High-throughput gene editing system and method

Abstract

This invention provides a high-throughput method for generating gene mutations in cells using CRISPR reagents, lipofection, and addressable array multiwell plates. [Solution] This disclosure provides a CRISPR-Cas system for generating mutant genes in cells within wells of an addressable well plate.

Description

Technical Field

[0001] The present disclosure generally relates to high-throughput methods of creating genetic mutations in cells using CRISPR reagents, lipofection, and an addressable array multiwell plate.

Background Art

[0002] Methods of using CRISPR reagents to create genetic mutations are known. See, e.g., International Publication No. 2018 / 057837, U.S. Patent No. 2019 / 0233820, International Publication No. 2019 / 025984, International Publication No. 2015 / 089465, U.S. Patent No. 2019 / 0153412, and International Publication No. 2015 / 089473. However, there is a need for methods of handling CRISPR reagents to improve their delivery into cells and increase CRISPR activity on high-throughput platforms.

Summary of the Invention

Problems to be Solved by the Invention

[0003] Aspects of the present disclosure relate to compositions and methods for multiplex gene editing using a CRISPR / Cas system comprising a Cas enzyme and a guide RNA known in the art for editing a target gene in target cells present in each of a plurality of wells within a well plate. Each well contains a guide RNA for a different target gene of the target cells. Thus, each well contains a CRISPR / Cas system targeting a single locus for the mutation. Considering each of the different single loci targeted for mutation within each well, multiple genes of the target cells are edited as a whole in the method, so that the genome of the target cells is efficiently multiplexed gene edited in parallel. Thus, a high-throughput screening method is provided for creating a single mutant gene in each well of an addressable or indexable multiwell plate using a CRISPR-Cas system.

Means for Solving the Problems

[0004] In one embodiment, the system comprises (1) a liposome containing guide RNA, wherein the guide RNA contains a specific spacer complementary to the target gene; (2) a nucleic acid sequence encoding a Cas enzyme or a liposome containing a Cas enzyme; and (3) a target cell. The guide RNA may be a dual guide RNA, i.e., a dual guide RNA containing separate crRNA molecules and separate tracrRNA molecules that hybridize together, as known in the art; or the guide RNA may be a single guide RNA, i.e., a single molecule having a crRNA portion and a tracrRNA portion that are linked together by a linker (e.g., a nucleic acid sequence), i.e., covalently linked to each other, as known in the art. Linkers such as GAAA are known to those skilled in the art. In a general embodiment, (1) a liposome containing guide RNA, (2) a nucleic acid sequence encoding a Cas enzyme or a liposome containing a Cas enzyme, and (3) a target cell are located in the wells of a well plate. The Cas enzyme and guide RNA form a co-localization complex with the target gene in the target cell. The Cas enzyme then generates a double-strand break in the target gene, which, when repaired by the cell, induces one or more mutations in the gene.

[0005] In one embodiment, the system includes (1) a liposome containing the crRNA portion of a guide RNA, wherein the crRNA portion contains a specific spacer complementary to the target gene and a segment complementary to the tracrRNA segment; (2) a liposome containing the tracrRNA portion of the guide RNA, wherein the tracrRNA portion hybridizes with the crRNA portion; (3) a liposome containing a nucleic acid sequence encoding a Cas enzyme or the Cas enzyme; and (4) target cells (which may be multiple identical cell types in a given well). In a general embodiment, (1) a liposome containing the crRNA portion of a guide RNA, (2) a liposome containing the tracrRNA portion of a guide RNA, (3) a liposome containing a nucleic acid sequence encoding a Cas enzyme or the Cas enzyme, and (4) target cells are located in the wells of a well plate. The crRNA portion and the tracrRNA portion hybridize to form the guide RNA. The Cas enzyme and guide RNA form a co-localization complex with the target gene in the target cell. The Cas enzyme then generates a double-strand break in the target gene, which, when repaired by the cell, induces one or more mutations in the gene.

[0006] In one embodiment, the method described herein is performed in multiple wells within a well plate, with the guide RNA in each well targeting a different target gene. In another embodiment, the method is performed simultaneously in multiple wells within a well plate, with the guide RNA in each well targeting a different target gene.

[0007] In one embodiment, (1) liposomes containing the crRNA portion of guide RNA, (2) liposomes containing the tracrRNA portion of the guide RNA, (3) liposomes containing a nucleic acid sequence encoding the Cas enzyme or the Cas enzyme, and (4) target cells may be introduced into the wells simultaneously. In another embodiment, (1) liposomes containing the crRNA portion of guide RNA, (2) liposomes containing the tracrRNA portion of the guide RNA, (3) liposomes containing a nucleic acid sequence encoding the Cas enzyme or the Cas enzyme, and (4) target cells may be introduced into the wells sequentially, or one or more, two or more, or three or more combinations of (1), (2), (3), or (4) may be introduced into the wells simultaneously. For example, (4) target cells may be introduced into a well, and then a mixture of (1) liposomes containing the crRNA portion of the guide RNA, (2) liposomes containing the tracrRNA portion of the guide RNA, and (3) liposomes containing a nucleic acid sequence encoding the Cas enzyme or the Cas enzyme may be introduced into the cell. For example, a mixture of (4) target cells, (2) liposomes containing the tracrRNA portion of the guide RNA, and (3) liposomes containing a nucleic acid sequence encoding the Cas enzyme or the Cas enzyme may be introduced into the cell, and then (1) liposomes containing the crRNA portion of the guide RNA may be introduced into the cell. Other embodiments for introducing (1) liposomes containing the crRNA portion of the guide RNA, (2) liposomes containing the tracrRNA portion of the guide RNA, (3) liposomes containing a nucleic acid sequence encoding the Cas enzyme or the Cas enzyme, and (4) target cells are envisioned and further described herein. Furthermore, embodiments are envisioned in which (1) liposomes containing guide RNA, (2) liposomes containing nucleic acid sequences encoding Cas enzymes or Cas enzymes, and (3) target cells are introduced simultaneously or separately, or a mixture of (1) and (2), a mixture of (2) and (3), or a mixture of (1) and (3) are introduced.

[0008] In another embodiment, the disclosure provides a nucleic acid encoding a Cas enzyme. In some embodiments, the nucleic acid encoding the Cas enzyme is located within a vector, for example, within an engineered DNA plasmid vector or a viral vector. The nucleic acid includes a promoter for the expression of the Cas enzyme in target cells. In some embodiments, the Cas enzyme is delivered to target cells by introducing a plasmid into the target cells and expressing the Cas enzyme. In some embodiments, the Cas enzyme may have one or more nuclear localization signals to facilitate the entry of the Cas enzyme into the nucleus of the target cell.

[0009] In one embodiment, target cells in the wells of a well plate (e.g., target cells of the same species type) are combined with CRISPR-Cas components in liposomes, thereby facilitating the entry of CRISPR-Cas components (i.e., guide RNA and Cas enzyme) into the target cells, resulting in gene cleavage and the generation of mutant genes. Cells with mutant genes are then analyzed. In one embodiment, the target cells are eukaryotic cells, such as yeast cells, plant cells, mammalian cells, or human cells. In another embodiment, the cells are prokaryotic cells. In one embodiment, the well plate is an addressable, indexable, or identifiable array, insofar as the identity and location of the guide RNA (e.g., having a unique spacer sequence) in each well of the array are known. Thus, the identity and location of the target gene to be mutated based on the identity of the spacer sequence are known within the array. The process may be repeated in a subsequent addressable array well plate, where each guide RNA is provided at a different well location than in the previous well plate. The above method can be repeated multiple times in an addressable array well plate, and the guide RNA is provided at different locations in the addressable array well plate.

[0010] Further features and advantages of specific embodiments of the present invention will become even more fully apparent in the following description of the embodiments and their drawings, and from the claims.

