Methods and systems for performing pooled genomic screens

Abstract

The present disclosure relates to methods, compositions, systems and kits for performing pooled genomic screens, e.g., using CRISPR-Cas systems. In particular, the disclosed subject matter relates to the use of a first lentivirus vector comprising a first nucleic acid sequence encoding a gRNA and a second nucleic acid sequence encoding a cell surface protein, and a second lentivirus vector comprising a first nucleic acid sequence encoding a Cas protein for performing a genomic screen.

Description

[0001] Attorney Ref. No. 00B206.1710

[0002] GNE Ref. No. P60053-WO METHODS AND SYSTEMS FOR PERFORMING POOLED GENOMIC SCREENS

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims priority to U.S. Provisional Application No. 63 / 779,981, filed March 28, 2025, the contents of which is incorporated by reference herein in its entirety.

[0005] FIELD

[0006] The subject matter disclosed herein relates to methods, compositions, systems and kits for performing pooled genomic screens, e.g., using CRISPR-Cas systems.

[0007] BACKGROUND

[0008] Pooled CRISPR screening approaches offer an opportunity to investigate the molecular mechanisms underlying normal and diseased cellular functions at an unprecedented scale. These CRISPR screens hinge on (1) the delivery of both a Cas9 and a guide RNA (gRNA) to any cell of interest, (2) robust perturbation efficiency, and (3) single-cell guide detection. Generally, cells are transduced by a lentiviral guide library either after the engineering of a Cas9-expressing cell line or prior to the transient transfection of Cas9-expressing DNA, RNA, or protein. Some studies have also utilized an all-in-one Cas9 and guide lentivirus. However, the predominant methods of Cas9 and gRNA delivery are time-consuming, laborious, and often extremely difficult or even impossible to implement across many cell types, especially primary and iPSC-derived cell type-types. For example, not all cells are amenable to Cas9 transfection, whether it be exposure to the Cas9 modality ( / .< ., lethality caused by DNA exposure) or transfection modality ( / .< ., nucleofection requires cells to be in suspension, and not all differentiated cells can be lifted from the plate). Likewise, creating a stable Cas9-expressing cell line presents challenges as well. Cas9 expression in iPSCs, for example, can be lethal or impair their subsequent differentiation, preventing CRISPR screening in the desired terminally differentiated cell models. If Cas9-effected perturbations are not restricted to terminally differentiation cells, the screen will identify regulators of development instead of perturbation effects. Chromatin rearrangement during iPSC differentiation can also prevent robust singlecell guide detection.

[0009] Thus, there is a need for tools and techniques to enable high throughput pooled CRISPR screens more broadly across more difficult, terminally differentiated cell types.

[0010] 516169085.1 1Attorney Ref. No. 00B206.1710

[0011] GNE Ref. No. P60053-WO SUMMARY

[0012] The purpose and advantages of the disclosed subject matter will be set forth in and are apparent from the description that follows, as well as will be learned by practice of the disclosed subject matter. Additional advantages of the disclosed subject matter will be realized and attained by the devices particularly pointed out in the written description and claims hereof, as well as from the appended drawings.

[0013] The present disclosure provides a method for performing a genomic screen. In certain embodiments, the method includes providing a plurality of cells, contacting the plurality of cells with (a) a first lentivirus vector comprising (i) a first nucleic acid sequence encoding a gRNA and (ii) a second nucleic acid sequence encoding a cell surface protein, and (b) a second lentivirus vector comprising (i) a first nucleic acid sequence encoding a Cas protein to obtain a plurality of transduced cells and contacting the plurality of transduced cells with magnetic microparticles coupled to an agent that binds to the cell surface protein to obtain an enriched population of cells that express the cell surface protein and the gRNA.

[0014] In certain embodiments, the method further includes culturing the transduced cells for a duration sufficient for expression of the gRNA, the cell surface protein and / or the Cas protein.

[0015] In certain embodiments, the method further includes contacting the enriched population of cells with one or more polynucleotides comprising a barcode to label each cell with a barcode.

[0016] In certain embodiments, the method further includes analyzing single-cell RNA expression data from the enriched population of cells. In certain embodiments, analyzing single-cell RNA expression data from the enriched population of cells comprises sequencing the RNAs of single cells obtained from the enriched population of cells.

[0017] In certain embodiments, the method further includes correlating a change in expression level of one or more RNAs to the gRNA expressed in a single cell of the enriched population of cells.

[0018] In certain embodiments, at least one of the first lentivirus vector and the second lentivirus vector further comprises a nucleic acid sequence encoding a reporter. In certain embodiments, the first lentivirus vector further comprises (iii) a third nucleic acid sequence encoding a reporter. In certain embodiments, the second lentivirus vector further comprises (ii) a second nucleic acid sequence encoding a reporter.

[0019] In certain embodiments, the cell surface protein is a receptor. In certain embodiments, the receptor is mThy 1.1.

[0020] 516169085.1 2Attorney Ref. No. 00B206.1710

[0021] GNE Ref. No. P60053-WO In certain embodiments, the agent is an antibody that specifically binds to the cell surface protein.

[0022] In certain embodiments, the expression of the cell surface protein and / or the Cas protein are controlled by a promoter that enables expression of the cell surface protein and / or the Cas protein in the plurality of cells.

[0023] In certain embodiments, the promoter is selected from the group consisting of an EFla, an Synl and an PGK promoter.

[0024] In certain embodiments, the expression of the gRNA is controlled by a first promoter and the expression of the cell surface protein is controlled by a second promoter. In certain embodiments, the first promoter is a U6 promoter, a T7 promoter or a combination thereof. In certain embodiments, the second promoter is an EF- la promoter, an Synl promoter or an PGK promoter.

[0025] In certain embodiments, the plurality of cells is contacted with the first lentivirus vector and the second lentivirus vector simultaneously. In certain embodiments, the plurality of cells is contacted with the first lentivirus vector, e.g., the lentivirus comprising (i) a first nucleic acid sequence encoding a gRNA, at an MOI of 0.3 or less.

[0026] In certain embodiments, the plurality of cells comprises cells of the nervous system or cancer cells. In certain embodiments, the plurality of cells comprises neurons. In certain embodiments, the plurality of cells comprises microglia.

[0027] In certain embodiments, the plurality of cells comprises from about 10 to about 1,000,000,000 cells.

[0028] In certain embodiments, contacting the plurality of cells with a first lentivirus vector comprises contacting the plurality of cells with a plurality of first lentivirus vectors, wherein each first lentivirus vector encodes a gRNA targeting a different target gene. In certain embodiments, each first lentivirus vector encodes from about 1 to about 5 gRNAs targeting a single target gene, e.g., about 3 gRNAs. In certain embodiments, the plurality of first lentivirus vectors comprises from about 10 to about 1,000 first lentivirus vectors.

[0029] The present disclosure further provides systems and kits for performing the methods described herein. In certain embodiments, a kit of the present disclosure is for use in performing a genomic screen described herein. In certain embodiments, a system of the present disclosure is for use in performing a genomic screen described herein.

[0030] The present disclosure further provides nucleic acid constructs described herein, lentivirus vectors comprising nucleic acid constructs described herein and compositions

[0031] 516169085.1 3Attorney Ref. No. 00B206.1710

[0032] GNE Ref. No. P60053-WO thereof. For example, but not by way of limitation, the present disclosure provides compositions for use in a genomic screen. In certain embodiments, the composition includes (a) a first nucleic acid construct comprising (i) a first nucleic acid sequence encoding a gRNA and (ii) a second nucleic acid sequence encoding a cell surface protein; and (b) a second nucleic acid construct comprising (i) a first nucleic acid sequence encoding a Cas protein. In certain embodiments, at least one of the first nucleic acid construct and the second nucleic acid construct further comprises a nucleic acid sequence encoding a reporter. In certain embodiments, the first nucleic acid construct comprises (i) a first nucleic acid sequence encoding a gRNA, (ii) a second nucleic acid sequence encoding a cell surface protein and (iii) a third nucleic acid sequence encoding a reporter. In certain embodiments, the second nucleic acid construct comprises (i) a first nucleic acid sequence encoding a Cas protein and (ii) a second nucleic acid sequence encoding a reporter.

[0033] In certain embodiments, a composition for use in performing a genomic screen includes (a) a first lentivirus vector comprising (i) a first nucleic acid sequence encoding a gRNA and (ii) a second nucleic acid sequence encoding a cell surface protein; and (b) a second lentivirus vector comprising (i) a first nucleic acid sequence encoding a Cas protein. In certain embodiments, at least one of the first lentivirus vector and the second lentivirus vector further comprises a nucleic acid sequence encoding a reporter. In certain embodiments, the first lentivirus vector comprises (i) a first nucleic acid sequence encoding a gRNA, (ii) a second nucleic acid sequence encoding a cell surface protein and (iii) a third nucleic acid sequence encoding a reporter. In certain embodiments, the second lentivirus vector comprises (i) a first nucleic acid sequence encoding a Cas protein and (ii) a second nucleic acid sequence encoding a reporter.

[0034] In certain embodiments of the composition, the first nucleic acid sequence encoding the gRNA is coupled to a first promoter and the second nucleic acid sequence encoding the cell surface protein is coupled to a second promoter. In certain embodiments, the first promoter is a U6 promoter, a T7 promoter or a combination thereof. In certain embodiments, the second promoter is an EF-la promoter, an Synl promoter or an PGK promoter. In certain embodiments, the first nucleic acid sequence encoding the Cas protein is coupled to a promoter. In certain embodiments, the promoter is selected from the group consisting of an EF-la, an Synl and an PGK promoter. In certain embodiments, the cell surface protein is a receptor. In certain embodiments, the receptor is m Thy 1.1. In certain embodiments, the Cas protein is a Cas9 protein.

[0035] 516169085.1 4Attorney Ref. No. 00B206.1710

[0036] GNE Ref. No. P60053-WO The present disclosure further provides systems and kits containing the nucleic acid constructs, lentivirus vectors comprising the nucleic acid constructs and compositions thereof. In certain embodiments, a kit of the present disclosure includes nucleic acid constructs described herein, lentivirus vectors comprising nucleic acid constructs described herein and compositions thereof. In certain embodiments, a system of the present disclosure includes nucleic acid constructs described herein, lentivirus vectors comprising nucleic acid constructs described herein and compositions thereof.

[0037] BRIEF DESCIPTION OF THE DRAWINGS

[0038] The following figures are included to illustrate certain aspects of the present disclosure and should not be viewed as exclusive embodiments. The subject matter disclosed is capable of considerable modifications, alterations, combinations and equivalents in form and function, without departing from the scope of this disclosure.

[0039] Fig- 1 illustrates an exemplary double lentivirus system, with a CROPseq lentiviral vector comprising a polynucleotide encoding a guide RNA (gRNA) and a second lentiviral vector comprising a polynucleotide encoding Cas9.

[0040] Fig- 2 illustrates the editing efficiency of double lentivirus transduction in cancer cells. A lentivirus vector for expression of Cas9 and lentivirus vector for expression of a gRNA targeting Target Gene 1 (“Tl”) were delivered to A549 cells serially, where the lentivirus vector for expression of a gRNA was delivered first and the lentivirus vector for expression of Cas9 was delivered second to the cancer cells. A Tl knock-out of 95.6% (left peak) was observed when measured by flow cytometry compared to a non-targeting control (NTC) gRNA (right peak, 10.1% background knock-out).

[0041] Fig. 3A illustrates an exemplary experimental approach to assay the editing of double lentivirus efficiency in iNeurons. iNeurons were transduced with the double lentivirus system on Day 1 and were sorted and analyzed by FACS on Day 21.

[0042] Fig. 3B illustrates an exemplary 4-part sort-gating FACS strategy, where (1) neurons were isolated from debris (SSC-A v. FSC-A), (2) singlets were isolated from multiplets (FSC-H vs. FSC-A), (3) mCherry+ expressing neurons were isolated (FL3-A), and finally (4) mTurq2+ expressing neurons were isolated.

[0043] Fig. 3C illustrates the knock-out of gene targets T2 and T3 of >80% and >90%, respectively.

[0044] Figs. 4A-4B illustrate the ability of the double lentivirus system to efficiently edit two

[0045] 516169085.1 5Attorney Ref. No. 00B206.1710

[0046] GNE Ref. No. P60053-WO iNeuron types. The double lentivirus system achieved 87.8% knock-out of gene target T4 in iNeuron type 1 (Fig. 4A) and 60% knock-out of gene target T4 in iNeuron type 2 (Fig. 4B).

[0047] Figs. 5A-5C illustrate the impact of transduction timing. Fig. 5A illustrates two significant populations of iNeurons transduced at DIV1: iNeurons expressing the reporter (mTurq2) from the gRNA vector with loss of the target protein as expected (cells in the top left quadrant that are mTurq2 -positive with editing), and a significant population of iNeurons that display loss of the target protein with no reporter (mTurq2) from the gRNA vector due to silencing (cells in the bottom left quadrant that are mTurq2 -negative with editing). All cells expressed the reporter (mCherry) from the Cas9 vector. Fig. 5B illustrates that transducing at DIV7 and DIV14 vastly reduced the percentage of edited mTurq- cells, with transduction at DIV7 showing the greatest reduction (2.13% silenced cells in the bottom left quadrant). Fig.

