturbations that confer resistance to drugs, toxins or pathogens. In such cases, the drug, toxin, or pathogen is introduced into each cell-containing compartment after the cells undergo changes to the target nucleic acid. Cells that continue to grow in the presence of drugs, toxins, or pathogens are classified as containing protective mutations attributable to changes in the target nucleic acid. Such target nucleic acids attributable to the phenotype of interest are identified and further characterized, for example, using high-throughput sequencing techniques (eg, next-generation sequencing techniques), as discussed above.

In some cases, the subject systems and methods are used to perform negative selection under selection pressure. For example, in some applications, cells that have undergone nucleic acid editing are selected for a desired cellular function, such as prolonged growth. In such cases, exhausted cells that are unable to grow are classified as carrying agents that target nucleic acids necessary for cell proliferation. Such negative screens identify gene perturbations that selectively target cancer cells with known oncogenic mutations. Genes identified in this negative screen may serve as possible cancer drug targets.

The subject systems and methods are also useful for large-scale production of cells containing genetic alterations of interest. For example, the subject systems and methods can be used to efficiently create large numbers of cells containing alterations of known oncogenes. Such mutant cells can then be used to screen for other genes that inhibit growth. Genes that are critical for growth are identified as putative drug targets. In some cases, the methods are used to produce cells that contain a mutation in a target nucleic acid that affects a biological pathway of interest. Such cells can then be used to identify other genes in biological pathways. The method can also be used to repair a mutated gene of interest. For example, the method can be used to replace a mutant allele with a wild-type allele in cells isolated from a subject having a disease associated with the mutant allele. The repaired cells can then be transplanted back into the subject as part of treatment for the disease.

The subject systems and methods can also be used for large-scale production of non-human transgenic animals or plants. In some cases, the subject methods can be used to generate transgenic animals that are mammals, such as mice, rats, or rabbits. The subject methods can also be used for the large-scale production of crops containing nucleic acid mutations of interest, such as drought-resistant crops. See, eg, Lawlor, 64(1):83-108 (2013), which is hereby incorporated by reference in its entirety for all purposes and in particular for all teachings relating to drought resistance-conferring mutations.

The systems and methods provided herein can be used to create plants, animals or cells that can be used as disease models. For example, the subject methods provided herein can be used to generate modified animals or cells that can comprise one or more target nucleic acids associated with a disease, or animals wherein expression of one or more target nucleic acids associated with a disease is altered or cell. Such target nucleic acid sequences may encode disease-associated protein sequences or may be disease-associated control sequences. Such disease models can be used to study the development and/or progression of a disease using standards commonly used to study the disease. This disease model is also useful for studying the effects of pharmaceutically active compounds on disease.

Example

Genome-wide screens for tumor enhancers and suppressor genes

Human KBM7CML cells were screened for mutations that play a role in DNA mismatch repair (MMR) using the systems and methods provided herein and CRISPR/Cas9 reagents. In the presence of the nucleotide analog 6-thioguanine (6-TG), MMR-proficient cells cannot repair 6-TG-induced damage and are stuck at the G2-M cell cycle checkpoint, whereas MMR-deficient cells cannot Recognize damage and proceed with division.

CRISPR/Cas9 reagents for generating genome-wide mutations were constructed. A guide RNA (gRNA) library containing 50,000 different gRNAs targeting more than 5,000 different KBM7 genes was constructed. The gRNA library contained 10 gRNAs for each of the 5,000 genes. Each gRNA also includes an oligonucleotide barcode sequence for identification: gRNAs targeting the same gene have the same barcode sequence, while gRNAs targeting different genes have different sequences. The gRNAs from the gRNA library are chemically cross-linked to the gel beads such that each bead contains gRNAs targeting the same gene.

Gel beads carrying gRNA and Cas9 nuclease were introduced into the droplet compartments containing KBM7CML cells using a microfluidic device as schematically shown in Figure 1 . First partitions containing the full set of CRISPR/Cas9 reagents are generated, each first partition being a microdroplet containing a Cas9 nuclease and a gel bead carrying a guide RNA. Each first partition is then assigned to a second partition containing transfection reagent. Each second partition is then assigned to a third partition containing individual cells and a chemical reagent to dissolve the gel beads. In the third compartment, CRISPR/Cas9 reagents are released from the beads and taken up by KBM7 cells, allowing gene editing in KBM7 cells.