[0011] The patent or application file includes at least one drawing made in color. A copy of this patent or patent application publication, including the color drawing, will be provided by the Patent Office upon request and payment of the necessary fees. The above and other features and advantages of this embodiment will be understood more fully from the following detailed description of the exemplary embodiment, together with the accompanying drawings. [Brief explanation of the drawing]

[0012] [Figure 1] One embodiment of this disclosure is schematically shown. [Figure 2] Alternative embodiments of this disclosure are schematically shown below. [Figure 3] Alternative embodiments of this disclosure are schematically shown below. [Figure 4] Alternative embodiments of this disclosure are schematically shown below. [Figure 5] This shows the measurement of gene editing rates using Sanger sequencing of cellular genomic DNA. [Figure 6] The results of knockout of the MFN2 gene or the OPA1 gene are shown. [Figure 7] The results of knocking out the PLK1 gene are shown. [Modes for carrying out the invention]

[0013] Aspects of this disclosure provide a CRISPR protein endonuclease (e.g., Cas9) comprising at least one nuclear localization signal, at least one nuclease domain, and at least one domain that interacts with a guide RNA to target the endonuclease to a specific nucleotide sequence for cleavage. Also provided are nucleic acids encoding a CRISPR protein endonuclease and methods for modifying the chromosomal sequence of a cell, such as a eukaryotic cell or an embryonic cell, using a CRISPR protein endonuclease. The CRISPR protein endonuclease interacts with a specific guide RNA, each of which guides the endonuclease to a specific target site, where the CRISPR protein endonuclease introduces a double-strand break, which can be repaired by a DNA repair process so that the chromosomal sequence is modified or mutated. Because the specificity is provided by the guide RNA (or crRNA), the CRISPR protein endonuclease is universal and can be used with a variety of guide RNAs to target a variety of genomic sequences. The methods disclosed herein may be used to target and modify specific chromosomal sequences at target locations in the genome of a cell.

[0014] The method described herein, utilizing the CRISPR-Cas system, provides a platform for highly efficient genome editing of cells. Cells and CRISPR-Cas system components are delivered to the wells of a multi-well plate, where the CRISPR-Cas system components are delivered to the cells, for example, by liposome-mediated transfection, and used to cleave target nucleic acids in the cell's genome. The use of multi-well plates and CRISPR-Cas system components results in high-throughput, multi-genome-wide cell editing.

[0015] Aspects of this disclosure relate to a method for mutating a target gene in target cells within wells of an addressable array well plate. Multiple target cells are contained within an addressable array well plate. The multiple cells are brought into contact with a CRISPR-Cas system, the components of which are located in one or more liposomes for entry into the target cells. An exemplary CRISPR-Cas system, comprising guide RNA and a Cas enzyme (e.g., Cas9), is known to those skilled in the art and is commercially available. The spacer sequence of the guide RNA can be designed as is commonly known in the art to target a target gene. Guide RNA having designed spacer sequences is commercially available. The CRISPR-Cas system components enter the cells, and the target gene is mutated within the multiple target cells. The well plate is addressable as long as the identity of the guide RNA spacer sequence, and therefore the identity of the target gene, is located in a known position within the well plate. According to one aspect, each cell of the multiple cells is of the same species type. According to another aspect, each cell of the multiple cells contains a different mutant gene. This results in mutations in multiple genes in the target cell. In summary, multiple mutated genes represent multiple mutations in the target cell, and their analysis provides a high-throughput screening method. According to one embodiment, the disclosure includes the use of a multi-well plate and a high-throughput method using such a plate, where different wells contain the Cas9 protein and transfection reagents. Furthermore, different wells contain different gRNA molecules. Such a plate can be used in a high-throughput method for altering multiple gene sites within a cell.

[0016] In one embodiment, the disclosure provides a ready-to-use reagent. For example, the ready-to-use reagent may comprise a mixture of a nucleic acid encoding a Cas protein (e.g., Cas9) or a Cas protein and a nucleic acid encoding the tracrRNA portion of a guide RNA or the tracrRNA portion of a guide RNA. The mixture may then be combined with cells and a nucleic acid encoding crRNA or crRNA to produce CRISPR activity.

[0017] CRISPR system Embodiments of this disclosure relate to CRISPR / Cas-based systems, the components of which may be encapsulated in liposomes to facilitate entry into target cells. CRISPR systems, i.e., CRISPR systems comprising a Cas enzyme having nuclease activity and guide RNA, are known to those skilled in the art. Such Cas enzymes include Cas enzymes of type II CRISPR systems, i.e., Cas enzymes that function as part of a type II CRISPR system. Examples of such Cas enzymes include, for example, the natural or wild-type Cas enzymes in type II CRISPR systems, or their variants or modifiers. Cas9 is an exemplary Cas enzyme. Cas enzymes are commercially available as described herein and may be modified from their wild-type versions as described in Vakuraskas et al., Nature Medicine, vol.24, August 2018, pp.1216-1224, which describes an exemplary single-point mutation R691A in Cas9, the entire work of which is incorporated herein by reference. For example, the Cas9 used herein is commercially available from Integrated DNA Technologies, Inc. (Coralville, Iowa, USA). Exemplary Cas enzyme nickases containing one or more point mutations are known in the art. The Cas9 protein and the type II CRISPR system are well documented in the art. See Makarova et al., Nature Reviews, Microbiology, Vol. 9, June 2011, pp. 467–477, for all supplementary information, which is incorporated entirely herein by reference. Some commercially available and useful Cas enzymes, such as Cas9, possess sequence identity or homology of 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, or 70% or more compared to wild-type Cas enzymes (such as Cas9) described in the art, such as Vakuraskas et al., Nature Medicine, vol.24, August 2018, pp.1216-1224, but retain the enzymatic function of producing double-strand breaks in target nucleic acids. Methods for determining sequence identity are known to those skilled in the art.In one embodiment, the Cas enzyme can be delivered within a liposome.

[0018] As used herein, the term “CRISPR activity” refers to activity associated with the CRISPR system. Examples of such activity include cleavage or nicking of target nucleic acids. As used herein, the term “CRISPR system” refers to an assembly of CRISPR proteins and nucleic acids that, when combined, produce at least CRISPR-related activity (e.g., target locus-specific double-strand breaks in double-stranded DNA). As used herein, the term “CRISPR complex” refers to CRISPR proteins and nucleic acids (e.g., guide RNA) that associate with each other to form functionally active aggregates. Examples of CRISPR proteins include wild-type, modified, or mutant proteins, which may be commercially available in any of these forms. An example of a CRISPR complex is the Cas9 (sometimes called Csn1) protein, which binds to a target locus-specific guide RNA. In many cases, CRISPR proteins will contain nuclear localization signals (NLS) that enable them to be transported to the nucleus. As used herein, the term “target locus” refers to a site within a nucleic acid molecule for CRISPR system interactions (e.g., binding and cleavage). When a single CRISPR complex is designed to cleave a double-stranded nucleic acid, the target locus is the cleavage site and surrounding region recognized by the CRISPR complex. When two CRISPR complexes are designed to nick adjacent double-stranded nucleic acids to create a double-strand break, the region surrounding the cleavage site, and containing the cleavage site, is called the target locus.

[0019] Generally, the CRISPR-Cas systems described herein rely on guide RNA that forms a complex with a Cas protein to target nucleic acid sequences. For a description of the CRISPR-Cas system and methods for introducing the CRISPR / Cas system into cells, see U.S. Patent No. 2018 / 0195089, which is incorporated herein by reference in its entirety. The CRISPR system does not require the creation of customized proteins to target specific sequences, but rather requires a single Cas enzyme that can be guided to the target nucleotide sequence (target locus) by a short RNA molecule with sequence complementarity to the target.