[0048] 5C illustrates the editing efficiency for transduction at DIV7 and DIV14. Transduction at DIV7 showed 54% editing efficiency (middle histogram), which was significantly more effective than the 28% editing efficiency observed for transduction at DIV14 (bottom histogram).

[0049] Fig- 6 illustrates the strength of reporter expression of the Cas9 lentivirus vector under the control of three different promoters (Efl a, hPGK, and hSynl). The greatest expression was observed with hPGK (60%, middle histogram), followed by hSynl (46%, bottom histogram), and then Efla (30%, top histogram).

[0050] Figs. 7A-7D illustrate the transduction of a pool of sgRNAs to iNeurons. Fig. 7A illustrates the exemplary experimental schematic. Fig. 7B illustrates an example distribution of NTC counts for a single guide. Many cells have a single count of multiple guides but high counts for a single NTC guide. This bimodal distribution of counts enables robust singlet assignment. Figs. 7C-7D illustrate the assignment of 386 singlets containing 171 distinct guides.

[0051] Figs. 8A-8H illustrate a pooled CRISPR screen in iNeurons. A perturb-seq pilot targeting 150 genes was performed, transducing on DIV7 and enriching for guide-expressing cells via FACS on DIV21. Fig. 8A illustrates iNeurons from the perturb-seq pilot projected onto a reference atlas space. Fig. 8B illustrates the distribution of 8,893 guide-assigned iNeurons (~30 cells per guide) across iNeuron subtypes. The preponderance of a single subtype is not representative of iNeuron heterogeneity. Fig. 8C illustrates the impact of 78 gRNAs targeting 49 distinct genes on their target transcript, with the strength of the signal along the diagonal indicating on-target knock-down. Fig. 8F summarizes the regulatory model. The regulatory matrix (Fig. 8D) shows the impact of knocking out 50 genes (columns) on 865

[0052] 516169085.1 6Attorney Ref. No. 00B206.1710

[0053] GNE Ref. No. P60053-WO affected genes (rows). Horizontal and vertical black lines delineate co-regulated gene programs (Fig. 8E) and their regulatory modules (Fig. 8F). Genes are clustered by module (Fig. 8F) or by program (Fig. 8E). Fig. 8G illustrates a schematic of functional gene networks identified in Figs. 8D-8F. A selected subset of genes in each module or program are listed as examples. Point arrows and blunt arrows denote activation and inhibition respectively. Fig.

[0054] 8H illustrates the exemplary experimental schematic and amounts of cells involved.

[0055] Fig.9A illustrates the exemplary experimental schematic for nuclei isolation and FACS enrichment. iNeurons were transduced one week after thaw with a lentivirus encoding a gRNA. At DIV21, iNeurons were dissociated and frozen down before lysing with TST buffer. The subsequent nuclei retain expression of H2B-GFP, enabling FACS-based enrichment of transduced neurons. Fig. 9B illustrates the same data two ways. One histogram (left) depicts the distribution of gRNA counts across cells versus nuclei; the other (right) shows the same data as a weighted histogram showing the distribution of reads over gRNA counts in the two different preps. Together, they show that reads are concentrated in cells with high gRNA expression compared to nuclei. Fig. 9C illustrates the distribution of guide-assigned nuclei across iNeuron subtypes.

[0056] Fig. 10A illustrates the exemplary experimental schematic for magnetic enrichment. iNeurons were transduced one week after thaw with a gRNA-encoding lentivirus that also expresses GFP2xNLS and mThy 1.1. mThy 1.1 -positive cells were isolated via commercially available magnetic bead-based positive selection kits. Fig. 10B illustrates mThy 1.1 expression measured by GFP signal. 56% of cells were mThy 1.1 -positive after magnetic enrichment, compared to 23% of mThy 1.1 -positive cells before magnetic enrichment. Fig. IOC illustrates the proportion of gRNA-assigned cells and their distribution across iNeuron subtypes, with -40% of cells assigned to a single gRNA. Fig. 10D illustrates the distribution of iNeuron subtypes using magnetic enrichment versus FACS enrichment. Magnetic enrichment better preserved iNeuron heterogeneity (compare the distribution of subtypes between “Magnetic Miltenyi” and “Magnetic Stemcell EasySep” versus “FACS Perturbseq” and internal reference datasets “reference...”).

[0057] Fig. HA illustrates the exemplary experimental schematic for magnetic enrichment.

[0058] Fig. 11B illustrates that cells remained intact through the enrichment protocol using either Miltenyi and StemCell Tech (EasySep™) magnetic beads. Figs. 11C-11E illustrate gRNA counts in cells enriched using EasySep™ beads compared to Miltenyi. Fig. HF illustrates the proportion of gRNA-assigned cells observed in cell and nuclei samples using EasySep

[0059] 516169085.1 7Attorney Ref. No. 00B206.1710

[0060] GNE Ref. No. P60053-WO compared to Miltenyi. The proportion of assigned singlets is overlaid on the bar plot. Fig.

[0061] 11G illustrates the enrichment of cells using Miltenyi (LD or LS columns; 30.3% and 38.1%, respectively) compared to StemCell Tech (EasySep™; 60.2 %) magnetic beads. The shaded histogram illustrates the starting population with 28.7% GFP-positive cells.

[0062] Fig. 12A illustrates the distribution of recovered doublets (top bar) and singlets (middle bar) across 3 10X v4 channels overloaded with 60-70k cells. Fig. 12B illustrates UMAP barcode space (color-coded as in Fig. 12A) showing the intermingled distribution of unknowns, doublets, and singlets across the barcode clusters. Fig. 12C shows the expression of MULTIseq barcodes across demultiplexed cells (rows correspond to individual barcodes, columns correspond to barcode assignments).

[0063] Fig. 13A illustrates nucleic acid constructs encoding a gRNA for use in the methods of the present disclosure. Fig. 13B illustrates nucleic acid constructs encoding Cas9 for use in the methods of the present disclosure.

[0064] Fig. 14 illustrates a nucleic acid construct encoding a gRNA, a fluorescent reporter and a cell surface protein for use in the methods of the present disclosure.

[0065] Fig. 15 illustrates the enrichment efficiency of cells at different MOI transduction rates.

[0066] Fig. 16 illustrates the enrichment efficiency of cell suspensions with different iNeuron cell concentrations following transduction.

[0067] DETAILED DESCRIPTION

[0068] The present disclosure provides improved methods of CRISPR screening in cells such as neuronal cells. The present disclosure is based, in part, on double lentiviral infection for expression of a Cas protein and a gRNA that are compatible with pooled CRISPR screening, allowing for greater temporal control over the timing of the perturbation and pooled CRISPR screening in any cell type amenable to lentiviral infections. The present disclosure is further based, in part, on streamlining the process of screen enablement by eliminating the need to create and test multiple Cas-expressing cells within or across cell lines, as the same virus can be applied to multiple cell types at once. The present disclosure is further based, in part, on stably integrating the Cas protein and gRNA into the cells, which may enable additional, future perturbations in the same cells if desired, a feature not possible with transient transfection. The present disclosure is further based, in part, on the use of magnetic enrichment of gRNA-expressing cells, e.g., neuronal cells, through the overexpression of a cell surface receptor, e.g., m Thy 1.1, offering a significantly faster and higher throughput approach to FACS, in addition

[0069] 516169085.1 8Attorney Ref. No. 00B206.1710

[0070] GNE Ref. No. P60053-WO to allowing for a more even distribution of cell types within the tested sample.

[0071] For clarity, but not by way of limitation, the detailed description of the presently disclosed subject matter is divided into the following subsections:

[0072] I. Definitions;

[0073] II. Nucleic Acid Constructs, Vectors and Compositions Thereof;

[0074] III. Cells;

[0075] IV. Methods;

[0076] V. Systems;

[0077] VI. Kits; and

[0078] VII. Exemplary Embodiments.

[0079] I. DEFINITIONS

[0080] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which the subject matter of the present disclosure belongs. The following references provide one of skill with a general definition of many of the terms used in the present disclosure: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.

[0081] As used herein, the use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification can mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[0082] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 3 or more than 3 standard deviations, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value.

[0083] The term “antibody” herein is used in the broadest sense and encompasses various

[0084] 516169085.1 9Attorney Ref. No. 00B206.1710

[0085] GNE Ref. No. P60053-WO antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) and antibody fragments so long as they exhibit the desired antigen-binding activity.

[0086] The terms “comprise(s),” “comprise(s),” “having,” “has,” “can,” “contain(s)” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms or words that do not preclude additional acts or structures. The present disclosure also contemplates other embodiments “comprising,” “consisting of’ and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0087] The terms “detect” or “detection,” as used herein, indicate the determination of the existence and / or presence of a target, e.g., a nucleic acid target (e.g., a variant target nucleic acid), in a limited portion of space, including but not limited to a sample. The terms “detect” or “detection,” as used herein, can comprise determination of chemical and / or biological properties of the target, including but not limited to ability to interact, and in particular bind, other compounds, ability to activate another compound and additional properties identifiable by a skilled person upon reading of the present disclosure. The detection can be quantitative or qualitative. A detection is “quantitative” when it refers, relates to, or involves the measurement of quantity or amount of the target or signal (also referred as quantitation), which comprises but is not limited to any analysis designed to determine the amounts or proportions of the target or signal. A detection is “qualitative” when it refers, relates to, or involves identification of a quality or kind of the target or signal in terms of relative abundance to another target or signal, which is not quantified.

[0088] The term “editing efficiency,” as used herein, refers to the total number of sequence reads with insertions or deletions of nucleotides into a target region of interest over the total number of sequence reads following cleavage by an RNA-guided nuclease.

[0089] The terms “expression” or “expresses,” as used herein, refer to transcription and translation occurring within a cell. The level of expression of a gene and / or nucleic acid in a cell can be determined on the basis of either the amount of corresponding mRNA that is present in the cell or the amount of the protein encoded by the gene and / or nucleic acid that is produced by the cell. For example, mRNA transcribed from a gene and / or nucleic acid is desirably quantitated by northern hybridization. Sambrook et al., Molecular Cloning: A Laboratory Manual, pp. 7.3-7.57 (Cold Spring Harbor Laboratory Press, 1989). Protein encoded by a gene and / or nucleic acid can be quantitated either by assaying for the biological activity of the protein or by employing assays that are independent of such activity, such as western blotting

[0090] 516169085.1 10Attorney Ref. No. 00B206.1710

[0091] GNE Ref. No. P60053-WO or radioimmunoassay using antibodies that are capable of reacting with the protein. Sambrook et al., Molecular Cloning: A Laboratory Manual, pp. 18.1-18.88 (Cold Spring Harbor Laboratory Press, 1989).

[0092] The terms “guide RNA,” “sgRNA,” “gRNA” or “gRNA molecule,” as used interchangeably herein, refer to a nucleic acid that promotes the specific targeting or homing of an RNA-guided nuclease to a target nucleic acid.

[0093] As used herein, the term “individual” or “subject” refers to a vertebrate or an invertebrate, such as a human or non-human animal, for example, a mammal. Mammals comprise, but are not limited to, humans, non-human primates, farm animals, sport animals, rodents and pets. Non-limiting examples of non-human animal subjects comprise rodents such as mice, rats, hamsters, guinea pigs, rabbits, dogs, cats, sheep, pigs, goats, cattle, horses, apes and monkeys. In certain embodiments, the individual or subject is a human.

[0094] As used herein, the term “zw vitro" refers to an artificial environment and to processes or reactions that occur within an artificial environment. In vitro environments exemplified, but are not limited to, test tubes and cell cultures.

[0095] An “isolated nucleic acid” refers to a nucleic acid molecule that has been separated from a component of its environment, e.g., natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0096] As used herein, a “reporter,” “label” or “marker” refers to an agent that allows for direct or indirect detection or for isolation. Labels comprise, but are not limited to, fluorescent labels, chromogenic labels, electron dense labels, chemiluminescent labels and radioactive labels. Non-limiting examples of labels comprise green fluorescent protein (“GFP”), mCherry, dtTomato, or other fluorescent proteins known in the art (e.g., Shaner et al., A Guide to Choosing Fluorescent Proteins, Nature Methods 2(12):905-909 (2005) incorporated by reference herein, 32P, 14C, 1251, 3H and 1311, fluorogens (such as Rare Earth Chelate or lucifer yellow and its derivatives), Rhodamine (rhodamine) and its derivatives, dansyl, umbelliferone, luciferase (such as firefly luciferase and bacterial fluorescence plain enzyme) (U.S. Patent number 4,737,456), fluorescein, 2,3-dihydros phthalazine diketone, as well as enzymes producing detectable signals, e.g., horseradish peroxidase (HRP), alkaline phosphorus sour enzyme, beta galactosidase, glucoamylase, lysozyme, carbohydrate oxidase (such as glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase (G6PD)) and heterocyclic

[0097] 516169085.1 11Attorney Ref. No. 00B206.1710

[0098] GNE Ref. No. P60053-WO oxidases (such as uricase and xanthine oxidase). In certain embodiments, a reporter can be used for the isolation of a cell.