Cells from each partition were then pooled and grown in the presence of 6-TG. Cells capable of proliferating under these conditions may contain disruptions in genes affecting MMR. These cells were isolated and sequenced to identify genes involved in MMR. Unique barcode identifiers included in each gRNA facilitate sequencing.

This specification provides a complete description of methods, systems and/or structures, and uses thereof, in terms of embodiments of the presently described technology. Although various aspects of this technology have been described above with a certain degree of particularity or with reference to one or more unique aspects, those skilled in the art can make changes to the disclosed aspects without departing from the spirit or scope of this technology. Lots of changes. Since many aspects can be devised without departing from the spirit and scope of the presently described technology, the proper scope resides in the hereinafter appended claims. Thus covering other aspects. Furthermore, it should be understood that any operations may be performed in any order, unless otherwise expressly required or required by the language itself. It is intended that all matter contained in the above description and shown in the accompanying drawings be interpreted as illustrative of certain aspects only and not as limitations of the illustrated embodiments. Unless otherwise clear or expressly stated from the context, any concentration values provided herein are generally given in terms of mixture values or percentages, without regard to any transformations that occur upon or after the addition of specific components of the mixture. All published references and patent documents referred to in this disclosure are hereby incorporated by reference in their entirety for all purposes, if not already expressly incorporated herein. Changes in detail or structure may be made without departing from the essential elements of the technology as defined in the following claims.

Claims (60)

1. a kind of being delivered to the method in respective cells by reagent, the method includes：

(a) multiple capsules are provided, wherein the capsule includes the expression for changing at least one of cell gene outcome Reagent；

(b) by the capsule delivery to discrete partition, wherein the discrete partition further includes respective cells；

(c) cause content of the capsule by the capsule under conditions of enabling the respective cells to absorb the reagent It is discharged into the discrete partition,

To which the reagent is delivered in the respective cells.

2. the method as described in claim 1, wherein described for changing at least one gene outcome encoded by DNA molecular The reagent of expression includes：

(i) the first controlling element, first controlling element are operably coupled at least one coding CRISPR System guides The nucleotide sequence of RNA, the CRISPR System guides RNA and the target sequence in the DNA molecular in the respective cells Hybridization,

(ii) the second controlling element, second controlling element is operably coupled to the nuclease of coding RNA guiding or RNA draws The nucleotide sequence for the Nuclease fusion protein led.

Wherein component (i) and (ii) is located on identical or different carrier, also, the nuclease of the wherein described RNA guiding and institute It is naturally occurring not together to state guiding RNA.

3. method as claimed in claim 2, wherein second controlling element is operably coupled to the core of coding RNA guiding The nucleotide sequence of sour enzyme, thus the guiding RNA target is to described in the target sequence and the nuclease cleavage of RNA guiding Thus DNA molecular changes the expression of at least one gene outcome.

4. method as claimed in claim 3 is drawn wherein the reagent for changing gene expression is also included in by the RNA The donor nucleic acid being inserted into after DNA molecular described in the nuclease cleavage led in the DNA molecular.

5. method as claimed in claim 2, wherein second controlling element is operably coupled to the RNA that coding deactivates The nucleotide sequence of the nuclease of guiding, it is thus described to guide RNA target to the target sequence and the RNA guiding deactivated Nuclease interference encode at least one gene outcome nucleic acid transcription, thus change at least one gene outcome Expression.

6. method as claimed in claim 2, wherein second controlling element is operably coupled to the core of coding RNA guiding The nucleotide sequence of sour enzyme fusion proteins, the nuclease that thus the guiding RNA target is guided to the target sequence and the RNA Fusion protein interferes the expression of at least one gene outcome, thus changes the expression of at least one gene outcome.

7. method as claimed in claim 6, wherein the Nuclease fusion protein of RNA guiding is drawn comprising the RNA to deactivate The nuclease and transcriptional activators or transcription repressor led.