[0020] Generally, three classes of CRISPR systems are commonly known, referred to as type I, type II, or type III. In one embodiment, the enzyme particularly useful in this disclosure for cleaving dsDNA is the single-effector enzyme Cas9, common to type II. See KS Makarova et al., Evolution and classification of the CRISPR-Cas systems. Nature reviews. Microbiology 9, 467 (Jun, 2011), which is incorporated herein by reference in its entirety. In one embodiment, Cas9 unwinds the DNA double strand and searches for a sequence that matches the crRNA in order to cleave it. Target recognition occurs when complementarity is detected between the “protospacer” sequence in the target DNA and the rest of the spacer sequence in the crRNA. Cas9 cleaves the DNA only if the correct protospacer facies motif (PAM) is also present at the 3' end. In certain embodiments, different protospacer facies motifs may be utilized. For example, the S. pyogenes system requires an NGG sequence, where N can be any nucleotide.The S. thermophilus type II system requires NGGNG (see P. Horvath, R. Barrangou, CRISPR / Cas, the immune system of bacteria and archaea. Science 327,167 (Jan 8, 2010), which is incorporated herein by reference in its entirety) and NNAGAAW (see H. Deveau et al., Phage response to CRISPR-encoded resistance in Streptococcus thermophilus. Journal of bacteriology 190,1390 (Feb, 2008), which is incorporated herein by reference in its entirety), respectively, while a different S. mutans system tolerates NGG or NAAR (see JR van der Ploeg, Analysis of CRISPR in Streptococcus mutans suggests frequent occurrence of acquired immunity against infection by M102-like bacteriophages. Microbiology 155,1966 (Jun, 2009), which is incorporated herein by reference in its entirety).Bioinformatics analysis has generated a comprehensive database of CRISPR loci in various bacteria that can be useful for identifying additional useful PAMs and expanding the set of CRISPR-targetable sequences (see M. Rho, Y. W. Wu, H. Tang, T. G. Doak, Y. Ye, Diverse CRISPRs evolving in human microbiomes. PloS genetics 8, e1002441 (2012) and D. T. Pride et al., Analysis of streptococcal CRISPRs from human saliva reveals substantial sequence diversity within and between subjects over time. Genome research 21, 126 (Jan, 2011), each of which is incorporated herein by reference in its entirety).

[0021] Non-limiting examples of suitable CRISPR proteins include Cas3, Cas4, Cas5, Cas5e (or CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9, Cas10, and Cas1. Examples include Od, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1 (or CasA), Cse2 (or CasB), Cse3 (or CasE), Cse4 (or CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csz1, Csx15, Csf1, Csf2, Csf3, Csf4, and Cu1966. The Cas9 protein was isolated from Methanococcus maripaludis C7;Corynebacterium diphtheriae;Corynebacterium efficiens YS-314;Corynebacterium glutamicum ATCC 13032, as identified in the supplementary information of Makarova et al., Nature Reviews, Microbiology, Vol.9, June 2011, pp.467-477. Kitasato;Corynebacterium glutamicum ATCC 13032 Bielefeld;Corynebacterium glutamicum R;Corynebacterium kroppenstedtii DSM 44385;Mycobacterium abscessus ATCC 19977;Nocardia farcinica IFM10152;Rhodococcus erythropolis PR4;Rhodococcus jostii RHA1;Rhodococcus opacus B4 uid36573;Acidothermus cellulolyticus 11B;Arthrobacter chlorophenolicus A6;Kribbella flavida DSM 17836 uid43465;Thermomonospora curvata DSM 43183; Bifidobacterium dentium Bd1; Bifidobacterium longum DJO10A; Slackia heliotrinireducens DSM 20476; Persephonella marina EX H1; Bacteroides fragilis NCTC 9434; Capnocytophaga ochracea DSM 7271; Flavobacterium psychrophilum JIP02 86; Akkermansia muciniphila ATCC BAA 835; Roseiflexus castenholzii DSM 13941; Roseiflexus RS1; Synechocystis PCC6803; Elusimicrobium minutum Pei191; uncultured Termite group 1 bacterium phylotype Rs D17; Fibrobacter succinogenes S85; Bacillus cereus ATCC 10987; Listeria innocua; Lactobacillus casei; Lactobacillus rhamnosus GG; Lactobacillus salivarius UCC118; Streptococcus agalactiae A909; Streptococcus agalactiae NEM316; Streptococcus agalactiae 2603; Streptococcus dysgalactiae equisimilis GGS 124; Streptococcus equi zooepidemicus MGCS10565; Streptococcus gallolyticus UCN34 uid46061; Streptococcus gordonii Challis subst CH1; Streptococcus mutans NN2025 uid46353; Streptococcus mutans; Streptococcus pyogenes M1 GAS; Streptococcus pyogenes MGAS5005;Streptococcus pyogenes MGAS2096;Streptococcus pyogenes MGAS9429;Streptococcus pyogenes MGAS10270;Streptococcus pyogenes MGAS6180;Streptococcus pyogenes MGAS315;Streptococcus pyogenes SSI-1;Streptococcus pyogenes MGAS10750;Streptococcus pyogenes NZ131;Streptococcus thermophiles CNRZ1066;Streptococcus thermophiles LMD-9;Streptococcus thermophiles LMG 18311;Clostridium botulinum A3 Loch Maree;Clostridium botulinum B Eklund 17B;Clostridium botulinum Ba4 657;Clostridium botulinum F Langeland;Clostridium cellulolyticum H10;Finegoldia magna ATCC 29328;Eubacterium rectale ATCC 33656;Mycoplasma gallisepticum;Mycoplasma mobile 163K;Mycoplasma penetrans;Mycoplasma synoviae 53;Streptobacillus moniliformis DSM 12112;Bradyrhizobium BTAi1;Nitrobacter hamburgensis X14;Rhodopseudomonas palustris BisB18;Rhodopseudomonas palustris BisB5;Parvibaculum lavamentivorans DS-1;Dinoroseobacter shibae DFL 12;Gluconacetobacter diazotrophicus Pal 5 FAPERJ;Gluconacetobacter diazotrophicus Pal 5 JGI;Azospirillum B510 uid46085;Rhodospirillum rubrum ATCC 11170;Diaphorobacter TPSY uid29975;Verminephrobacter eiseniae EF01-2;Neisseria meningitides 053442;Neisseria meningitides alpha14;Neisseria meningitides Z2491;Desulfovibrio salexigens DSM 2638;Campylobacter jejuni doylei 269 97;Campylobacter jejuni 81116;Campylobacter jejuni;Campylobacter lari RM2100;Helicobacter hepaticus;Wolinella succinogenes;Tolumonas auensis DSM 9187;Pseudoalteromonas atlantica T6c;Shewanella It is known to be present in many type II CRISPR systems, including Peeleana ATCC 700345; Legionella pneumophila Paris; Actinobacillus succinogenes 130Z; Pasteurella multocida; Francisella tularensis novicida U112; Francisella tularensis holarctica; Francisella tularensis FSC 198; Francisella tularensis tularensis; Francisella tularensis WY96-3418; and Treponema denticola ATCC 35405. The Cas9 protein is sometimes referred to as Csn1 by those skilled in the art in the literature. An exemplary S. pyogenes Cas9 protein sequence is provided in its entirety in Deltcheva et al., Nature 471, 602-607 (2011), which is incorporated herein by reference.

[0022] CRISPR systems and methods of delivery to cells useful in the present disclosure are also described in International Publication No. WO 2018 / 057837, U.S. Patent Application Publication No. 2019 / 0233820, International Publication No. WO 2019 / 25984, International Publication No. WO 2015 / 089465, U.S. Patent Application Publication No. 2019 / 0153412, International Publication No. WO 2015 / 089473, International Publication No. WO 2018 / 094356, and U.S. Patent Application Publication No. 2019 / 0390229, each of which is incorporated herein by reference in its entirety.

[0023] According to certain embodiments, a CRISPR protein having two or more nuclease domains may be modified or altered to inactivate all but one of the nuclease domains. Such modified or altered CRISPR proteins are referred to as nickases as long as the CRISPR protein cleaves or nicks only one strand of double-stranded DNA. Exemplary CRISPR proteins are those of type II CRISPR systems, e.g., Cas9 protein, modified Cas9, homologs of Cas9, or variants of Cas9. Exemplary Cas9 protein nickases are known in the art. See Jinek et al., Science 337, 816-821 (2012). According to certain embodiments, Cas9 protein or a Cas9 protein nickase includes homologs and orthologs thereof that bind to DNA and retain the ability of the protein to be guided by RNA. According to certain embodiments, Cas9 protein includes sequences described for naturally occurring Cas9 from S. thermophiles, S. aureus, or S. pyogenes, and protein sequences having 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology thereto.