[0099] The term “nucleic acid” or “polynucleotide” comprises any compound and / or substance that comprises a polymer of nucleotides. Each nucleotide is composed of a base, specifically a purine- or pyrimidine base ( / .< ., cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar ( / .< ., deoxyribose or ribose), and a phosphate group. Often, the nucleic acid molecule is described by the sequence of bases, whereby said bases represent the primary structure (linear structure) of a nucleic acid molecule. The sequence of bases is typically represented from 5’ to 3’. The term nucleic acid encompasses deoxyribonucleic acid (DNA) including, e.g., complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), e.g., messenger RNA (mRNA), synthetic forms of DNA or RNA, and mixed polymers comprising two or more of these molecules. The nucleic acid molecule can be linear or circular. In addition, the term nucleic acid comprises both, sense and antisense strands, as well as single stranded and double stranded forms. Moreover, the herein described nucleic acid can contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides comprise modified nucleotide bases with derivatized sugars or phosphate backbone linkages or chemically modified residues. The terms “nucleic acid” and “polynucleotide” as used herein refer to a polymeric form of nucleotides of any length, either ribonucleotides (RNA) or deoxyribonucleotides (DNA). These terms refer to the primary structure of the molecule, and thus include double- and single-stranded DNA, and double- and single-stranded RNA. The terms include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs and modified polynucleotides such as, for example, methylated and / or capped polynucleotides. Nucleic acids are typically linked via phosphate bonds to form nucleic acid sequences or polynucleotides, though many other linkages are known in the art (e.g., phosphorothioates, boranophosphates, and the like).

[0100] The nucleic acids and / or polynucleotides described herein can be prepared using any suitable method, a variety of which are known in the art (see, for example, Sambrook et al., Molecular Cloning. A Laboratory Manual, 1989, 2. Supp. Ed., Cold Spring Harbour Laboratory Press: New York, N.Y.; M. A. Innis (Ed.), PCR Protocols. A Guide to Methods and Applications, Academic Press: New York, N.Y. (1990); P. Tijssen, Hybridization with Nucleic Acid Probes - Laboratory Techniques in Biochemistry and Molecular Biology (Parts I and II), Elsevier Science (1993); M. A. Innis (Ed.), PCR Strategies, Academic Press: New York, N.Y. (1995); and F. M. Ausubel (Ed.), Short Protocols in Molecular Biology, John Wiley & Sons:

[0101] 516169085.1 12Attorney Ref. No. 00B206.1710

[0102] GNE Ref. No. P60053-WO Secaucus, N.J. (2002); Narang et al., Meth. Enzymol., 68:90-98 (1979); Brown et al., Meth. Enzymol., 68:109-151 (1979); and Belousov et al., Nucleic Acids Res., 25: 3440-3444 (1997), each of which is incorporated herein by reference in its entirety). Polynucleotide synthesis can be performed on synthesizers such as those commercially available from Perkin Elmer / Applied Biosystems, Inc. (Foster City, CA), DuPont (Wilmington, DE), or Milligen (Bedford, MA). Alternatively, polynucleotides can be custom made and obtained from a variety of commercial sources well-known in the art, including, for example, the Midland Certified Reagent Company (Midland, TX), Eurofins Scientific (Louisville, KY), BioSearch Technologies, Inc. (Novato, CA), and the like. Polynucleotides can be purified using any suitable method known in the art, such as, for example, native acrylamide gel electrophoresis, anion-exchange HPLC (see, e.g., Pearson et al., J. Chrom., 255:137-149 (1983), incorporated herein by reference), and reverse phase HPLC (see, e.g., McFarland et al., Nucleic Acids Res., 7: 1067-1080 (1979), incorporated herein by reference).

[0103] The sequence of the polynucleotides can be verified using any suitable sequencing method known in the art, including, but not limited to, chemical degradation (see, e.g., Maxam et al., Methods of Enzymology, 65:499-560 (1980), incorporated herein by reference), matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (see, e.g., Pieles et al., Nucleic Acids Res., 21:3191 -3196 (1993), incorporated herein by reference), mass spectrometry following a combination of alkaline phosphatase and exonuclease digestions (Wu et al., Anal. Biochem., 29).

[0104] The term “operative connection,” “operatively coupled,” “operably coupled,” “operably linked” or “operatively linked,” as used herein, with regard to regulatory sequences of a nucleic acid construct indicate an arrangement of elements in a combination enabling production of an appropriate effect. With respect to nucleic acid constructs and regulatory sequences, an operative connection indicates a configuration of the nucleic acid constructs (or a nucleic acid sequence within the nucleic acid construct) with respect to the regulatory sequence allowing the regulatory sequences to directly or indirectly increase or decrease transcription or translation of the nucleic acid construct (or a nucleic acid sequence within the nucleic acid construct). In particular, in certain embodiments, regulatory sequences directly increasing transcription of the operatively linked nucleic acid comprise promoters typically located on a same strand and upstream on a nucleic acid sequence (towards the 5’ region of the sense strand), adjacent to the transcription start site of the nucleic acid construct (or a nucleic acid sequence within the nucleic acid construct) whose transcription they initiate.

[0105] 516169085.1 13Attorney Ref. No. 00B206.1710

[0106] GNE Ref. No. P60053-WO The term “plurality” refers to a number larger than one. In certain embodiments, the term “plurality of cells” refers to a number of cells larger than one. For example, but not by way of limitation, a plurality of cells includes at least two cells. In certain embodiments, a plurality of cells includes about 10,000 or more cells, about 20,000 or more cells, about 30,000 or more cells, about 40,000 or more cells, about 50,000 or more cells, about 100,000 or more cells, about 150,000 or more cells, about 200,000 or more cells, about 300,000 or more cells, about 400,000 or more cells or 500,000 or more cells. In certain embodiments, the term “plurality of gRNA-expressing vectors,” e.g., lentivirus vectors, refers to a number of vectors larger than one. For example, but not by way of limitation, a plurality of plurality of gRNA-expressing vectors comprises at least two gRNA-expressing vectors. In certain embodiments, the term “plurality of particles” refers to a number of particles larger than one. For example, but not by way of limitation, a plurality of particles comprises at least two particles.

[0107] The term “reverse-transcription process” refers to a process of generating a complementary strand of DNA using an enzyme called a reverse transcriptase.

[0108] The term “sample,” as used herein, refers to any sample containing one or more individual cells. In certain embodiments, “sample” refers to a sample of biological material obtained from a subject, e.g., a tissue biopsy or a tissue sample. In certain embodiments, the subject can be a human, non-human primate, e.g., an ape or a monkey, a farm animal, a mouse, a rat, a hamster, a guinea pig, a rabbit, a dog, cat, a sheep, a pig, a goat, a cow or a horse. In certain embodiments, the subject is a human. In certain embodiments, the sample can be obtained from preserved tissue, e.g., fixed tissue, from frozen tissue or from fresh tissue, e.g., tissue samples. In certain embodiments, the sample can be obtained from a frozen sample. In certain embodiments, a sample that can be analyzed using the methods of the present disclosure comprise at least two or more cells and / or nuclei. For example, but not by way of limitation, a sample can comprise about 10 or more cells and / or nuclei, about 100 or more cells and / or nuclei, about 1,000 or more cells and / or nuclei, about 5,000 or more cells and / or nuclei, about 10,000 or more cells and / or nuclei, about 20,000 or more cells and / or nuclei, about 30,000 or more cells and / or nuclei, about 40,000 or more cells and / or nuclei, about 50,000 or more cells and / or nuclei, about 100,000 or more cells and / or nuclei, about 150,000 or more cells and / or nuclei, about 200,000 or more cells and / or nuclei, about 300,000 or more cells and / or nuclei, about 400,000 or more cells and / or nuclei, about 500,000 or more cells and / or nuclei, about 1,000,000 or more cells and / or nuclei, about 2,000,000 or more cells and / or nuclei, about 3,000,000 or more cells and / or nuclei, about 4,000,000 or more cells and / or nuclei or about

[0109] 516169085.1 14Attorney Ref. No. 00B206.1710

[0110] GNE Ref. No. P60053-WO 5,000,000 or more cells and / or nuclei. In certain embodiments, the cells and / or nuclei of a sample are obtained from (e.g., isolated from) a tissue. In certain embodiments, the cells and / or nuclei of a sample are obtained from (e.g, isolated from) a cell sample.

[0111] The term “specifically binds,” as used herein, refers to the preferential binding to a target molecule, e.g, a protein or nucleic acid, relative to other molecules, e.g, proteins or nucleic acids, in a sample.

[0112] As used herein, the term “subset” refers to a small portion of a larger quantity of material.

[0113] The term “vector,” as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term comprises the vector as a selfreplicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors.”

[0114] II. NUCLEIC ACID CONSTRUCTS, VECTORS AND COMPOSITIONS THEREOF

[0115] The present disclosure provides nucleic acid constructs for use in the methods of the present disclosure. The present disclosure further provides compositions (e.g., nucleic acid compositions) that comprise one or more nucleic acid constructs for use in the methods of the present disclosure. In certain embodiments, a composition of the present disclosure comprises two nucleic acid constructs, e.g., a first nucleic acid construct and a second nucleic acid construct.

[0116] In certain embodiments, a nucleic acid construct, e.g., a first nucleic acid construct, of the present disclosure comprises a nucleotide sequence (e.g, a polynucleotide) that encodes a gRNA. Non-limiting examples of a first nucleic acid construct comprising a nucleic acid sequence (e.g., a polynucleotide) that encodes a gRNA are shown in Fig. 13A and Fig. 14.

[0117] In certain embodiments, a gRNA that is encoded by a nucleic acid construct, e.g., a first nucleic acid construct, of the present disclosure has a length from about 20 to about 200 nucleotides, e.g., from about 20 to about 190, from about 20 to about 180, from about 20 to about 170, from about 20 to about 160, from about 20 to about 150, from about 20 to about 140, from about 20 to about 130, from about 20 to about 120, from about 20 to about 110, from about 20 to about 100, from about 30 to about 200, from about 40 to about 200, from about 50

[0118] 516169085.1 15Attorney Ref. No. 00B206.1710

[0119] GNE Ref. No. P60053-WO to about 200, from about 60 to about 200, from about 70 to about 200, from about 80 to about 200, from about 90 to about 200, from about 50 to about 150, from about 80 to about 120 or from about 90 to about 100 nucleotides. In certain embodiments, a gRNA of the present disclosure has a length from about 80 to about 120 nucleotides. In certain embodiments, a gRNA of the present disclosure is about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 155, about 160, about 165, about 170, about 175, about 180, about 185, about 190, about 195 or about 200 or more nucleotides in length.

[0120] In certain embodiments, the gRNA comprises a targeting domain that is complementary to, e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% complementary to, a genomic nucleotide sequence, e.g., of a target gene. In certain embodiments, the targeting domain is from about 15 to about 25 nucleotides in length. In certain embodiments, the targeting domain is 18 nucleotides in length. In certain embodiments, the targeting domain is 19 nucleotides in length. In certain embodiments, the targeting domain is 20 nucleotides in length. In certain embodiments, the targeting domain is 21 nucleotides in length. In certain embodiments, the targeting domain is 22 nucleotides in length. In certain embodiments, the targeting domain is 23 nucleotides in length. In certain embodiments, the targeting domain is 24 nucleotides in length. In certain embodiments, the targeting domain is 25 nucleotides in length.

[0121] In certain embodiments, a gRNA of the present disclosure can have a scaffold as disclosed in Dang et al., Genome Biology 16:280 (2015), the contents of which are incorporated by reference herein in their entirety. For example, but not by way of limitation, the duplex region of the gRNA scaffold can be extended by at least 1 base pair, 2 base pairs, 3 base pairs, 4 base pairs, 5 base pairs, 6 base pairs, 7 base pairs, 8 base pairs, 9 base pairs or 10 base pairs (see Figure 1 of Dang et al. (2015)). In certain embodiments, the duplex region of the gRNA scaffold can be extended by at least 5 base pairs, e.g., to improve editing efficiency of the gRNA (see Figure 7 of Dang et al. (2015)). In certain embodiments, the gRNA of the present disclosure can further include one or more mutations in the duplex (e.g., in the lower stem of the duplex). For example, but not by way of limitation, the gRNA scaffold of the

[0122] 516169085.1 16Attorney Ref. No. 00B206.1710

[0123] GNE Ref. No. P60053-WO present disclosure can include a mutation in the continuous sequence of Ts present in the duplex (e.g., present in the lower stem of the duplex), as shown in Figure 7 of Dang et al. (2015), to improve editing efficiency of the gRNA. In certain embodiments, position 4 of the continuous sequence of Ts in the lower stem of the duplex is mutated to a C or G).