8. method as claimed in claim 6, wherein the Nuclease fusion protein includes the nuclease for the RNA guiding deactivated With the epigenetic modification factor.

9. such as method described in any item of the claim 1 to 8, wherein the nuclease of RNA guiding be Cas9 albumen or Cpf1 albumen.

10. method as claimed in any one of claims 1-9 wherein, wherein the capsule is configured as discharging when applying stimulation Its content.

11. method as claimed in claim 10, wherein the stimulation is selected from chemical stimulation, electro photoluminescence, thermostimulation, magnetic thorn Swash, the variation of pH value, the variation of ion concentration, the reduction of disulfide bond, light stimulus with and combinations thereof.

12. method as claimed in claim 11, wherein the stimulation is thermostimulation.

13. the method as described in any one of claim 1 to 12, wherein the multiple capsule includes about 100 to 100,000 kind For changing the different reagents of the expression of at least one gene outcome, so that different respective cells receive different examinations Agent.

14. the method as described in any one of claim 1 to 13, wherein the capsule also includes one or more for described The additive of capsule or its content and the compatibility of the respective cells.

15. method as claimed in claim 14, wherein one or more additives include transfection agents.

16. the method as described in any one of claim 1 to 15, wherein the table for changing at least one gene outcome The reagent reached also includes oligonucleotides, and the oligonucleotides includes nucleic acid bar code sequence, and wherein different individual cells Receive different nucleic acid bar code sequences.

17. the method as described in any one of claim 2 to 16, wherein the table for changing at least one gene outcome The reagent reached includes also a pair of Cas9 nickases or Cas9 fusion proteins, when the nuclease guided using RNA compared with, it is described Cas9 nickases or Cas9 fusion proteins improve the specificity of the CRISPR systems.

18. the method as described in any one of claim 2 to 17, wherein the target sequence has in the cellular genome Little or no affiliation.

19. the method as described in any one of claim 2 to 18, wherein the table for changing at least one gene outcome The reagent reached also includes homologous in the cell to increase by the gene of inhibition participation non-homologous end joining (NHEJ) approach The reagent of the frequency of recombination.

20. method as claimed in claim 19, wherein the reagent include with the coding CRISPR System guides RNA extremely The Cas9 albumen of few nucleotide sequence coded a Cas9 nucleases or nuclease free.

21. the method as described in any one of claim 2 to 20, wherein the guiding RNA also includes and the cytogene The identical spacer region of targeting protospacer sequence in group.

22. the method as described in any one of claim 2 to 21, wherein second controlling element is inducible promoter.

23. method as claimed in claim 22, wherein the inducible promoter is selected from the group being made of the following terms：Light Inducible promoter, heat-inducible promoter and chemical inducible promoter.

24. a kind of method for reagent to be delivered to cell, the method includes：

(a) reagent for being releasedly coupled to microcapsules is provided；

(b) microcapsules are separated into discrete partition, wherein the discrete partition further includes respective cells；And

(c) reagent is discharged under conditions of so that the reagent is absorbed in the cell.

25. method as claimed in claim 24, wherein the reagent includes between the nuclease of coding RNA guiding, Regularity Every short palindrome repetitive sequence (CRISPR) or can hybridize with one or more of the DNA molecular of cell target sequence CRISPR guides the carrier of at least one of RNA and one or more condition inducible promoters.

26. method as claimed in claim 25, wherein the reagent includes：

(i) operable first controlling element in eukaryocyte, first controlling element are operably coupled at least one The nucleotide sequence of a coding CRISPR System guides RNA, the CRISPR System guides RNA and the intracellular target sequence Hybridization,

(ii) the second controlling element, second controlling element is operably coupled to the nuclease of coding RNA guiding or RNA draws The nucleotide sequence for the Nuclease fusion protein led.

Wherein component (i) and (ii) is located on identical or different carrier, also, the nuclease of the wherein described RNA guiding and institute It is naturally occurring not together to state guiding RNA.

27. method as claimed in claim 26, wherein second controlling element is operably coupled to coding RNA guiding The nucleotide sequence of nuclease, the nuclease cleavage institute that thus the guiding RNA target is guided to the target sequence and the RNA DNA molecular is stated, the expression of at least one gene outcome is thus changed.