[0024] Examples of CRISPR systems include S. thermophiles Cas9 nuclease or S. aureus Cas9 nuclease (ST1 Cas9, Sa Cas9) (see Esvelt KM, et al., Orthogonal Cas9 proteins for RNA-guided gene regulation and editing, Nature Methods., (2013), which is incorporated herein by reference in its entirety). An example of a CRISPR system is the S.pyogenes Cas9 nuclease (Sp.Cas9), a programmable DNA-binding protein isolated from a type II CRISPR-related system (see Sternberg, SH, Redding, S., Jinek, M., Greene, EC & Doudna, JA, DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature 507, 62-67 (2014), which is incorporated herein by reference in its entirety). In one embodiment, the Cas9 protein is the enzymatically active Cas9 protein, the wild-type Cas9 protein, or the Cas9 protein niccasse.

[0025] In certain embodiments, the Cas9 protein may, for example, be introduced into a cell or produced within the cell. Furthermore, the duration of cell uptake or intracellular production of the Cas9 protein, and the amount present within the cell, can be controlled or regulated. For example, the Cas9 protein coding sequence integrated into the chromosome may be operably ligated to a regulated promoter. Furthermore, the amount of mRNA encoding the Cas9 protein introduced into the cell may be regulated.

[0026] Guide RNA Embodiments of this disclosure relate to the use of the CRISPR / Cas system, and more particularly to the use of a guide RNA which may comprise one or more of a spacer sequence, a tracr mate sequence, and a tracr sequence. The guide RNA may be provided within a liposome. The term spacer sequence is understood by those skilled in the art and may comprise any polynucleotide having sufficient complementarity to the target nucleic acid sequence to hybridize with the target nucleic acid sequence and guide sequence-specific binding of the CRISPR complex to the target sequence. The guide RNA molecule may have a sequence complementarity region (known in the art as a spacer) of at least 10 (e.g., about 10 to about 50, about 10 to about 40, about 10 to about 35, about 10 to about 30, about 10 to about 25, about 15 to about 25, about 17 to about 22, about 20) nucleotides relative to the target locus. Often, the target locus is a naturally occurring chromosomal locus in eukaryotic cells. Spacer sequences can be designed, for example, by using bioinformatics data, to target nucleic acid sequences within a cell's genome. Guide RNA may be formed from a tracr-mate sequence (which may be called crRNA) and a spacer sequence covalently bonded to a separate tracr sequence, where the tracr-mate sequence hybridizes to a portion of the tracr sequence. In certain embodiments, the tracr-mate sequence and the tracr sequence are connected or linked by covalent bonding via a linker sequence, and this construct may be called a fusion of the tracr-mate sequence and the tracr sequence. Thus, the crRNA portion of the guide RNA and the tracrRNA portion of the guide RNA are linked and provided as a single guide RNA within a liposome. A linker sequence, as referred herein, is a nucleotide sequence, referred herein as a nucleic acid sequence, that connects the tracr-mate sequence and the tracr sequence. Therefore, guide RNA can be a two-component species (i.e., separate crRNA and tracr RNA that hybridize with each other) or a single-molecule species (i.e., a crRNA-tracr RNA fusion, often called sgRNA).If the guide RNA is a two-component species, a single tracrRNA molecule / segment attached to a target site-specific crRNA molecule / segment can be used to generate guide RNA molecules with specificity for different target sites. For example, different crRNA molecules / segments targeting different nucleic acids in the genome can be designed, and a single tracrRNA molecule / segment can be hybridized to each of the different crRNA molecules / segments to form functional guide RNAs targeting different nucleic acids in the genome. In this embodiment, the tracrRNA molecules can be introduced into each well, for example, as a mixture, along with cells and Cas proteins. Then, different target-specific crRNAs can be added to each well, thereby creating different gene mutations in the cell genome within each well.

[0027] Guide RNA molecules can be designed and synthesized using methods known to those skilled in the art. For example, a target nucleic acid sequence suitable for CRISPR editing (known as a protospacer sequence) may be identified in the cellular genome. A complementary sequence (known as a spacer sequence) may be synthesized. The spacer sequence may be linked to a scaffold sequence known to those skilled in the art for binding to the Cas protein. In this way, a functional guide RNA containing a crRNA segment and a tracrRNA segment can be constructed. As an example, the spacer sequence (about 15-30 nucleotides) is a target locus recognition sequence of crRNA containing a portion (about 10-30 nucleotides) that hybridizes to tracrRNA. The tracrRNA (containing 10-50 nucleotides) hybridizes to the crRNA to form the guide RNA. The crRNA may be linked to the tracrRNA via a linker nucleic acid segment.

[0028] In one embodiment, an assembly of crRNA molecules having specificity for individual target sites is provided. For example, an assembly of crRNA molecules having specificity for target sites within a particular type of cell (e.g., human cells) is provided. Members of such an assembly of cells may be generated based on sequence information of these particular types of cells. As an example, one such assembly can be generated using the complete genome sequence of a particular type of cell. Using genome sequence data, a library of crRNA molecules having specificity for the coding region of each gene in the human genome can be generated. Parameters that may be used to generate such a library may include the location of protospacer-adjacent motif (PAM) sites, off-target effects (e.g., sequences specific to the target region), and, if gene "knockout" is desired, locations within the coding region that are likely to completely or partially render the gene expression product nonfunctional (e.g., active site coding region, intron / exon junctions, etc.). The aggregates or libraries of crRNA molecules range from approximately 5 to approximately 100,000, for example, approximately 50 to approximately 100,000, approximately 200 to approximately 100,000, approximately 500 to approximately 100,000, approximately 800 to approximately 100,000, approximately 1,000 to approximately 100,000, approximately 2,000 to approximately 100,000, approximately 4,000 to approximately 100,000, approximately 5,000 to approximately 100,000, approximately 50 to approximately 50,000, approximately 100 to approximately 50,000, approximately 500 to approximately 50,000, approximately 1,000 to approximately 50,000, and approximately 2,000 to approximately 5 It can contain a wide variety of individual molecules, such as 0,000, approximately 4,000 to 50,000, approximately 50 to 10,000, approximately 100 to 10,000, approximately 200 to 10,000, approximately 500 to 10,000, approximately 1,000 to 10,000, approximately 2,000 to 10,000, approximately 4,000 to 10,000, approximately 50 to 5,000, approximately 100 to 5,000, approximately 500 to 5,000, approximately 1,000 to 5,000, approximately 50 to 2,000, approximately 100 to 2,000, and approximately 500 to 2,000. The number of crRNA molecules may be determined by the number of genes to be mutated in a given genome of a given target cell.

[0029] In certain embodiments, the guide RNA may be delivered directly to cells as a natural species or transcribed from its congener DNA by methods known to those skilled in the art, including injection or lipofection, the congener DNA being introduced into cells by electroporation, transient and stable transfection (including lipofection), and viral transduction. In exemplary embodiments, the guide RNA coding sequence is packaged in liposomes and delivered to cells. In one embodiment, different crRNAs are delivered to each well of a group of wells in order to produce different genomic mutations in each well.

[0030] cell Cells as described herein include any cells capable of introducing and expressing the disclosed nucleic acids as described herein. It should be understood that the basic concepts of this disclosure as described herein are not limited by cell type. Representative cells that may be used in carrying out the present invention include, but are not limited to, bacterial cells, yeast cells, plant cells, and animal cells (such as mammalian cells and human cells). In some embodiments, cells are derived from an embryo. Cells may be stem cells, zygotes, or germline cells. In embodiments where cells are stem cells, the stem cells are embryonic stem cells or pluripotent stem cells. In other embodiments, cells are somatic cells. In embodiments where cells are somatic cells, the somatic cells are eukaryotic or prokaryotic cells. Eukaryotic cells may be animal cells derived from pig cells, mouse cells, rat cells, rabbit cells, dog cells, horse cells, bovine cells, non-human primate cells, or human cells, etc.

[0031] The present invention further includes cells containing one or more CRISPR system components, and cells produced by the methods described herein. For example, the present invention includes cells into which a CRISPR complex has been introduced (e.g., cells containing (1) a plasmid encoding Cas9 and guide RNA, (2) Cas9 mRNA and guide RNA, etc.). The present invention further includes cells modified by the methods of the present invention, which may or may not contain one or more CRISPR system components (e.g., cells that have undergone cleavage and exclusion of cellular DNA, which may or may not have insertions at cleavage sites).