[0124] In certain embodiments, a gRNA of the present disclosure can have an editing efficiency of about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% (e.g., when expressed using a nucleic acid construct of the present disclosure).

[0125] In certain embodiments, the gRNA has an editing efficiency of about 60% or greater (e.g., when expressed using a nucleic acid construct of the present disclosure). In certain embodiments, the gRNA has an editing efficiency of about 65% or greater (e.g., when expressed using a nucleic acid construct of the present disclosure). In certain embodiments, the gRNA has an editing efficiency of about 70% or greater (e.g., when expressed using a nucleic acid construct of the present disclosure). In certain embodiments, the gRNA has an editing efficiency of about 75% or greater (e.g., when expressed using a nucleic acid construct of the present disclosure). In certain embodiments, the gRNA has an editing efficiency of about 80% or greater (e.g., when expressed using a nucleic acid construct of the present disclosure). In certain embodiments, the gRNA has an editing efficiency of about 85% or greater (e.g., when expressed using a nucleic acid construct of the present disclosure). In certain embodiments, the gRNA has an editing efficiency of about 90% or greater (e.g., when expressed using a nucleic acid construct of the present disclosure). In certain embodiments, the gRNA has an editing efficiency of about 95% or greater (e.g., when expressed using a

[0126] 516169085.1 17Attorney Ref. No. 00B206.1710

[0127] GNE Ref. No. P60053-WO nucleic acid construct of the present disclosure).

[0128] In certain embodiments, the methods of the present disclosure can be used to identify gRNAs that have an editing efficiency of about 5% or greater, about 10% or greater, about 15% or greater, about 20% or greater, about 25% or greater, about 30% or greater, about 35% or greater, about 40% or greater, about 45% or greater, about 50% or greater, about 55% or greater, about 60% or greater, about 65% or greater, about 70% or greater, about 75% or greater, about 80% or greater, about 85% or greater, about 90% or greater or about 95% or greater. In certain embodiments, the methods of the present disclosure can be used to identify gRNAs that have an editing efficiency of about 30% or greater. In certain embodiments, the methods of the present disclosure can be used to identify gRNAs that have an editing efficiency of about 50% or greater.

[0129] In certain embodiments, a nucleic acid construct of the present disclosure, e.g., a first nucleic acid construct, comprises at least one promoter. For example, but not by way of limitation, a nucleic acid construct of the present disclosure, e.g., a first nucleic acid construct, comprises at least one promoter located upstream of the gRNA, e.g., as shown in Fig. 13A and Fig. 14 In certain embodiments, a nucleic acid construct of the present disclosure, e.g., a first nucleic acid construct, comprises a first promoter located upstream of the gRNA, e.g., operably linked to the gRNA. In certain embodiments, the promoter for use in the present disclosure is configured to express the gRNA in cells. In certain embodiments, the promoter is a regulated promoter (e.g., inducible promoter). In certain embodiments, the promoter is a constitutive promoter. In certain embodiments, the promoter is a viral promoter. In certain embodiments, the promoter is a mammalian promoter. In certain embodiments, the promoter is a non-viral promoter. In certain embodiments, the promoter for use in the present disclosure is configured to express the target nucleic acid, e.g., gRNA, in neuronal cells. Non-limiting examples of promoters for expressing the gRNA in a cell comprise U6, U3, U2, U5, Hl, 75J, EF-la, CMV, tRNA promoters, pGK, SV40, CAG, TRE, 7SK, VAI, Synl, T7, and PGK. In certain embodiment, the promoter for expressing the gRNA is U6. In certain embodiment, the promoter for expressing the gRNA is T7, e.g., for use in the Perturb View method (see, e.g., WO 2025 / 137335, the contents of which are incorporated by reference herein in their entirety). In certain embodiments, the promoter is a T7 promoter incorporated into a U6 promoter as disclosed in WO 2025 / 137335 (the contents of which are incorporated by reference herein in their entirety).

[0130] In certain embodiments, the promoter for expressing a gRNA in cells of the nervous

[0131] 516169085.1 18Attorney Ref. No. 00B206.1710

[0132] GNE Ref. No. P60053-WO system using a nucleic acid construct of the present disclosure, e.g., a first nucleic acid construct, is a U6 promoter.

[0133] In certain embodiments, the promoter for expressing a gRNA in cells of the nervous system using a nucleic acid construct of the present disclosure, e.g., a first nucleic acid construct, is a T7 promoter incorporated into a U6 promoter.

[0134] In certain embodiments, the promoter for expressing a gRNA in neurons using a nucleic acid construct of the present disclosure, e.g., a first nucleic acid construct, is a U6 promoter.

[0135] In certain embodiments, the promoter for expressing a gRNA in neurons using a nucleic acid construct of the present disclosure, e.g., a first nucleic acid construct, is a T7 promoter incorporated into a U6 promoter.

[0136] In certain embodiments, the present disclosure further provides a second nucleic acid construct, or compositions thereof, that comprises a nucleotide sequence (e.g., a polynucleotide) that encodes a nuclease. Non-limiting examples of a second nucleic acid construct comprising a nucleic acid sequence (e.g., a polynucleotide) that encodes a nuclease are shown in Fig. 13B In certain embodiments, the nucleotide sequence that encodes a nuclease is operatively coupled to a promoter. Non-limiting examples of promoters for expressing the nuclease in a cell comprise U6, U3, U2, U5, Hl, 75J, EF-la, CMV, tRNA promoters, pGK, SV40, CAG, TRE, 7SK, VAI, UbiC (Ubiquitin C) promoter, CD68 promoter, 134 bp microglia-specific HexB promoter, Synl, and PGK. In certain embodiments, the promoter is selected from the group of EF-la, hSynl, and hPGK promoters. In certain embodiments, the promoter for expressing a nuclease in cells using a nucleic acid construct of the present disclosure, e.g., a second nucleic acid construct, is an EF-la promoter. In certain embodiments, the EF-la promoter is the full-length EF-la promoter that is about 1,200 bp in length. In certain embodiments, the EF-la promoter is the core EF-la promoter that is about 200-300 bp in length and is derived from the full-length EF-la promoter. In certain embodiments, the promoter for expressing a nuclease in cells using a nucleic acid construct of the present disclosure, e.g., a second nucleic acid construct, is an hSynl promoter. In certain embodiments, the promoter for expressing a nuclease in cells using a nucleic acid construct of the present disclosure, e.g., a second nucleic acid construct, is an hPGK promoter.

[0137] In certain embodiments, the promoter for expressing a nuclease in cells of the nervous system using a nucleic acid construct of the present disclosure, e.g., a second nucleic acid construct, is an hPGK promoter. In certain embodiments, the promoter for expressing a nuclease in neurons using a nucleic acid construct of the present disclosure, e.g., a second

[0138] 516169085.1 19Attorney Ref. No. 00B206.1710

[0139] GNE Ref. No. P60053-WO nucleic acid construct, is an hPGK promoter. As shown in Fig. 6, use of a hPGK promoter resulted in significant expression of a Cas protein in neuronal cells.

[0140] In certain embodiments, the promoter for expressing a nuclease in cells of the nervous system using a nucleic acid construct of the present disclosure, e.g., a second nucleic acid construct, is an hSynl promoter.

[0141] In certain embodiments, the promoter for expressing a nuclease in neurons using a nucleic acid construct of the present disclosure, e.g., a second nucleic acid construct, is an hSynl promoter.

[0142] Non-limiting examples of nucleases include RNA-guided nucleases. In certain embodiments, the RNA-guided nuclease is a Cas protein. Non-limiting examples of Cas proteins are disclosed in Makarova and Koonin, Methods Mol. Biol. 1311:47-75 (2015), the contents of which are incorporated herein by reference in their entirety. In certain embodiments, Cas proteins comprise Casl, Cas2, Cas3, Cas3-HD, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, CaslO, Casl2a (Cpfl), Casl3, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, CsaX, Csm2, Csm3, Csm4, Csm5, Csm6, Csn2, Csbl, Csb2, Csb3, Csxl, Csx3, CsxlO, Csxl4, Csxl5, Csxl6, Csxl7, Csfl, Csf2, Csf3, Csf4, C2cl, C2c2 and C2c3. In certain embodiments, the Cas protein is selected from the group consisting of a Cas9, a Cas 12a, a Cas 13 and a combination thereof. In certain embodiments, the Cas protein is a Cas9 protein.

[0143] In certain embodiments, the Cas protein is an engineered Cas protein that differs from a reference Cas protein, e.g., a wild-type Cas protein. In certain embodiments, the reference Cas protein is a naturally occurring Cas protein. In certain embodiments, the Cas protein comprises one or more amino acid variations compared to a reference Cas protein, e.g., a wildtype Cas protein. In certain embodiments, an engineered Cas protein retains or substantially retains the nuclease (e.g., endonuclease) activity of the reference Cas protein. In certain embodiments, the engineered Cas protein retains at least about 70%, about 80%, about 90%, about 95% or about 99% nuclease activity of the reference Cas protein. In certain embodiments, an engineered Cas protein has no or no substantial cleavage activity. In certain embodiments, a Cas protein can lack cleavage activity or have substantially less, e.g., less than 20%, about 10%, about 5% or about 1% of the cleavage activity of a reference Cas protein.

[0144] In certain embodiments, the engineered Cas protein comprises one or more deletions that reduces the size of the Cas protein while at least partially retaining the nuclease activity of the Cas protein. In certain embodiments, the reduced size of the engineered Cas protein can

[0145] 516169085.1 20Attorney Ref. No. 00B206.1710

[0146] GNE Ref. No. P60053-WO allow flexibility with respect to the methods for delivering such engineered Cas proteins.

[0147] In certain embodiments, a Cas protein (e.g., expressed from a second nucleic acid construct of the present disclosure) interacts with a gRNA molecule of the present disclosure (e.g., expressed from a first nucleic acid construct of the present disclosure) and, in concert with the gRNA molecule, localizes to a target genomic sequence (e.g., a sequence that is complementary to the targeting domain sequence of the gRNA molecule) and a PAM sequence. In certain embodiments, the ability of a Cas protein to interact with and cleave a target genomic sequence is PAM sequence dependent. In certain embodiments, cleavage of the target genomic sequence occurs upstream from the PAM sequence. In certain embodiments, cleavage of the target genomic sequence occurs downstream from the PAM sequence. Cas molecules from different species, e.g., bacterial species, can recognize different PAM sequences.

[0148] In certain embodiments, a Cas protein for use in the present disclosure can be derived from any one of the following species: Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus thermophilus, Streptococcus agalactiae, Streptococcus parasanguinis, Streptococcus oralis, Streptococcus salivarius, Streptococcus macacae, Streptococcus dysgalactiae, Streptococcus anginosus, Streptococcus constellatus, Streptococcus pseudoporcinus, Streptococcus mutans, Listeria innocua, Spiroplasma apis, Spiroplasma syrphidicola, Porphyromonas catoniae, Prevotella intermedia, Treponema socranskii, Finegoldia magna, Pasteurella bettyae, Olivibacter sitiensis, Epilithonimonas tenax, Mesonia mobilis, Lactobacillus plantarum, Coriobacteriaceae bacterium, Olsenella profusa, Haemophilus sputorum, Bacillus cereus, Aquimarina muellen, Chryseobacterium palustre, Bacteroides graminisolvens, Neisseria meningitidis, Francisella novicida, Haemophilus pittmaniae, Pasteurella bettyae, Olivibacter sitiensis, Epil ithonimonas tenax, Mesonia mobilis, Lactobacillus plantarum, Bacillus cereus, Aquimarina muellen, Chryseobacterium palustre, Bacteroides graminisolvens, Neisseria meningitidis, Francisella novicida, Flavobacterium frigidarium, Flavobacterium soli and / or Treponema denticola.

[0149] In certain embodiments, a Cas protein for use in the present disclosure directs cleavage of one or both strands at a genomic location. For example, but not by way of limitation, a Cas protein for use in the present disclosure directs cleavage of one or both strands within a genomic location. Alternatively, the Cas protein directs cleavage of one or both strands within about 500 base pairs (e.g., within about 400, about 300, about 200, about 100, about 80, about 60, about 40, about 20, about 10 or about 5 base pairs) from the targeted genomic location.

[0150] 516169085.1 21Attorney Ref. No. 00B206.1710

[0151] GNE Ref. No. P60053-WO In certain embodiments, a Cas protein, e.g., a Cas9, that comprises functional RuvC and HNH nuclease domains can cleave both strands of a target nucleic acid sequence. In certain embodiments, the Cas protein, e.g., Cas9, comprises one functional endonuclease domain that allows the Cas protein to cleave only one strand (z.e., nick) of a target nucleic acid sequence. For example, but not by way of limitation, a Cas9 nickase can comprise (i) a non-functional RuvC domain (e.g., a mutant RuvC domain) and (ii) a functional HNH domain (e.g., a wild type HNH domain). In certain embodiments, a Cas9 nickase can comprise (i) a functional RuvC domain (e.g., wild type RuvC domain) and (ii) a non-functional HNH domain (e.g., a mutant HNH domain). In certain embodiments, a Cas9 nickase comprises a functional HNH-like and comprise a mutation at DIO, e.g., D10A. In certain embodiments, a Cas9 nickase comprises a functional RuvC domain and comprises a mutation at H840, e.g., H840A. In certain embodiments, a Cas9 nickase comprises a functional RuvC domain and comprises a mutation atN863, e.g., N863A.