28. method as claimed in claim 26, wherein second controlling element is operably coupled to what coding deactivated The nucleotide sequence of the nuclease of RNA guiding, thus the guiding RNA target is to the target sequence and the RNA to deactivate The nuclease interference of guiding encodes the transcription of the nucleic acid of at least one gene outcome, thus changes at least one gene The expression of product.

29. method as claimed in claim 26, wherein second controlling element is operably coupled to coding RNA guiding The nucleotide sequence of Nuclease fusion protein, the nucleic acid that thus the guiding RNA target is guided to the target sequence and the RNA Enzyme fusion proteins interfere the expression of at least one gene outcome, thus change the expression of at least one gene outcome.

30. method as claimed in claim 29, wherein the Nuclease fusion protein of RNA guiding includes the RNA to deactivate The nuclease and transcriptional activators or transcription repressor of guiding.

31. method as claimed in claim 30, wherein the Nuclease fusion protein includes the nucleic acid for the RNA guiding deactivated Enzyme and the epigenetic modification factor.

32. the method as described in any one of claim 26 to 31, wherein the nuclease of RNA guiding be Cas9 albumen or Cpf1 albumen.

33. the method as described in any one of claim 26 to 32, wherein one or more carriers being capable of stable integration Into the genome of the cell.

34. the method as described in claim 24 to 33, wherein the release steps include to the microcapsules apply stimulation with Discharge the reagent.

35. method as claimed in claim 34, wherein the stimulation is selected from chemical stimulation, electro photoluminescence, thermostimulation, magnetic thorn Swash, the variation of pH value, the variation of ion concentration, the reduction of disulfide bond, light stimulus with and combinations thereof.

36. the method as described in any one of claim 24 to 35, wherein promoting the reagent intake to institute by electroporation It states in cell.

37. the method as described in any one of claim 24 to 36, wherein the microcapsules also include one or more additions Agent is to improve the reagent intake to the compatibility in the cell.

38. method as claimed in claim 37, wherein one or more additives include transfection reagent.

39. the method as described in any one of claim 24 to 38, wherein the microcapsules include the droplet in lotion With the member of cross-linked polymer.

40. the method as described in any one of claim 24 to 39, wherein the microcapsules include bead.

41. method as claimed in claim 40, wherein the bead is gel bead.

42. the method as described in any one of claim 24 to 42, wherein the microcapsules also include releasedly with its idol The group of the nucleic acid bar code sequence of connection, wherein it includes identical bar code sequence that the bar code sequence is substantially all.

43. method as claimed in claim 42, wherein the bar code sequence also includes hairpin.

44. the method as described in claim 25 to 43 merges wherein the reagent also includes a pair of Cas9 nickases or Cas9 Albumen, when the nuclease guided using RNA compared with, the Cas9 nickases or Cas9 fusion proteins improve the CRISPR The specificity of system.

45. a kind of method for changing the gene expression in multiple cells, the method includes：

(a) multiple capsules are provided, wherein capsule includes the reagent of the expression for changing at least one gene outcome, the reagent Including engineering, non-naturally occurring Regularity interval short palindrome repetitive sequence (CRISPR) system, the system packet Containing one or more carriers, one or more carriers include：

(i) the first controlling element, first controlling element are operably coupled at least one coding CRISPR-Cas systems The nucleotide sequence of RNA is guided, the CRISPR-Cas System guides RNA can be with the target sequence in the DNA molecular of the cell Row hybridization, and

(ii) the second controlling element, second controlling element are operably coupled to the nuclease protein of coding RNA guiding Nucleotide sequence, wherein component (i) and (ii) are located on the identical or different carrier of the system,

It (b) will be in the capsule delivery to the discrete partition containing respective cells；

(c) stimulation is provided to cause the capsule to be discharged in it under conditions of making reagent be delivered in the respective cells It is tolerant,

After it relays the application stimulation, the guiding RNA hybridizes with the target sequence, and the nucleic acid of RNA guiding The DNA molecular of the zymoprotein cracking containing the target sequence, thus changes the expression of at least one gene outcome.