[0032] vector Vectors for use with the methods and constructs described herein are intended. The term “vector” includes a nucleic acid molecule capable of transporting another nucleic acid to which it is ligated. Vectors used to deliver nucleic acids to cells as described herein include vectors known to those skilled in the art and used for such purposes. Certain exemplary vectors may be plasmids, lentiviruses, or adeno-associated viruses known to those skilled in the art. Vectors include, but are not limited to, single-stranded nucleic acid molecules, double-stranded nucleic acid molecules, or partially double-stranded nucleic acid molecules; nucleic acid molecules with one or more free ends, nucleic acid molecules without free ends (e.g., circular); nucleic acid molecules containing DNA, RNA, or both; and various other polynucleotides known in the art. One type of vector is a “plasmid,” which refers to a circular double-stranded DNA loop into which additional DNA segments can be inserted by standard molecular cloning techniques, etc. Another type of vector is the viral vector, in which a viral DNA or RNA sequence is present within the vector for packaging into a virus (e.g., retroviruses, lentiviruses, replication-deficient retroviruses, adenoviruses, replication-deficient adenoviruses, and adeno-associated viruses). Viral vectors also contain polynucleotides carried by the virus for transfection into host cells. Certain vectors can autonomously replicate within the host cell into which they are introduced (e.g., bacterial vectors with bacterial origins of replication, and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the host cell's genome upon introduction into the host cell and thereby replicate together with the host genome. Furthermore, certain vectors can induce the expression of genes to which they are operably linked. Such vectors are referred to herein as “expression vectors.” Common expression vectors useful in recombinant DNA technology are often in the form of plasmids.Recombinant expression vectors may contain the nucleic acids of the present invention in a form suitable for nucleic acid expression within host cells, meaning that the recombinant expression vector may contain one or more regulatory elements operably ligated to the nucleic acid sequence to be expressed, which may be selected based on the host cell used for expression. Within a recombinant expression vector, "operably ligated" is intended to mean that the nucleotide sequence of interest is ligated to a regulatory element to enable the expression of the nucleotide sequence (for example, in an in vitro transcription / translation system, or within the host cell if the vector is introduced into a host cell).

[0033] Nonviral delivery methods for nucleic acids, or natural DNA-binding proteins, natural guide RNAs, or other natural species include lipofection, microinjection, particulate guns, virosomes, liposomes, immunoliposomes, polycations, or lipid-nucleic acid complexes, naked DNA, artificial virions, and drug-enhanced DNA uptake. Lipofection is described, for example, in U.S. Patents 5,049,386, 4,946,787, and 4,897,355, and lipofection reagents are commercially available (e.g., Transfectam® and Lipofectin®). Cationic and neutral lipids suitable for efficient receptor recognition lipofection of polynucleotides include those described in Felgner, International Publication No. 91 / 17424; International Publication No. 91 / 16024. Delivery may be to cells (e.g., in vitro or ex vivo administration) or target tissue (e.g., in vivo administration). The term "natural" includes the protein species, enzyme species, or guide RNA species themselves, but does not include the nucleic acid that codes for that species.

[0034] Adjustment element, terminator, and tag Regulatory elements for use with the methods and constructs described herein are intended. The term “regulatory element” is intended to include promoters, enhancers, internal ribosome entry sites (IRESs), and other expression regulatory elements (e.g., transcription termination signals such as polyadenylation signals and poly-U sequences). Such regulatory elements are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatory elements include those that lead to constitutive expression of nucleotide sequences in many types of host cells, and those that lead to expression of nucleotide sequences only in specific host cells (e.g., tissue-specific regulatory elements). Tissue-specific promoters may primarily lead to expression in desired tissues of interest, e.g., muscle, neurons, bone, skin, blood, specific organs (e.g., liver, pancreas), or specific cell types (e.g., lymphocytes). The regulatory elements may also lead to time-dependent expression, for example, in a cell cycle-dependent or developmental stage-dependent manner, which may or may not be tissue-specific or cell type-specific. In some embodiments, the vector may include one or more pol III promoters (e.g., one, two, three, four, five, or more pol III promoters), one or more pol II promoters (e.g., one, two, three, four, five, or more pol II promoters), one or more pol I promoters (e.g., one, two, three, four, five, or more pol I promoters), or a combination thereof. Examples of pol III promoters include, but are not limited to, the U6 promoter and the H1 promoter.Examples of Pol II promoters include, but are not limited to, the retroviral Roussarcoma virus (RSV) LTR promoter (optionally having an RSV enhancer), the cytomegalovirus (CMV) promoter (optionally having a CMV enhancer) (see, e.g., Boshart et al, Cell, 41:521-530 (1985)), the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1α promoter, as well as the Pol II promoter described herein. The term “regulatory element” includes WPRE; CMV enhancer; the R-U5' segment in the LTR of HTLV-I (Mol. Cell. Biol., Vol. 8(1), p.466-472, 1988); the SV40 enhancer; and the intron sequence between exon 2 and exon 3 of rabbit β-globin (Proc. Natl. Acad. Sci. USA., Vol.78(3)). Enhancer elements such as those described in pp. 1527-31, 1981 are also included. It will be understood by those skilled in the art that the design of the expression vector may depend on factors such as the selection of the host cell to be transformed and the desired expression level. The vector can be introduced into host cells to produce transcripts, proteins, or peptides including fusion proteins or fusion peptides encoded by the nucleic acids described herein (e.g., transcripts, proteins, enzymes, variant forms thereof, and fusion proteins thereof of clustered and regularly arranged short palindromic sequence repeats (CRISPR)).

[0035] Embodiments of the methods described herein may utilize terminator sequences. Terminator sequences include portions of nucleic acid sequences that mark the ends of genes or operons within genomic DNA during transcription. These sequences mediate transcription termination by providing a signal to newly synthesized mRNA that triggers a process of releasing mRNA from the transcription complex. These processes include direct interaction between the mRNA secondary structure and the complex, and / or indirect activation of recruited termination factors. The release of the transcription complex releases RNA polymerase and associated transcriptional mechanisms to initiate transcription of new mRNA. Terminator sequences include those known in the art and identified and described herein.

[0036] Embodiments of the methods described herein may utilize epitope tags and reporter gene sequences. Non-exclusive examples of epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Examples of reporter genes, but not limited to, include glutathione-S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT) beta-galactosidase, beta-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and blue fluorescent protein (BFP).

[0037] Wellplate Well plates useful for the methods described herein include multiple wells known in the art. A well plate is generally a flat plate having multiple “wells” in which reagents are placed. The wells function as test tubes or containers for performing genome editing using a CRISPR / Cas system. A well plate may have 6, 12, 24, 48, 96, 384, 1536, 3456, or 9600 wells arranged in a rectangular matrix that may form an array, which may be an addressable array. Each well may hold tens of nanoliters to several milliliters of liquid. The shape of the wells may vary from circular to square. Well plates useful in this disclosure are commercially available.

[0038] In certain embodiments, the multiwell plate includes 500 or more wells, 1000 or more wells, or 3000 or more wells. In one embodiment, the well plate includes edge wells, which are either empty or not used during the high-throughput method described herein.

[0039] Introduction of the CRISPR system into cells In certain embodiments, components of the CRISPR / Cas system, nucleic acids encoding components of the CRISPR / Cas system, or vectors containing nucleic acids encoding components of the CRISPR / Cas system can be delivered to cells by methods known to those skilled in the art. Many standard laboratory manuals, including those described in Davis et al., BASIC METHODS IN MOLECULAR BIOLOGY, (1986) and Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbour Laboratory Press, Cold Spring Harbour, NY (1989), provide compositions and methods for introducing CRISPR system components into cells, which include calcium phosphate transfection, DEAE-dextran-mediated transfection, transfection, microinjection, cationic lipid-mediated transfection, electroporation, nucleofection, transduction, scrape loading, ballistic introduction, nucleoporation, hydrodynamic shock, and infection.