[0152] In certain embodiments, the nucleotide sequence (e.g, polynucleotide) encoding a Cas protein and present in a nucleic acid construct of the present disclosure is codon optimized. For example, but not by way of limitation, the nucleotide sequence encoding a Cas protein can be codon optimized, e.g, where at least one non-common codon or less-common codon has been replaced by a common codon, for optimized expression in a particular cell type, e.g., a mammalian cell.

[0153] In certain embodiments, the Cas protein is a fusion protein that comprises one or more heterologous protein domains. In certain embodiments, a Cas fusion protein can comprise any additional protein domains, e.g., epitope tags, reporter sequences and protein domains having one or more of the following activities: methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity and nucleic acid binding activity.

[0154] In certain embodiments, the Cas protein comprises one or more nuclear localization sequences to promote accumulation of the Cas protein in a detectable amount in the nucleus of a cell. Nuclear localization sequences are known in the art. For example, but not by way of limitation, a Cas protein can comprise a nuclear localization sequence (NLS) (e.g., from SV40) at its N-terminus and / or C-terminus. In certain embodiments, a Cas protein can include two or more NLSs.

[0155] In certain embodiments, a nucleic acid construct of the present disclosure, e.g., a first nucleic acid construct and / or a second nucleic acid construct, can further comprise one or more

[0156] 516169085.1 22Attorney Ref. No. 00B206.1710

[0157] GNE Ref. No. P60053-WO additional polynucleotide sequences. For example, but not by way of limitation, a nucleic acid construct of the present disclosure can further include a nucleic acid sequence (e.g., a polynucleotide) that encodes a reporter. In certain embodiments, a nucleic acid construct of the present disclosure can further comprise one or more nucleic acid sequences (e.g., polynucleotides) encoding one or more fluorescent reporters. Non-limiting examples of fluorescent reporters for use in the present disclosure are shown in Figs. 13A-13B and 14. For example, but not by way of limitation, examples of fluorescent reporters include mCherry, mKate2, mScarlet2, mTurq2, green fluorescent protein (GFP), mNEON and blue fluorescent protein (BFP). In certain embodiments, the fluorescent reporter is BFP. In certain embodiments, the fluorescent reporter is GFP. In certain embodiments, the fluorescent reporter is mTurq2. In certain embodiments where the cells are microglia, the fluorescent reporter is mTurq2. In certain embodiments, the fluorescent reporter is mScarlet. In certain embodiments, the fluorescent reporter is mCherry. In certain embodiments, the fluorescent reporter can be coupled to a nuclear localization sequence (NLS), e.g, one or two copies of an NLS. In certain embodiments, the fluorescent reporter is GFP comprising one or more copies of an NLS (e.g., two NLS copies). In certain embodiments, the fluorescent reporter is BFP comprising one or more copies of an NLS (e.g., two NLS copies).

[0158] In certain embodiments, the fluorescent reporter can be under control of (e.g., operatively coupled to) one or more of the promoters disclosed herein, e.g., a promoter selected from U6, U3, U2, U5, Hl, 75J, EF-la, CMV, tRNA promoters, pGK, SV40, CAG, TRE, 7SK, VAI, UbiC (Ubiquitin C) promoter, CD68 promoter, 134 bp microgliaspecific HexB promoter, Synl, and PGK. For example, but not by way of limitation, the fluorescent reporter can be under the control of e.g., operatively coupled to) an EF-la promoter, e.g., a full-length EF-la promoter or a core EF-la promoter.

[0159] In certain embodiments, a first nucleic acid construct can comprise (i) a first nucleic acid sequence e.g., a first polynucleotide) encoding a gRNA and (ii) a second nucleic acid sequence e.g., a second polynucleotide) encoding a reporter, e.g., a fluorescent reporter. In certain embodiments, the fluorescent reporter of the first nucleic acid construct can be under control of e.g., operatively coupled to) one or more of the promoters disclosed herein. For example, but not by way of limitation, a first nucleic acid construct can comprise a fluorescent reporter under the control of e.g., operatively coupled to) an EF-la promoter, e.g., a full-length EF-la promoter or a core EF-la promoter, e.g., as shown in Fig. 14. In certain embodiments, the fluorescent reporter of the first nucleic acid construct can be under control of e.g.,

[0160] 516169085.1 23Attorney Ref. No. 00B206.1710

[0161] GNE Ref. No. P60053-WO operatively coupled to) one or more of the promoters disclosed herein. For example, but not by way of limitation, a first nucleic acid construct can comprise a fluorescent reporter under the control of (e.g., operatively coupled to) a full-length EF-la promoter as shown in Fig. 14. In certain embodiments, the fluorescent reporter is under control of (e.g., operatively coupled to) a promoter that is different than the promoter used for expression of the gRNA, as shown in Fig. 13A and Fig. 14. In certain embodiments, a first nucleic acid construct can comprise (i) a first promoter, (ii) a first nucleic acid sequence (e.g., a first polynucleotide) encoding a gRNA operatively coupled to the first promoter, (iii) a second promoter and (iv) a second nucleic acid sequence e.g., a second polynucleotide) encoding a reporter, e.g., a fluorescent reporter, operatively coupled to the second promoter, where the first promoter and second promoter are different. In certain embodiments, a first nucleic acid construct can comprise (i) a U6 promoter, (ii) a first nucleic acid sequence (e.g., a first polynucleotide) encoding a gRNA operatively coupled to the U6 promoter, (iii) an EF-la promoter and (iv) a second nucleic acid sequence (e.g., a second polynucleotide) encoding a reporter, e.g., a fluorescent reporter, operatively coupled to the EF-la promoter. Alternatively, the fluorescent reporter is under control of the same promoter used for expression of the gRNA. In certain embodiments, a first nucleic acid construct can comprise (i) a promoter, (ii) a first nucleic acid sequence (e.g., a first polynucleotide) encoding a gRNA and (iii) a second nucleic acid sequence (e.g., a second polynucleotide) encoding a reporter, e.g., a fluorescent reporter, where the first nucleic acid sequence and the second nucleic acid sequence are under control of (e.g., operatively coupled to) the promoter.

[0162] In certain embodiments, a second nucleic acid construct can comprise (i) a first nucleic acid sequence (e.g., a first polynucleotide) encoding a Cas protein and (ii) a second nucleic acid sequence (e.g., a second polynucleotide) encoding a reporter, e.g., a fluorescent reporter. In certain embodiments, the fluorescent reporter of the second nucleic acid construct can be under control of (e.g., operatively coupled to) one or more of the promoters disclosed herein. For example, but not by way of limitation, a second nucleic acid construct can comprise a fluorescent reporter under the control of (e.g., operatively coupled to) an EF-la promoter, e.g., a full-length EF-la promoter or a core EF-la promoter. In certain embodiments, the fluorescent reporter is under control of (e.g., operatively coupled to) the same promoter used for expression of the Cas protein, as shown in Fig. 13B. In certain embodiments, a second nucleic acid construct can comprise (i) a promoter, (ii) a first nucleic acid sequence (e.g., a first polynucleotide) encoding a Cas protein and (iii) a second nucleic acid sequence (e.g., a second

[0163] 516169085.1 24Attorney Ref. No. 00B206.1710

[0164] GNE Ref. No. P60053-WO polynucleotide) encoding a reporter, e.g., a fluorescent reporter, where the first nucleic acid sequence and the second nucleic acid sequence are under control of (e.g., operatively coupled to) the promoter. In certain embodiments, a second nucleic acid construct can comprise (i) an EF-la promoter, (ii) a first nucleic acid sequence (e.g., a first polynucleotide) encoding a Cas protein and (iii) a second nucleic acid sequence (e.g., a second polynucleotide) encoding a reporter, e.g., a fluorescent reporter, where the first nucleic acid sequence and the second nucleic acid sequence are under control of (e.g., operatively coupled to) the EF-la promoter. In certain embodiments, a second nucleic acid construct can comprise (i) a hPGK promoter, (ii) a first nucleic acid sequence (e.g., a first polynucleotide) encoding a Cas protein and (iii) a second nucleic acid sequence (e.g., a second polynucleotide) encoding a reporter, e.g., a fluorescent reporter, where the first nucleic acid sequence and the second nucleic acid sequence are under control of (e.g., operatively coupled to) the hPGK promoter. In certain embodiments, a second nucleic acid construct can comprise (i) a hSynl promoter, (ii) a first nucleic acid sequence (e.g., a first polynucleotide) encoding a Cas protein and (iii) a second nucleic acid sequence (e.g., a second polynucleotide) encoding a reporter, e.g., a fluorescent reporter, where the first nucleic acid sequence and the second nucleic acid sequence are under control of (e.g., operatively coupled to) the hSynl promoter. Alternatively, the fluorescent reporter is under control of a promoter that is different than the promoter used for expression of the Cas protein. In certain embodiments, a second nucleic acid construct can comprise (i) a first promoter, (ii) a first nucleic acid sequence (e.g., a first polynucleotide) encoding a Cas protein operatively coupled to the first promoter, (iii) a second promoter and (iv) a second nucleic acid sequence (e.g., a second polynucleotide) encoding a reporter, e.g., a fluorescent reporter, operatively coupled to the second promoter, where the first promoter and second promoter are different.

[0165] In certain embodiments, each of the first nucleic acid construct and the second nucleic acid construct includes a nucleic acid sequence (e.g., a polynucleotide) that encodes a reporter. For example, but not by way of limitation, the first nucleic acid construct includes a nucleic acid sequence (e.g., a polynucleotide) that encodes a first reporter, and the second nucleic acid construct includes a nucleic acid sequence (e.g., a polynucleotide) that encodes a second reporter. In certain embodiments, the first reporter and the second reporter are different, e.g., different fluorescent reporters.

[0166] In certain embodiments, a nucleic acid construct of the present disclosure, e.g., a first nucleic acid construct and / or a second nucleic acid construct, can further comprise a nucleic acid sequence (e.g., a polynucleotide) that encodes a cell surface protein, e.g., a heterologous

[0167] 516169085.1 25Attorney Ref. No. 00B206.1710

[0168] GNE Ref. No. P60053-WO cell surface protein. Non-limiting examples of cell surface proteins that can be expressed using a nucleic acid construct of the present disclosure include adhesion proteins, receptor proteins, transporter proteins and enzymes. In certain embodiments, the cell surface protein, e.g., a receptor protein, is encoded by a nucleic acid sequence that has a length of about 5 kb or less. In certain embodiments, the cell surface protein, e.g. , a receptor protein, is encoded by a nucleic acid sequence that has a length of about 2 kb to about 5 kb, e.g., about 3 kb to about 4 kb. As shown in Example 2, the cell surface protein is a cell surface protein that is not endogenously expressed in the plurality of cells that is being transduced with the nucleic acid construct that encodes the cell surface protein.

[0169] In certain embodiments, the cell surface protein expressed using a nucleic acid construct of the present disclosure is a receptor. In certain embodiments, the receptor is a mThyl allelic variant. For example, but not by way of limitation, the receptor is mThyl.1, as shown in Fig.

[0170] 14. In certain embodiments, the receptor is mThyl.2. As disclosed herein, the cell surface protein, e.g., receptor, can be used to enrich for cells expressing the cell surface protein, as shown in Example 2. In certain embodiments, expression of mThyl.1 using a nucleic acid construct of the present disclosure can be used to enrich for cells expressing mThyl.1.

[0171] As shown in Fig. 13A and Fig. 14, in certain embodiments, a nucleic acid construct comprising a nucleic acid sequence (e.g., a polynucleotide) encoding a gRNA can further include a nucleic acid sequence (e.g., a polynucleotide, e.g., a second polynucleotide) encoding a cell surface protein, e.g., a receptor. In certain embodiments, expression of the receptor can be used to enrich for cells expressing the gRNA. In certain embodiments, a nucleic acid construct of the present disclosure (e.g., a first nucleic acid construct) can comprise (i) a first nucleic acid sequence (e.g., a first polynucleotide) encoding a gRNA and (ii) a second nucleic acid sequence (e.g., a second polynucleotide) encoding a cell surface protein, e.g., a receptor. In certain embodiments, a nucleic acid construct of the present disclosure (e.g., a first nucleic acid construct) can comprise (i) a first nucleic acid sequence (e.g., a first polynucleotide) encoding a gRNA, (ii) a second nucleic acid sequence (e.g., a second polynucleotide) encoding a cell surface protein, e.g., a receptor, and (iii) a third nucleic acid sequence (e.g., a third polynucleotide) encoding a reporter, e.g., a fluorescent reporter. In certain embodiments, the first polynucleotide, the second polynucleotide and / or the third polynucleotide are coupled to a promoter capable of promoting expression of the gRNA, cell surface protein and / or reporter in a cell type disclosed herein, e.g., in neuronal cells. In certain embodiments, the promoter is selected from the group consisting of an EF-la, an Synl and an PGK promoter. In certain

[0172] 516169085.1 26Attorney Ref. No. 00B206.1710

[0173] GNE Ref. No. P60053-WO embodiments, the promoter is an EF-la promoter.