46. method as claimed in claim 45, wherein the reagent for changing gene expression is also included in by the RNA The donor nucleic acid being inserted into after DNA molecular described in the nuclease cleavage of guiding in the DNA molecular.

47. a kind of method for changing the gene expression in multiple cells, the method includes：

(a) multiple capsules are provided, wherein capsule includes the reagent of the expression for changing at least one gene outcome, the reagent Including engineering, non-naturally occurring Regularity interval short palindrome repetitive sequence (CRISPR) system, the system packet Containing one or more carriers, one or more carriers include：

(i) the first controlling element, first controlling element are operably coupled at least one coding CRISPR-Cas systems The nucleotide sequence of RNA is guided, the CRISPR-Cas System guides RNA can be with the target sequence in the DNA molecular of the cell Row hybridization, and

(ii) the second controlling element, second controlling element are operably coupled to the nucleic acid for the RNA guiding that coding deactivates The nucleotide sequence of enzyme, wherein component (i) and (ii) are located on the identical or different carrier of the system,

It (b) will be in the capsule delivery to the discrete partition containing respective cells；

(c) stimulation is provided to cause the capsule to be discharged in it under conditions of making reagent be delivered in the respective cells It is tolerant,

After it relays the application stimulation, the guiding RNA hybridizes with the target sequence, and the RNA to deactivate draws The nuclease interference led encodes the transcription of the nucleic acid of at least one gene outcome, thus changes at least one gene production The expression of object.

48. a kind of method for changing the gene expression in multiple cells, the method includes：

(a) multiple capsules are provided, wherein capsule includes the reagent of the expression for changing at least one gene outcome, the reagent Including engineering, non-naturally occurring Regularity interval short palindrome repetitive sequence (CRISPR) system, the system packet Containing one or more carriers, one or more carriers include：

(i) the first controlling element, first controlling element are operably coupled at least one coding CRISPR-Cas systems The nucleotide sequence of RNA is guided, the CRISPR-Cas System guides RNA can be with the target sequence in the DNA molecular of the cell Row hybridization, and

(ii) the second controlling element, second controlling element are operably coupled to the histone-nuclease fusion egg of coding RNA guiding White nucleotide sequence, wherein component (i) and (ii) are located on the identical or different carrier of the system,

It (b) will be in the capsule delivery to the discrete partition containing respective cells；

(c) stimulation is provided to cause the capsule to be discharged in it under conditions of making reagent be delivered in the respective cells It is tolerant,

After it relays the application stimulation, the guiding RNA hybridizes with the target sequence, and the nucleic acid of RNA guiding Enzyme fusion proteins interfere the expression of at least one gene outcome, thus change the expression of at least one gene outcome.

49. method as claimed in claim 48, wherein the Nuclease fusion protein of RNA guiding includes the RNA to deactivate The nuclease and transcriptional activators or transcription repressor of guiding.

50. method as claimed in claim 48, wherein the Nuclease fusion protein includes the nucleic acid for the RNA guiding deactivated Enzyme and the epigenetic modification factor.

51. the method as described in any one of claim 45 to 50, wherein the nuclease of RNA guiding be Cas9 albumen or Cpf1 albumen.

52. the method as described in any one of claim 45 to 51, wherein different capsules include can with it is described individual thin The guiding RNA of the different target sequences hybridization of intracellular, so that the expression of different genes product changes in different cells.

53. the method as described in any one of claim 45 to 53, wherein the multiple capsule includes about 500 to about 100, 000 capsule.

54. the method as described in any one of claim 45 to 53, wherein the multiple capsule includes about 10,000 to about 50, 000 capsule.

55. the method as described in any one of claim 45 to 53, wherein the multiple capsule includes about 15,000 to about 30, 000 capsule.

56. the method as described in any one of claim 45 to 53, wherein only by single capsule delivery to each discrete partition In.

57. the method as described in any one of claim 45 to 56, wherein the capsule includes the droplet in lotion.

58. the method as described in any one of claim 45 to 56, wherein the capsule includes polymer gel.

59. method as claimed in claim 58, wherein the polymer gel is polyacrylamide.

60. the method as described in any one of claim 45 to 56, wherein the capsule includes gel bead.