[0040] Suitable transfection agents for use in conjunction with the present invention include transfection agents that promote the introduction of RNA, DNA, and proteins into cells. Exemplary transfection reagents include TurboFect Transfection Reagent (Thermo Fisher Scientific), Lipofectamine CRISPRMAX (Thermo Fisher Scientific), Pro-Ject Reagent (Thermo Fisher Scientific), TRANSPASS P Protein Transfection Reagent (New England Biolabs), CHARIOT Protein Delivery Reagent (Active Motif), PROTEOJUICE Protein Transfection Reagent (EMD Millipore), 293fectin, LIPOFECTAMINE 2000, LIPOFECTAMINE 3000 (Thermo Fisher Scientific), LIPOFECTAMINE (Thermo Fisher Scientific), LIPOFECTIN (Thermo Fisher Scientific), DMRIE-C, CELLFECTIN (Thermo Fisher Scientific), OLIGOFECTAMINE (Thermo Fisher Scientific), LIPOFECTACE, FUGENE (Roche, Basel, Switzerland), FUGENE HD (Roche), TRANSFECTAM (Transfectam, Promega, Madison, Wis.), TFX-10 (Promega), TFX-20. (Promega), TFX-50 (Promega) , TRANSFECTIN(BioRad,Hercules,Calif.), SILENTFECT(Bio-Rad), EFFECTENE(Qiagen,Valencia,Calif.), DC-chol(Avanti Polar Lipids), GENEPORTER (Gene Therapy Systems, San Diego, Calif.)Examples include DHARMAFECT 1 (Dharmacon, Lafayette, Colo.), DHARMAFECT 2 (Dharmacon), DHARMAFECT 3 (Dharmacon), DHARMAFECT 4 (Dharmacon), ESCORT III (Sigma, St. Louis, Mo.), and ESCORT IV (Sigma Chemical Co.).

[0041] In one embodiment, lipofection is a lipid-based transfection technique that involves mixing cationic lipids with a material to be introduced into cells to create liposomes that fuse with the cell membrane and deposit the material inside the cell. Reagents for creating liposomes for lipofection are commercially available, including LIPOFECTAMINE RNAIMAX and LIPOFECTAMINE CRISPRMAX, both sold by ThermoFisher Scientific.

[0042] Conditions are typically adjusted, for example, for each cell type, to introduce the desired level of CRISPR system components into the cells. Any number of conditions may be varied to enhance the introduction of CRISPR system components into the cells. Exemplary incubation conditions include pH, ionic strength, cell type, cell energy charge, specific CRISPR system components present, ratio of CRISPR system components (if multiple CRISPR system components are present), CRISPR system component / cell ratio, cell and CRISPR system component concentrations, and incubation time.

[0043] In one embodiment, the CRISPR components described herein are provided in wells containing cells, which are then incubated or cultured for a period of time (e.g., about 2 minutes to about 8 hours, about 10 minutes to about 8 hours, about 20 minutes to about 8 hours, about 30 minutes to about 8 hours, about 60 minutes to about 8 hours, about 20 minutes to about 6 hours, about 20 minutes to about 3 hours, about 20 minutes to about 2 hours, about 45 minutes to about 3 hours, etc.), and the intracellular CRISPR activity, e.g., cleavage of target nucleic acids, is then measured using methods known to those skilled in the art. For example, the entire nucleic acid may be isolated from the cells being tested for CRISPR system activity, and then analyzed for the amount of nucleic acid that was so at the target locus, e.g., by measuring mutation induction as a substitute for cleavage at the target locus of the nucleic acid.

[0044] Delivery of system components to the wells of a well plate. In one embodiment, one or more cells or components of the CRISPR / Cas system are delivered to the wells of a well plate using methods known to those skilled in the art. In one embodiment, a microfluidics system is used in which, for each well of a multiwell plate, one or more conduits connected to one or more reservoirs containing one or more reagents are transported into the well, and one or more components of the CRISPR / Cas system are transported into the well. Systems for transporting fluids to the wells of a multiwell plate, such as the MultiDrop Dispenser (Thermo Fisher Scientific), are commercially available. Once the cells and CRISPR components have been transported to the multiwell plate, the multiwell plate is incubated or otherwise configured under conditions in which the components of the CRISPR / Cas system are provided to the cells, a co-localization complex is formed between the guide RNA, Cas enzyme, and target nucleic acid, the Cas enzyme cleaves the target nucleic acid, and a mutant gene is produced.

[0045] In one embodiment, cells and / or components of the CRISPR / Cas system are delivered to the wells of a well plate using acoustic droplet ejection of reagents from a source plate located below the well plate to the wells of the well plate located above the source plate. Sound waves eject precisely sized droplets from the source to the wells suspended above the source. Exemplary delivery systems are commercially available from LABCYTE, including ECHO liquid handling technology. In one embodiment, one or more or all of the following are delivered to the wells by droplet transfer from a source container to the wells using sound waves: liposomes containing the crRNA portion of guide RNA, liposomes containing the tracrRNA portion of the guide RNA, nucleic acid sequences encoding the Cas enzyme or liposomes containing the Cas enzyme, and target cells. [Examples]

[0046] The following embodiments are described as representative of the Disclosure. These embodiments should not be construed as limiting the scope of the Disclosure, as these and other equivalent embodiments become apparent from the Disclosure, the drawings and the appended claims.

[0047] Example I General transfection protocols for CRISPR / Cas9 reagents Referring to the schematic diagram shown in Figure 1, cells are transfected as follows: Cas9 mRNA and a guide RNA complementary to the target nucleic acid are combined with a lipofection reagent to create a mixture of liposomes containing Cas9 mRNA and guide RNA ("reagent mixture"). crRNA and tracrRNA can be included in separate liposomes as separate reagents added to the mixture. Suitable lipofection reagents include RNAMAX, CRISPRMAX, or LIPOFECTAMINE 2000. The reagent mixture is then delivered to the wells of a multiwell plate using acoustic droplet ejection. In this method, a fluid source containing the reagent (or cells) is operably connected to an acoustic generator. The multiwell plate is placed above the fluid source in an inverted position so that the wells face the fluid source. Sound waves are generated to eject droplets from the fluid source onto and into the wells of the multiwell plate, and the droplets are retained in the multiwell plate despite the multiwell plate being inverted. Multiple fluid sources may be placed below the multiwell plate so that reagents are delivered to multiple wells simultaneously. Alternatively, different fluid sources can be placed beneath a particular well to deliver specific reagents to that well, such as a unique crRNA sequence for targeting a specific gene in a target cell. Thus, each well in a group of wells may contain a different unique crRNA. Furthermore, different fluid sources can be placed beneath a particular well to deliver two or more specific reagents to that well, such as two or more unique crRNA sequences for targeting different genes in a target cell. In addition, different fluid sources can be placed beneath a particular well to deliver two or more specific reagents to that well for targeting two or more specific genes in a target cell, such as two or more unique crRNA sequences in a mixture. Therefore, this disclosure intends not only to deliver a single unique crRNA to a particular well, but also to deliver multiple unique crRNAs to a particular well to target multiple genes in one or more cells within a single well. The well concentration of the guide RNA may be 10 nm to 1 μmol, for example, 25 pmol to 125 pmol.The well concentration of the Cas enzyme is 1 nM to 150 nM, for example, 60 nM to 80 nM. Then, cells are added to each well containing the reagent mixture using either an acoustic droplet injection system or a multi-drop dispenser (Thermo Fisher Scientific). The cells are incubated with the reagent mixture for 24 hours. Then, the medium is changed and the cells are incubated for a further 96 hours. Then, the cells are removed from the wells, fixed, stained, and imaged.

[0048] Example II General transfection protocols for CRISPR / Cas9 reagents (Cas9 pretreatment) Referring to Figure 2, Cas9 is added to the cells by combining them with Cas9 mRNA in a lipofection reagent before contacting the cells with the guide RNA. The cells transfected to contain Cas9 are then mixed with the guide RNA in the lipofection reagent, and the mixture is added to the wells of a multi-well plate using either a multi-drop dispenser or acoustic droplet ejection. Alternatively, cells transfected to contain Cas9 may be added to the wells, and then the guide RNA reagent, which has been treated with the lipofection reagent to create liposomes containing the guide RNA, may be added to the wells. Suitable lipofection reagents include RNAMAX, CRISPRMAX, or LIPOFECTAMINE 2000. The well concentration of guide RNA may be 10 nm to 1 μmol, for example, 25 pmol to 125 pmol. The cells are incubated with the reagent mixture for 24 hours. The medium is then changed, and the cells are incubated for a further 96 hours. The cells are then removed from the wells, fixed, stained, and imaged.