[0174] In certain embodiments, the cell surface protein is under control of a promoter that is different than the promoter used for expression of the gRNA, as shown in Fig. 14. In certain embodiments, a first nucleic acid construct can comprise (i) a first promoter, (ii) a first nucleic acid sequence (e.g., a first polynucleotide) encoding a gRNA operatively coupled to the first promoter, (iii) a second promoter and (iv) a second nucleic acid sequence (e.g., a second polynucleotide) encoding a cell surface protein operatively coupled to the second promoter, where the first promoter and second promoter are different. In certain embodiments, a first nucleic acid construct can comprise (i) a U6 promoter, (ii) a first nucleic acid sequence (e.g., a first polynucleotide) encoding a gRNA operatively coupled to the U6 promoter, (iii) an EF-la promoter and (iv) a second nucleic acid sequence e.g., a second polynucleotide) encoding a cell surface protein operatively coupled to the EF-la promoter. Alternatively, the cell surface protein is under control of the same promoter used for expression of the gRNA. In certain embodiments, a first nucleic acid construct can comprise (i) a promoter, (ii) a first nucleic acid sequence (e.g., a first polynucleotide) encoding a gRNA and (iii) a second nucleic acid sequence (e.g., a second polynucleotide) encoding a cell surface protein, where the first nucleic acid sequence and the second nucleic acid sequence are under control of (e.g., operatively coupled to) the promoter.

[0175] In certain embodiments, a nucleic acid construct comprising a nucleic acid sequence (e.g., a polynucleotide) encoding a Cas protein can further include a nucleic acid sequence (e.g., a polynucleotide) encoding a cell surface protein, e.g., a receptor. In certain embodiments, expression of the reporter can be used to enrich for cells expressing the Cas protein. In certain embodiments, a nucleic acid construct of the present disclosure (e.g., a second nucleic acid construct) can comprise (i) a first nucleic acid sequence (e.g., a first polynucleotide) encoding a Cas protein and (ii) a second nucleic acid sequence (e.g., a second polynucleotide) encoding a cell surface protein, e.g., a receptor. In certain embodiments, a nucleic acid construct of the present disclosure (e.g., a second nucleic acid construct) can comprise (i) a first nucleic acid sequence (e.g., a first polynucleotide) encoding a Cas protein, (ii) a second nucleic acid sequence (e.g., a second polynucleotide) encoding a cell surface protein, e.g., a receptor, and (iii) a third nucleic acid sequence (e.g., a third polynucleotide) encoding a reporter, e.g., a fluorescent reporter. In certain embodiments, the first polynucleotide, the second polynucleotide and / or the second polynucleotide are coupled to a promoter capable of promoting expression of the Cas protein, cell surface protein and / or

[0176] 516169085.1 27Attorney Ref. No. 00B206.1710

[0177] GNE Ref. No. P60053-WO reporter in a cell type disclosed herein, e.g., in neuronal cells. In certain embodiments, the promoter is selected from the group consisting of an EF-la, an Synl and an PGK promoter. In certain embodiments, the promoter is an EF-la promoter.

[0178] In certain embodiments, the cell surface protein is under control of a promoter that is different than the promoter used for expression of the Cas protein. In certain embodiments, a second nucleic acid construct can comprise (i) a first promoter, (ii) a first nucleic acid sequence (e.g., a first polynucleotide) encoding a Cas protein operatively coupled to the first promoter, (iii) a second promoter and (iv) a second nucleic acid sequence (e.g., a second polynucleotide) encoding a cell surface protein operatively coupled to the second promoter, where the first promoter and second promoter are two separate promoters. In certain embodiments, a second nucleic acid construct can comprise (i) a first EF-la promoter, (ii) a first nucleic acid sequence (e.g., a first polynucleotide) encoding a Cas protein coupled to the first EF-la promoter, (iii) a second EF-la promoter and (iv) a second nucleic acid sequence (e.g., a second polynucleotide) encoding a cell surface protein coupled to the second EF-la promoter. Alternatively, the cell surface protein is under control of the same promoter used for expression of the Cas protein. In certain embodiments, a second nucleic acid construct can comprise (i) a promoter, (ii) a first nucleic acid sequence (e.g., a first polynucleotide) encoding a Cas protein and (iii) a second nucleic acid sequence (e.g., a second polynucleotide) encoding a cell surface protein, where the first nucleic acid sequence and the second nucleic acid sequence are under control of the promoter. In certain embodiments, a second nucleic acid construct can comprise (i) an EF-la promoter, (ii) a first nucleic acid sequence (e.g., a first polynucleotide) encoding a Cas protein and (iii) a second nucleic acid sequence (e.g., a second polynucleotide) encoding a cell surface protein, where the first nucleic acid sequence and the second nucleic acid sequence are coupled to the EF-la promoter. In certain embodiments, a second nucleic acid construct can comprise (i) a hPGK promoter, (ii) a first nucleic acid sequence (e.g., a first polynucleotide) encoding a Cas protein and (iii) a second nucleic acid sequence (e.g., a second polynucleotide) encoding a cell surface protein, where the first nucleic acid sequence and the second nucleic acid sequence are coupled to the hPGK promoter. In certain embodiments, a second nucleic acid construct can comprise (i) a hSynl promoter, (ii) a first nucleic acid sequence (e.g. , a first polynucleotide) encoding a Cas protein and (iii) a second nucleic acid sequence (e.g, a second polynucleotide) encoding a cell surface protein, where the first nucleic acid sequence and the second nucleic acid sequence are coupled to the hSynl promoter.

[0179] In certain embodiments, the vector or delivery vehicle for a nucleic acid construct of

[0180] 516169085.1 28Attorney Ref. No. 00B206.1710

[0181] GNE Ref. No. P60053-WO the present disclosure is a viral vector (e.g., for generation of recombinant viruses). In certain embodiments, the virus is a DNA virus (e.g, dsDNA or ssDNA virus). In certain embodiments, the virus is an RNA virus (e.g., an ssRNA virus). Exemplary viral vectors / viruses comprise, e.g., retroviruses, lentiviruses, adenovirus, adeno-associated virus (AAV), vaccinia viruses, poxviruses, and herpes simplex viruses. In certain embodiments, the virus infects dividing and / or non-dividing cells. In certain embodiments, the virus can integrate into the host genome. In certain embodiments, the virus does not integrate into the host genome. In certain embodiments, the virus is replication-competent. In certain embodiments, the virus is replication-defective, e.g., having one or more coding regions for the genes necessary for additional rounds of virion replication and / or packaging replaced with other genes or deleted. In certain embodiments, the nucleic acid constructs of the present disclosure are delivered into a cell by a lentivirus, e.g., an integration-deficient lentivirus. In certain embodiments, the lentivirus vectors can be concentrated prior to delivery of the lentivirus vectors to the cells, e.g., concentration of the lentivirus vectors can be performed by ultracentrifugation or by use of a Lenti-X Concentrator (e.g, Lenti-X™ Concentrator (Takara Bio)). In certain embodiments, the lentivirus for expression of Cas9 can include one or more features that results in higher virus titers (e.g., 10-fold higher virus titers). In certain embodiments, the lentivirus for expression of the gRNA can have a CROPseq backbone.

[0182] In certain embodiments, a nucleic acid construct of the present disclosure is contained within a lentivirus. For example, but not by way of limitation, the present disclosure provides a lentivirus comprising a nucleic acid construct described herein.

[0183] In certain embodiments, the present disclosure provides a first lentivirus vector comprising a first nucleic acid construct disclosed herein. In certain embodiments, a first lentivirus vector of the present disclosure comprises (i) a first nucleic acid sequence (e.g., a first polynucleotide) encoding a gRNA and (ii) a second nucleic acid sequence (e.g., a second polynucleotide) encoding a cell surface protein, e.g., a receptor. In certain embodiments, a first lentivirus vector of the present disclosure comprises (i) a first nucleic acid sequence (e.g., a first polynucleotide) encoding a gRNA and (ii) a second nucleic acid sequence (e.g., a second polynucleotide) encoding a reporter, e.g., a fluorescent reporter. In certain embodiments, a first lentivirus vector of the present disclosure comprises (i) a first nucleic acid sequence (e.g., a first polynucleotide) encoding a gRNA, (ii) a second nucleic acid sequence (e.g., a second polynucleotide) encoding a cell surface protein, e.g., a receptor, and (iii) a third nucleic acid sequence (e.g., a third polynucleotide) encoding a reporter, e.g., a fluorescent reporter. In

[0184] 516169085.1 29Attorney Ref. No. 00B206.1710

[0185] GNE Ref. No. P60053-WO certain embodiments, the second nucleic acid sequence (e.g., a second polynucleotide) and the third nucleic acid sequence (e.g., a third polynucleotide) are under control of (e.g., operatively coupled to) the same promoter, as shown in Fig. 14. In certain embodiments, the first nucleic acid sequence (e.g, a first polynucleotide) encoding a gRNA is operatively coupled to a different promoter from the second nucleic acid sequence (e.g, a second polynucleotide) encoding a cell surface protein and the third nucleic acid sequence (e.g., a third polynucleotide) encoding a reporter. For example, but by way of limitation, a first lentivirus vector of the present disclosure comprises (i) a first promoter, (ii) a first nucleic acid sequence (e.g., a first polynucleotide) encoding a gRNA operatively coupled to the first promoter, (iii) a second promoter, (iv) a second nucleic acid sequence (e.g., a second polynucleotide) encoding a cell surface protein, e.g., a receptor, operatively coupled to the second promoter and (v) a third nucleic acid sequence (e.g., a third polynucleotide) encoding a reporter, e.g., a fluorescent reporter, operatively coupled to the second promoter. In certain embodiments, the first promoter is a U6, a T7 or a combination thereof. In certain embodiments, the second promoter is an EF- la promoter.

[0186] In certain embodiments, the present disclosure provides a second lentivirus vector comprising a second nucleic acid construct disclosed herein. In certain embodiments, a second lentivirus vector of the present disclosure comprises (i) a first nucleic acid sequence (e.g., a first polynucleotide) encoding a Cas protein and (ii) a second nucleic acid sequence (e.g., a second polynucleotide) encoding a cell surface protein, e.g., a receptor. In certain embodiments, a second lentivirus vector of the present disclosure comprises (i) a first nucleic acid sequence (e.g., a first polynucleotide) encoding a Cas protein and (ii) a second nucleic acid sequence (e.g., a second polynucleotide) encoding a reporter, e.g., a fluorescent reporter. In certain embodiments, the first nucleic acid sequence (e.g., a second polynucleotide) encoding the Cas protein and the second nucleic acid sequence (e.g., a third polynucleotide) encoding the reporter or cell surface protein are under control of (e.g., operatively coupled to) the same promoter. In certain embodiments, a second lentivirus vector of the present disclosure comprises (i) a first nucleic acid sequence (e.g., a first polynucleotide) encoding a Cas protein, (ii) a second nucleic acid sequence (e.g., a second polynucleotide) encoding a cell surface protein, e.g., a receptor, and (iii) a third nucleic acid sequence (e.g., a third polynucleotide) encoding a reporter, e.g., a fluorescent reporter. In certain embodiments, the first nucleic acid sequence (e.g., a second polynucleotide) encoding the Cas protein and the third nucleic acid sequence (e.g., a third polynucleotide) encoding the reporter are under control of (e.g.,

[0187] 516169085.1 30Attorney Ref. No. 00B206.1710

[0188] GNE Ref. No. P60053-WO operatively coupled to) the same promoter, as shown in Fig. 13B. For example, but by way of limitation, a second lentivirus vector of the present disclosure comprises (i) a promoter, (ii) a first nucleic acid sequence (e.g., a first polynucleotide) encoding a Cas protein operatively coupled to the promoter and (iii) a second nucleic acid sequence (e.g., a third polynucleotide) encoding a reporter, e.g., a fluorescent reporter, operatively coupled to the promoter. In certain embodiments, the second nucleic acid sequence (e.g., a second polynucleotide) encoding a cell surface protein, e.g., a receptor, is operatively coupled to a different promoter (e.g., a second promoter). For example, but not by way of limitation, the promoter coupled to the first nucleic acid sequence (e.g., a first polynucleotide) encoding a Cas protein is inducible and the promoter coupled to the second nucleic acid sequence (e.g., a second polynucleotide) encoding a cell surface protein, e.g., a receptor, is a constitutive promoter. In certain embodiments, the first nucleic acid sequence (e.g., a second polynucleotide), second nucleic acid sequence (e.g., a second polynucleotide) and the third nucleic acid sequence (e.g., a third polynucleotide) are under control of the same promoter. For example, but by way of limitation, a second lentivirus vector of the present disclosure comprises (i) a promoter, (ii) a first nucleic acid sequence (e.g., a first polynucleotide) encoding a Cas protein operatively coupled to the promoter, (iii) a second nucleic acid sequence (e.g., a second polynucleotide) encoding a cell surface protein, e.g., a receptor, operatively coupled to the promoter and (iv) a third nucleic acid sequence (e.g., a third polynucleotide) encoding a reporter, e.g., a fluorescent reporter, operatively coupled to the promoter. In certain embodiments, the promoter is an EF-la promoter. In certain embodiments, the promoter is a Synl promoter. In certain embodiments, the promoter is a PGK promoter. In certain embodiments, the fluorescent reporter is mScarlet or mCherry.

[0189] The present disclosure further provides compositions comprising a nucleic acid construct or a vector comprising a nucleic acid construct described herein. In certain embodiments, a composition of the present disclosure comprises one or more nucleic acid constructs and / or one or more vectors comprising a nucleic acid construct described herein. The compositions disclosed herein can be used for performing the methods, e.g., a pooled genomic screen, of the present disclosure.

[0190] In certain embodiments, a composition of the present disclosure comprises a first lentivirus vector disclosed herein. In certain embodiments, a composition of the present disclosure comprises a first lentivirus vector comprising (i) a first nucleic acid sequence (e.g., a first polynucleotide) encoding a gRNA and (ii) a second nucleic acid sequence (e.g., a second polynucleotide) encoding a cell surface protein, e.g., a receptor. In certain embodiments, a

[0191] 516169085.1 31Attorney Ref. No. 00B206.1710

[0192] GNE Ref. No. P60053-WO composition of the present disclosure comprises a first lentivirus vector comprising (i) a first nucleic acid sequence (e.g., a first polynucleotide) encoding a gRNA, (ii) a second nucleic acid sequence (e.g., a second polynucleotide) encoding a cell surface protein, e.g., a receptor, and (iii) a third nucleic acid sequence (e.g., a third polynucleotide) encoding a reporter, e.g., a fluorescent reporter. In certain embodiments, a composition of the present disclosure comprises a first lentivirus vector comprising (i) a nucleic acid sequence e.g., a first polynucleotide) encoding a gRNA, and (ii) a nucleic acid sequence (e.g., a third polynucleotide) encoding a reporter, e.g., a fluorescent reporter. In certain embodiments, a composition of the present disclosure comprises a first lentivirus vector comprising (i) a first promoter, (ii) a second promoter, (iii) a nucleic acid sequence (e.g., a polynucleotide) encoding a gRNA operatively coupled to the first promoter, and (iv) a nucleic acid sequence (e.g., a polynucleotide) encoding a reporter, e.g., a fluorescent reporter, operatively coupled to the second promoter. In certain embodiments, a composition of the present disclosure comprises a first lentivirus vector comprising (i) a first promoter, (ii) a first nucleic acid sequence (e.g., a first polynucleotide) encoding a gRNA operatively coupled to the first promoter, (iii) a second promoter and (iv) a second nucleic acid sequence (e.g., a second polynucleotide) encoding a cell surface protein, e.g., a receptor, operatively coupled to the second promoter. In certain embodiments, a composition of the present disclosure comprises a first lentivirus vector comprising (i) a first promoter, (ii) a first nucleic acid sequence (e.g., a first polynucleotide) encoding a gRNA operatively coupled to the first promoter, (iii) a second promoter, (iv) a second nucleic acid sequence (e.g., a second polynucleotide) encoding a cell surface protein, e.g., a receptor, operatively coupled to the second promoter and (v) a third nucleic acid sequence (e.g., a third polynucleotide) encoding a reporter, e.g., a fluorescent reporter, operatively coupled to the second promoter. In certain embodiments, the first promoter is a U6, a T7 or a combination thereof and the second promoter is an EF-la promoter.

[0193] In certain embodiments, a composition of the present disclosure comprises a plurality of first lentivirus vectors, where each first lentivirus vector present in the compositions encodes a gRNA targeting a different target gene. In certain embodiments, a composition of the present disclosure can include a plurality of lentivirus vectors that encode from about 10 to about 100,000 gRNAs, e.g., from about 100 to about 50,000 gRNAs, from about 1,000 to about 50,000 gRNAs or about 10,000 to about 50,000 gRNAs. For example, but not by way of limitation, a composition of the present disclosure comprises from about 10 to about 1,000 first lentivirus vectors, where each first lentivirus vector present in the compositions encodes a

[0194] 516169085.1 32Attorney Ref. No. 00B206.1710

[0195] GNE Ref. No. P60053-WO gRNA targeting a different target gene. In certain embodiments, a composition of the present disclosure includes from about 50 to about 1,000 first lentivirus vectors, where each first lentivirus vector present in the composition encodes a gRNA targeting a different target gene. In certain embodiments, a composition of the present disclosure includes from about 100 to about 1,000 first lentivirus vectors, where each first lentivirus vector present in the compositions encodes a gRNA targeting a different target gene. In certain embodiments, a composition of the present disclosure includes from about 500 to about 1,000 first lentivirus vectors, where each first lentivirus vector present in the compositions encodes a gRNA targeting a different target gene. In certain embodiments, about 1 to about 5 gRNAs are used per target gene. In certain embodiments, a composition of the present disclosure includes a plurality of first lentivirus vectors, e.g., from about 10 to about 100,000 first lentivirus vectors, where each first lentivirus vector present in the composition encodes 1 to about 5 gRNAs targeting a different target gene. For example, but not by way of limitation, each first lentivirus vector can include nucleic acid sequences that encode from about 1 to about 5 gRNAs, e.g., from about 2 to about 5 gRNAs, from about 2 to about 4 gRNAs, targeting a single gene. Alternatively or additionally, each first lentivirus vector can include nucleic acid sequences that encode from about 1 to about 5 gRNAs, e.g., from about 2 to about 5 gRNAs, from about 2 to about 4 gRNAs, where each gRNA targets a different gene.

[0196] In certain embodiments, a composition of the present disclosure comprises a second lentivirus vector disclosed herein. In certain embodiments, a composition of the present disclosure comprises a second lentivirus vector comprising (i) a first nucleic acid sequence (e.g., a first polynucleotide) encoding a Cas protein and (ii) a second nucleic acid sequence (e.g., a second polynucleotide) encoding a cell surface protein, e.g., a receptor. In certain embodiments, a composition of the present disclosure comprises a second lentivirus vector comprising (i) a first nucleic acid sequence (e.g., a first polynucleotide) encoding a Cas protein and (ii) a second nucleic acid sequence (e.g., a second polynucleotide) encoding a reporter, e.g., a fluorescent reporter. In certain embodiments, a composition of the present disclosure comprises a second lentivirus vector comprising (i) a first nucleic acid sequence (e.g., a first polynucleotide) encoding a Cas protein, (ii) a second nucleic acid sequence (e.g., a second polynucleotide) encoding a cell surface protein, e.g., a receptor, and (iii) a third nucleic acid sequence (e.g., a third polynucleotide) encoding a reporter, e.g., a fluorescent reporter. In certain embodiments, a composition of the present disclosure comprises a second lentivirus vector comprising (i) a promoter, (ii) a first nucleic acid sequence (e.g., a first polynucleotide)

[0197] 516169085.1 33Attorney Ref. No. 00B206.1710

[0198] GNE Ref. No. P60053-WO encoding a Cas protein operatively coupled to the promoter and (iii) a second nucleic acid sequence (e.g., a second polynucleotide) encoding a reporter, e.g., a fluorescent reporter, operatively coupled to the promoter. In certain embodiments, a composition of the present disclosure comprises a second lentivirus vector comprising (i) a promoter, (ii) a first nucleic acid sequence (e.g., a first polynucleotide) encoding a Cas protein operatively coupled to the promoter, (iii) a second nucleic acid sequence e.g., a second polynucleotide) encoding a cell surface protein, e.g., a receptor, operatively coupled to the promoter and (iv) a third nucleic acid sequence e.g., a third polynucleotide) encoding a reporter, e.g., a fluorescent reporter, operatively coupled to the promoter. In certain embodiments, a composition of the present disclosure comprises a second lentivirus vector comprising (i) a first promoter, (ii) a second promoter, (iii) a first nucleic acid sequence e.g., a first polynucleotide) encoding a Cas protein operatively coupled to the first promoter, (iv) a second nucleic acid sequence e.g., a second polynucleotide) encoding a cell surface protein, e.g., a receptor, operatively coupled to the second promoter and (v) a third nucleic acid sequence e.g., a third polynucleotide) encoding a reporter, e.g., a fluorescent reporter, operatively coupled to the first promoter. In certain embodiments, the Cas protein is a Cas9 protein. In certain embodiments, the promoter or first promoter is an EF-la promoter. In certain embodiments, the promoter or first promoter is a Synl promoter. In certain embodiments, the promoter or first promoter is a PGK promoter.

[0199] In certain embodiments, a composition of the present disclosure comprises a first lentivirus vector disclosed herein and a second lentivirus vector disclosed herein. For example, but not by way of limitation, a composition of the present disclosure comprises (a) a first lentivirus vector comprising (i) a first nucleic acid sequence e.g., a first polynucleotide) encoding a gRNA and (ii) a second nucleic acid sequence e.g., a second polynucleotide) encoding a cell surface protein, e.g., a receptor, and (b) a second lentivirus vector comprising (i) a first nucleic acid sequence e.g., a first polynucleotide) encoding a Cas protein. In certain embodiments, a composition of the present disclosure comprises (a) a first lentivirus vector comprising (i) a first nucleic acid sequence e.g., a first polynucleotide) encoding a gRNA, (ii) a second nucleic acid sequence e.g., a second polynucleotide) encoding a cell surface protein, e.g., a receptor, and (iii) a third nucleic acid sequence e.g., a third polynucleotide) encoding a reporter, e.g., a fluorescent reporter, and (b) a second lentivirus vector comprising (i) a first nucleic acid sequence e.g., a first polynucleotide) encoding a Cas protein and (ii) a second nucleic acid sequence e.g., a second polynucleotide) encoding a reporter, e.g., a fluorescent reporter. In certain embodiments, a composition of the present disclosure includes (a) a first

[0200] 516169085.1 34Attorney Ref. No. 00B206.1710

[0201] GNE Ref. No. P60053-WO lentivirus vector comprising (i) a first promoter, (ii) a first nucleic acid sequence (e.g., a first polynucleotide) encoding a gRNA operatively coupled to the first promoter, (iii) a second promoter and (iv) a second nucleic acid sequence (e.g., a second polynucleotide) encoding a cell surface protein, e.g., a receptor, operatively coupled to the second promoter, and (b) a second lentivirus vector comprising (i) a promoter, (ii) a first nucleic acid sequence (e.g., a first polynucleotide) encoding a Cas protein coupled to the promoter and (iii) a second nucleic acid sequence (e.g., a second polynucleotide) encoding a reporter, e.g., a fluorescent reporter, operatively coupled to the promoter. In certain embodiments, a composition of the present disclosure includes (a) a first lentivirus vector comprising (i) a first promoter, (ii) a first nucleic acid sequence (e.g., a first polynucleotide) encoding a gRNA operatively coupled to the first promoter, (iii) a second promoter, (iv) a second nucleic acid sequence (e.g., a second polynucleotide) encoding a cell surface protein, e.g., a receptor, operatively coupled to the second promoter and (v) a third nucleic acid sequence (e.g., a third polynucleotide) encoding a reporter, e.g., a fluorescent reporter, operatively coupled to the second promoter, and (b) a second lentivirus vector comprising (i) a promoter, (ii) a first nucleic acid sequence (e.g., a first polynucleotide) encoding a Cas protein coupled to the promoter and (iii) a second nucleic acid sequence (e.g., a second polynucleotide) encoding a reporter, e.g., a fluorescent reporter, operatively coupled to the promoter. In certain embodiments, a composition of the present disclosure includes (a) a first lentivirus vector comprising (i) a first promoter, (ii) a second promoter, (iii) a nucleic acid sequence (e.g., a polynucleotide) encoding a gRNA coupled to the first promoter, and (iv) a nucleic acid sequence (e.g., a polynucleotide) encoding a reporter, e.g., a fluorescent reporter, operatively coupled to the second promoter, and (b) a second lentivirus vector comprising (i) a first promoter, (ii) a second promoter, (iii) a first nucleic acid sequence (e.g., a first polynucleotide) encoding a Cas protein operatively coupled to the first promoter, (iv) a second nucleic acid sequence (e.g., a second polynucleotide) encoding a cell surface protein, e.g., a receptor, operatively coupled to the second promoter and (v) a third nucleic acid sequence (e.g., a third polynucleotide) encoding a reporter, e.g., a fluorescent reporter, operatively coupled to the first promoter.