[0049] Example III Common transfection protocols for CRISPR / Cas9 reagents (different gRNA per well) Referencing the schematic diagram shown in Figure 3, transfect the cells as follows: Deliver the cells to the wells of a 1536 multiwell plate, for example, using a multidrop dispenser or acoustic droplet injection. Combine Cas9 mRNA with a lipofection reagent to create liposomes containing Cas9 mRNA, and then add these to the wells, for example, using a multidrop dispenser or acoustic droplet injection. Prepare reagents containing different guide RNAs complementary to the target nucleic acid by combining each different guide RNA with a lipofection reagent to create guide RNA-containing liposomes. Suitable lipofection reagents include RNAMAX, CRISPRMAX, or LIPOFECTAMINE 2000. Add each different guide RNA reagent to different wells, for example, using a multidrop dispenser or acoustic droplet injection. The well concentration of the guide RNA may be 10 nm to 1 μmol, for example, 25 pmol to 125 pmol. The well concentration of the Cas enzyme is 1 nM to 150 nM, for example, 60 nM to 80 nM. The cells are incubated with the reagent mixture for 24 hours. Then the medium is changed and the cells are incubated for a further 96 hours. The cells are then removed from the wells, fixed, stained, and imaged. Each well contains cells with a different genomic modification.

[0050] Representative genes targeted by guide RNA are shown in Figure 3.

[0051] Example IV General transfection protocols (reagent combinations) for CRISPR / Cas9 reagents According to aspects of this disclosure, various reagents can be added at various times and in various orders, for example, using a multidrop dispenser or acoustic droplet ejection. Generally, referring to Figure 4, the method for genetically modifying multiple target cells described herein includes the steps of (a) combining, in each of the multiple wells of a first well plate, (1) a liposome containing a crRNA portion of guide RNA, where the crRNA portion contains a unique spacer complementary to the target gene, (2) a liposome containing a tracrRNA portion of the guide RNA, (3) a liposome containing a nucleic acid sequence encoding a Cas enzyme or a Cas enzyme, and (4) target cells, and (b) incubating the multiple target cells in the multiple wells. Here, the crRNA portion and the tracrRNA portion form the guide RNA, the Cas enzyme and the guide RNA form a colocalization complex with the target gene of the target cell, the Cas enzyme cleaves the target gene and mutates the target gene, the unique spacer sequence of the crRNA portion in each well is complementary to a different target gene, the location of each unique spacer sequence in each well within the first well plate is known, and the target cell in each well has a different target gene mutation. The process is repeated for a given set of crRNAs, but the crRNAs may be provided at different well locations, or the process may be carried out for different sets of crRNAs corresponding to a portion of the genome or the entire genome. Thus, a high-throughput method using CRISPR reagents is provided using an addressable array and single knockout mutations per well at genome scale. Cells may be analyzed, for example, using cell painting and high-dimensional analysis.

[0052] According to one embodiment, liposomes containing the crRNA portion of guide RNA, liposomes containing the tracrRNA portion of the guide RNA, and liposomes containing a nucleic acid sequence encoding the Cas enzyme or the Cas enzyme are provided to the well before target cells are provided to the well. According to one embodiment, target cells are provided to the well before liposomes containing the crRNA portion of guide RNA, liposomes containing the tracrRNA portion of the guide RNA, and liposomes containing a nucleic acid sequence encoding the Cas enzyme or the Cas enzyme are provided to the well. According to one embodiment, liposomes containing the tracrRNA portion of guide RNA, liposomes containing a nucleic acid sequence encoding the Cas enzyme or the Cas enzyme, and target cells are provided to the well before liposomes containing the crRNA portion of guide RNA are provided to the well. According to one embodiment, liposomes containing the crRNA portion of guide RNA are provided to the well before liposomes containing the tracrRNA portion of guide RNA, liposomes containing a nucleic acid sequence encoding the Cas enzyme or the Cas enzyme, and target cells are provided to the well. In one embodiment, liposomes containing the crRNA portion of guide RNA are provided to the well after liposomes containing the tracrRNA portion of the guide RNA, a nucleic acid sequence encoding the Cas enzyme or liposomes containing the Cas enzyme, and target cells are provided to the well. In another embodiment, liposomes containing the tracrRNA portion of guide RNA and liposomes containing the nucleic acid sequence encoding the Cas enzyme or liposomes containing the Cas enzyme are provided to the well as a mixture. In yet another embodiment, liposomes containing the tracrRNA portion of guide RNA, a nucleic acid sequence encoding the Cas enzyme or liposomes containing the Cas enzyme, and target cells are provided to the well as a mixture. In yet another embodiment, cells are transfected with the nucleic acid encoding the Cas enzyme before target cells are provided to the well.In one embodiment, (1) liposomes containing the crRNA portion of guide RNA, (2) a mixture of liposomes containing the tracrRNA portion of the guide RNA and liposomes containing a nucleic acid sequence encoding a Cas enzyme or a Cas enzyme, and (3) target cells are provided separately in the wells. In one embodiment, steps (a) and (b) above for a general method are repeated for a second well plate, wherein each unique spacer sequence provided for the wells of the first well plate is provided at a different position in the second well plate than in the first well plate. In one embodiment, steps (a) and (b) above for a general method are repeated for a plurality of well plates, wherein each unique spacer sequence provided for the wells of the first well plate is provided at a different position in each of the plurality of well plates and the first well plate.

[0053] In one embodiment, CRISPR activity for each gene across the entire genome can be assayed or analyzed using high-throughput gene mutations for each gene. A crRNA is designed for each gene. Each crRNA is introduced into a single well, and the entire genome of target cells can be assayed for CRISPR activity using approximately 1200 1536 multiwell plates, as described herein. Such a whole-genome assay can be performed in approximately 3 weeks, processing approximately 400 plates per week.

[0054] In one embodiment, the methods provided herein and described above enable the randomization of CRISPR activity by generating data on specific gene cleavage obtained from various well locations. In this way, any influence or artifacts generated by well location can be minimized.

[0055] Example V Specific transfection protocols for CRISPR / Cas9 reagents To prepare for cell lipofection, reagents are repeatedly added to a 1536-well plate. First, crRNA is dispensed into the plate using an Echo Acoustic Liquid Handler. 10 μL of one or more types of crRNA is dispensed into each well at a concentration of 200 μM. After dispensing, these plates are sealed and stored at 4°C. These plates should be stored at 4°C for no more than one month.

[0056] Prepare the lipofectamine mixture according to the method described herein. Each 1536-well plate requires 3.2 mL of the lipofectamine mixture. Tables 1 and 2 below show the amounts per 1 mL. Those skilled in the art can adjust or increase the amounts based on the size of the desired experiment. Label two 15 mL conical tubes "A" and "B". Prepare tube A by combining the reagents listed in Table 1 in the order they are listed, adding tracrRNA and, if desired, components to assist Cas9 transfection into cells, such as Cas9 PLUS, commercially available from ThermoFisher, and then ensuring vortexing. Let the tube stand at room temperature for 5 minutes before using it in the next step.

[0057] [Table 1]

[0058] Prepare tube B by combining the reagents in the order listed in Table 2.

[0059] [Table 2]

[0060] Let this tube stand at room temperature for 5 minutes before using it in the next step. To complete the preparation of the lipofectamine mixture, pour the contents of tube A into tube B. Gently swirl them together until the mixture is combined.

[0061] Once the lipofectamine mixture is ready, add it to a 1536-well plate. Dispense 2 μL of the mixture per well using an automated liquid handler. Subsequently, dispense 8 μL of cells at 125,000 cells / mL into each well using the automated liquid handler. After 1 hour, transfer the plate with the lipofectamine and cells to a 37°C incubator. After 24 hours, change the medium on the plate to remove the lipofectamine mixture. This medium change should be appropriate for the cell type in question. Perform a second medium change after an additional 48 hours (or 72 hours after the start of the experiment). After 96 hours, the samples are ready for downstream analysis.

[0062] The gene knockout was validated using the procedure described herein. Fluorescence images of cells targeting specific gene knockouts were analyzed to confirm the success of cell editing using the method described herein. In the first example, the gene editing rate was measured using Sanger sequencing of cellular genomic DNA. This method allows for the detection of the percentage of cells edited using CRISPR relative to unedited cells in each sample. The first panel of Figure 5 details the results of using six different CRISPR reagents against the gene MFN2 to generate edited cells, demonstrating that the method can yield an editing rate of approximately 50%. The second panel of Figure 5 includes results performed on the genes PLK1 and OPA1 using the same procedure. Both panels demonstrate observable editing of cells.

[0063] To further confirm gene editing, the MFN2 and OPA1 genes were targeted for knockout, and cells were stained with fluorescent dyes to observe the mitochondrial morphology patterns. Both of these genes are necessary for regulating mitochondrial stability within cells, and removal of these genes leads to mitochondrial fragmentation. As a result, mitochondria change from a filamentous, spaghetti-like morphology to a punctate, dispersed morphology, as exemplified in the first panel of Figure 6. By analyzing sections of images obtained from cells treated with MFN2, OPA1, or a non-targeted control reagent, wells targeting MFN2 or OPA1 knockout contain the fragmented mitochondrial phenotype.

[0064] In the second example, cells were targeted for deletion of PLK1, a protein involved in the cell cycle. PLK1 knockout results in either cell death or multinucleated cells. The first panel of Figure 7 shows the distribution of cell numbers observed in a 1536-well plate when cells were treated with various reagents targeting the PLK1 gene. Compared to the control, four of the six PLK1 targeting reagents significantly reduced the observed cell number, which is a predicted effect of PLK1 knockout. Furthermore, multinucleated cells were also observed in the wells targeted by the cell-reducing PLK1 reagents. An example of this is shown in Figure 7, where multinucleated cells are circled in red. This phenotype is not observed in similar samples of unedited cells.

[0065] The above experiments demonstrate that cultured cells can be given a predictable visible phenotype.

[0066] All patents, published applications, and references cited herein are incorporated in their entirety by reference.

[0067] Although the present invention has been shown and described in particular with reference to its exemplary embodiments, it will be understood by those skilled in the art that various modifications of form and detail can be made without departing from the scope of the invention as encompassed by the appended claims.

Claims

1. A method for genetically modifying multiple target cells, (a) In each of the multiple wells of the first well plate, (1) a liposome containing the crRNA portion of the guide RNA, wherein the crRNA portion contains a unique spacer complementary to the target gene, (2) a liposome containing the tracrRNA portion of the guide RNA, (3) a liposome containing a nucleic acid sequence encoding the Cas enzyme or the Cas enzyme, and (4) a target cell, and (b) Incubating the plurality of target cells in the plurality of wells, wherein the crRNA portion and the tracrRNA portion form the guide RNA, the Cas enzyme and the guide RNA form a colocalization complex with the target gene of the target cell, the Cas enzyme cleaves the target gene and mutates the target gene. Includes, The unique spacer sequences of the crRNA portion in each well are complementary to different target genes, the location of each unique spacer sequence in each well within the first well plate is known, and the target cells in each well have different target gene mutations. The aforementioned method.

2. The method according to claim 1, wherein in step (a), at least two or more crRNA portions of a guide RNA, each having a unique spacer complementary to a different target gene, are separately provided within a liposome, and in step (b), at least two or more different target genes are knocked out in each target cell.

3. The method according to claim 1, wherein the plurality of wells include 500 or more wells.

4. The method according to claim 1, wherein the plurality of wells include 1,000 or more wells.

5. The method according to claim 1, wherein the plurality of wells include 1,536 or more wells.

6. The method according to claim 1, wherein the plurality of wells include 3,000 or more wells.

7. The method according to claim 1, wherein the first well plate includes an edge well, and the edge well is empty.

8. The method according to claim 1, wherein each of the target cells among the plurality of target cells is of the same cell type.

9. The method according to claim 1, wherein each of the target cells among the plurality of target cells is of a different cell type.

10. The method according to claim 1, wherein, before the target cells are provided to the well, the liposomes containing the crRNA portion of the guide RNA, the liposomes containing the tracrRNA portion of the guide RNA, and the liposomes containing the nucleic acid sequence encoding the Cas enzyme or the Cas enzyme are provided to the well.

11. The method according to claim 1, wherein the target cells are provided to the well before the liposomes containing the crRNA portion of the guide RNA, the liposomes containing the tracrRNA portion of the guide RNA, and the liposomes containing the nucleic acid sequence encoding the Cas enzyme or the Cas enzyme are provided to the well.

12. The method according to claim 1, wherein, before the liposomes containing the crRNA portion of the guide RNA are provided to the well, the liposomes containing the tracrRNA portion of the guide RNA, the nucleic acid sequence encoding the Cas enzyme, or the liposomes containing the Cas enzyme, and the target cells are provided to the well.

13. The method according to claim 1, wherein the liposome containing the tracrRNA portion of the guide RNA, the liposome containing the nucleic acid sequence encoding the Cas enzyme or the Cas enzyme, and the target cells are provided to the well before the liposome containing the crRNA portion of the guide RNA are provided to the well.

14. The method according to claim 1, wherein the liposome containing the tracrRNA portion of the guide RNA, the liposome containing the nucleic acid sequence encoding the Cas enzyme or the Cas enzyme, and the target cells are provided to the well, and then the liposome containing the crRNA portion of the guide RNA is provided to the well.

15. The method according to claim 1, wherein the liposome containing the tracrRNA portion of the guide RNA, and the liposome containing the nucleic acid sequence encoding the Cas enzyme or the Cas enzyme are provided in the well as a mixture.

16. The method according to claim 1, wherein the liposome containing the tracrRNA portion of the guide RNA, the liposome containing a nucleic acid sequence encoding the Cas enzyme or the Cas enzyme, and the target cells are provided in the well as a mixture.

17. The method according to claim 1, wherein the nucleic acid sequence encoding the Cas enzyme or the liposome containing the Cas enzyme, and the target cells are provided in the well as a mixture.

18. The method according to claim 1, wherein the nucleic acid encoding the Cas enzyme is transfected to the target cells before the target cells are provided to the well.

19. The method according to claim 1, wherein (1) the liposome containing the crRNA portion of the guide RNA, (2) a mixture of the liposome containing the tracrRNA portion of the guide RNA and the liposome containing a nucleic acid sequence encoding the Cas enzyme or the Cas enzyme, and (3) the target cells are provided separately in the well.

20. The method according to claim 1, wherein steps (a) and (b) are repeated for a second well plate, wherein each unique spacer arrangement provided for the wells of the first well plate is provided in the second well plate at a different position than that of the first well plate.

21. The method according to claim 1, wherein steps (a) and (b) are repeated for a plurality of well plates, wherein each unique spacer arrangement provided for the wells of the first well plate is provided at different positions in each of the plurality of well plates and the first well plate.

22. The method according to claim 21, wherein the plurality of well plates are more than 1,000 well plates.

23. The method according to claim 1, wherein one or fewer cells are present in each well.

24. The method according to claim 1, wherein in step (a), the crRNA portion of the guide RNA and the tracrRNA portion of the guide RNA are ligated together and provided as a single guide RNA in a liposome.

25. The method according to claim 1, wherein the well concentration of the guide RNA is 10 nM to 1 μM.

26. The method according to claim 1, wherein the well concentration of guide RNA is 25 pmol to 125 pmol.

27. The method according to claim 1, wherein the well concentration of guide RNA is 75 pmol to 125 pmol.

28. The method according to claim 1, wherein the well concentration of Cas enzyme is 1 nM to 150 nM.

29. The method according to claim 1, wherein the well concentration of Cas enzyme is 60 nM to 80 nM.

30. The method according to claim 1, wherein the liposomes containing the crRNA portion of the guide RNA, the liposomes containing the tracrRNA portion of the guide RNA, the liposomes containing a nucleic acid sequence encoding a Cas enzyme or a Cas enzyme, and the target cells are provided to the well by droplet transfer from a source container to the well using sound waves.

31. The method according to claim 1, wherein the Cas enzyme is a type II CRISPR system Cas enzyme.

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