[0202] III. CELLS

[0203] The present disclosure provides methods for CRISPR screening in different cell types. As discussed herein, current CRISPR screen techniques can be time-consuming, laborious, and incompatible with many cell types (e.g., neurons). The present disclosure provides improved

[0204] 516169085.1 35Attorney Ref. No. 00B206.1710

[0205] GNE Ref. No. P60053-WO high throughput methods for CRISPR screening.

[0206] In certain embodiments, cells that are to be analyzed by the methods of the present disclosure comprise one or more of the nucleic acid constructs disclosed herein or a vector comprising a nucleic acid construct disclosed herein. For example, but not by way of limitation, cells (e.g., a plurality of cells) that are to be analyzed by the methods of the present disclosure can comprise at least one cell comprising a first nucleic acid construct and a second nucleic acid construct.

[0207] In certain embodiments, a method of the present disclosure can comprise providing a cell or a plurality of cells that comprise a first nucleic acid construct and a second nucleic acid construct described herein. In certain embodiments, a method of the present disclosure can comprise introducing a first nucleic acid construct and a second nucleic acid construct described herein into a cell or a plurality of cells. For example, but not by way of limitation, a method of the present disclosure can comprise contacting a cell or a plurality of cells with a first nucleic acid construct and a second nucleic acid construct described herein (or one or more compositions thereof). In certain embodiments, the nucleic acid construct can be integrated into the genome of the cell or the genomes of the plurality of cells. In certain embodiments, at least two nucleic acid constructs are integrated in the genomes of the plurality of cells. In certain embodiments, at least one first nucleic acid construct comprising a nucleic acid sequence encoding a gRNA, and at least one second nucleic acid construct comprising a nucleic acid sequence encoding a Cas protein.

[0208] In certain embodiments, the cells (e.g., the plurality of cells) can be obtained from a subject. In certain embodiments, the subject can be a human, non-human primate, e.g, an ape or a monkey, a farm animal, a mouse, a rat, a hamster, a guinea pig, a rabbit, a dog, cat, a sheep, a pig, a goat, a cow or a horse. In certain embodiments, the subject is a human.

[0209] In certain embodiments, the cells (e.g., the plurality of cells) can be obtained from a tissue, e.g., a tissue sample, or are present in a tissue sample. In certain embodiments, the cells (e.g, the plurality of cells) can be obtained from a tissue, e.g, a tissue sample. In certain embodiments, the cells (e.g., the plurality of cells) are present in a tissue, e.g., in a tissue sample.

[0210] In certain embodiments, the cells (e.g., the plurality of cells) can comprise terminally differentiated cells.

[0211] In certain embodiments, the cells (e.g., the plurality of cells) can comprise cancer cells. In certain embodiments, the cells (e.g., the plurality of cells) can be obtained from and / or

[0212] 516169085.1 36Attorney Ref. No. 00B206.1710

[0213] GNE Ref. No. P60053-WO present in a malignancy of a tissue or a tumor. Non-limiting examples of such malignancies comprise carcinomas, adenocarcinomas, sarcomas and fibroadenomas. In certain embodiments, the cells are obtained from a cancer such as bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, head and neck cancer, kidney cancer, leukemia, lung cancer, lymphoma, melanoma, pancreatic cancer, parathyroid cancer, prostate cancer, stomach cancer, testicular cancer, thyroid cancer and uterine cancer.

[0214] In certain embodiments, the cells (e.g., the plurality of cells) can comprise cells from the nervous system, e.g., microglia, neurons, astrocytes, oligodendrocyte and ependymal cells.

[0215] In certain embodiments, the cells are derived from organoids or xenografts or stem cells, e.g., pluripotent stem cells (iPSCs) or embryonic stem cells. In certain embodiments, the cells for use in the present disclosure can be derived from stem cells, e.g., stem cells that have undergone natural differentiation or artificially induced reprogramming or transdifferentiation.

[0216] In certain embodiments, the cells (e.g., the plurality of cells) can be obtained from in vitro cell cultures.

[0217] In certain embodiments, the cells (e.g., the plurality of cells) can be immune cells. Nonlimiting examples of immune cells comprise neutrophils, eosinophils, basophils, mast cells, monocytes, macrophages, dendritic cells, natural killer cells (NK cells) and lymphocytes, e.g., B cells and T cells (e.g., cytotoxic T cells, natural killer T cells, regulatory T cells and helper T cells). In certain embodiments, the cells can be modified immune cells that have been genetically engineered to express a chimeric antigen receptor (CAR), e.g., CAR T cells and CAR NK cells.

[0218] In certain embodiments, the cells can comprise diseased cells and healthy cells. For example, but not by way of limitation, the cells can comprise cells obtained from a tumor or cancer and comprise non-cancerous cells (e.g., healthy cells that were located adjacent to the tumor or cancer or healthy cells obtained from a subject that does not have cancer).

[0219] In certain embodiments, the cells (e.g., the plurality of cells) can be enriched for cells of interest to produce an enriched cell sample, which can be subjected to the methods of the present disclosure. Any technique known in the art can be used to enrich for the cells of interest.

[0220] In certain embodiments, the cells (e.g., the plurality of cells) have been genetically modified to express and / or secrete an agent, e.g., a therapeutic agent. For example, but not by way of limitation, the cells to be used in the methods of the present disclosure express an antibody or an antibody fragment.

[0221] 516169085.1Attorney Ref. No. 00B206.1710

[0222] GNE Ref. No. P60053-WO In certain embodiments, the cells (e.g., the plurality of cells) for use in the present disclosure can be treated with an agent. For example, but not by way of limitation, the agent can be a therapeutic agent. In certain embodiments, methods of the present disclosure can be used in drug screening, e.g. , to determine the genomic and / or transcriptional changes associated with a test therapeutic agent, e.g. , a newly identified therapeutic agent. In certain embodiments, methods of the present disclosure can be used in determining the genomic and / or transcriptional changes associated with resistance to a therapeutic agent. Non-limiting examples of such therapeutics comprise polypeptide therapeutics, e.g., antibody -based therapeutics, oligonucleotides, and small molecule therapeutics. In certain embodiments, the therapeutic can be cell cycle regulators, kinase regulators (e.g., kinase inhibitors or activators), receptor regulators (e.g., receptor inhibitors or activators), chemotherapeutics and / or antibodies (e.g., agonist or antagonist antibodies).

[0223] IV. METHODS

[0224] The present disclosure provides high throughput methods for genomic screening of cells. The methods of the present disclosure can be used for a variety of purposes, e.g., for determining biological relationships between genetic perturbations, for identifying affected genes and their relationships to one another, and for determining the pathways and cellular functions affected by single or groups of related or disparate perturbations. The methods of present disclosure can also be used to identify regulators and biomarkers of transcriptionally defined cell states, such as healthy and diseased cells. Alternatively and / or additionally, methods of the present disclosure can be used to compare biological relationships between perturbations and their regulated genes across different cell populations. As disclosed herein, methods of the present disclosure can be used for screening greater than 130 million cells in a single experiment.

[0225] In certain embodiments, non-limiting examples of a method of the present disclosure are provided in Figs. 3A, 5A, 5B, 8H, 9A, 10A and 11 A.

[0226] In certain embodiments, a method of the present disclosure can include providing a plurality of cells. For example, but not by way of limitation, a method of the present disclosure can comprise providing a plurality of cells that include at least about 5, at least about 10, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000, at least about 5,000, at least about 10,000, at least about 100,000, at least about 1,000,000, at least

[0227] 516169085.1 38Attorney Ref. No. 00B206.1710

[0228] GNE Ref. No. P60053-WO about 10,000,000, at least about 100,000,000 or at least about 1,000,000,000 cells. In certain embodiments, a plurality of cells can include at least about 100,000,000 cells. In certain embodiments, a plurality of cells can include from about 10 to about 1,000,000,000 cells, e.g., about 1,000 to about 1,000,000,000 or about 5,000 to about 1,000,000,000.

[0229] In certain embodiments, the plurality of cells can comprise a variety of cells, e.g., cells of different types, cells of different lineages and / or cells with different genomic mutations. In certain embodiments, the plurality of cells can comprise a variety of cell types pooled together. In certain embodiments, a plurality of cells can comprise at least two or more cell types, at least three or more cell types, at least four or more cell types, at least five or more cell types, at least six or more cell types, at least seven or more cell types, at least eight or more cell types, at least nine or more cell types or at least ten or more cell types. Alternatively, in certain embodiments, the plurality of cells comprises a single cell type, e.g., a plurality of isolated cells. In certain embodiments, the plurality of cells comprising a cell type expressing specific marker.

[0230] In certain embodiments, the plurality of cells is obtained from a sample. For example, but not by way of limitation, the plurality of cells can be isolated from a tissue sample, e.g., a fresh sample, a frozen sample and / or a preserved sample. Non-limiting examples of cells and samples for use in a method of the present disclosure are provided in Section III. In certain embodiments, the plurality of cells can comprise neurons. In certain embodiments, the plurality of cells comprises microglia. In certain embodiments, the plurality of cells comprises cancer cells.

[0231] In certain embodiments, a method of the present disclosure can further include contacting the plurality of cells with one or more nucleic acid constructs disclosed herein or one or more vectors comprising one or more nucleic acid constructs disclosed herein. Nonlimiting examples of nucleic acid constructs for use in a method of the present disclosure are provided in Section II. In certain embodiments, a method of the present disclosure can further include contacting the plurality of cells with a first nucleic acid construct disclosed herein. In certain embodiments, a method of the present disclosure can further include contacting the plurality of cells with a second nucleic acid construct disclosed herein. In certain embodiments, a method of the present disclosure can include contacting the plurality of cells with a first nucleic acid construct disclosed herein and a second nucleic acid construct disclosed herein. In certain embodiments, the first nucleic acid and the second nucleic acid are contacted with the plurality of cells simultaneously, e.g., by being present in the same composition.

[0232] 516169085.1 39Attorney Ref. No. 00B206.1710

[0233] GNE Ref. No. P60053-WO In certain embodiments, the plurality of cells is contacted with the one or more nucleic acid constructs at a stage where the cells, e.g., neurons, are undergoing differentiation. In certain embodiments, the plurality of cells is contacted with the one or more nucleic acid constructs at a stage where the cells, e.g., neurons, have completed differentiation. In certain embodiments, the plurality of cells is contacted with the one or more nucleic acid constructs at about 1 day to about 14 days after initiation of the cell culture (e.g., after initial seeding of the cell culture). In certain embodiments, the plurality of cells is contacted with the one or more nucleic acid constructs at about 4 days to about 14 days, e.g., 7 days, after initiation of the culture, e.g., after thawing and seeding of the plurality of cells, as shown in Fig. 11 A. In certain embodiments, the plurality of cells is contacted with the one or more nucleic acid constructs at about 7 days to about 14 days after initiation of the culture, e.g., after the cells have completed differentiation, as shown in Fig. 11 A.

[0234] In certain embodiments, a method of the present disclosure includes contacting the plurality of cells, e.g., neurons, with (a) a first lentivirus vector comprising (i) a first nucleic acid sequence (e.g., a first polynucleotide) encoding a gRNA and (ii) a second nucleic acid sequence (e.g., a second polynucleotide) encoding a cell surface protein, e.g., a receptor, and (b) a second lentivirus vector comprising (i) a first nucleic acid sequence (e.g., a first polynucleotide) encoding a Cas protein. In certain embodiments, a method of the present disclosure includes contacting the plurality of cells, e.g., neurons, with (a) a first lentivirus vector comprising (i) a first nucleic acid sequence (e.g., a first polynucleotide) encoding a gRNA, (ii) a second nucleic acid sequence (e.g., a second polynucleotide) encoding a cell surface protein, e.g., a receptor, and (iii) a third nucleic acid sequence (e.g., a third polynucleotide) encoding a reporter, e.g., a fluorescent reporter, and (b) a second lentivirus vector comprising (i) a first nucleic acid sequence (e.g., a first polynucleotide) encoding a Cas protein and (ii) a second nucleic acid sequence (e.g., a second polynucleotide) encoding a reporter, e.g., a fluorescent reporter, e.g., as shown in Fig. 11 A. In certain embodiments, a method of the present disclosure includes contacting the plurality of cells, e.g., neurons, with (a) a first lentivirus vector comprising (i) a first promoter, (ii) a second promoter, (iii) a first nucleic acid sequence (e.g., a first polynucleotide) encoding a gRNA operatively coupled to the first promoter and (iv) a second nucleic acid sequence (e.g., a second polynucleotide) encoding a cell surface protein, e.g., a receptor, operatively coupled to the second promoter, and (b) a second lentivirus vector comprising (i) a promoter, (ii) a first nucleic acid sequence (e.g., a first polynucleotide) encoding a Cas protein coupled to the promoter and (iii) a second

[0235] 516169085.1 40Attorney Ref. No. 00B206.1710

[0236] GNE Ref. No. P60053-WO nucleic acid sequence (e.g., a second polynucleotide) encoding a reporter, e.g., a fluorescent reporter, coupled to the promoter. In certain embodiments, a method of the present disclosure includes contacting the plurality of cells, e.g, neurons, with (a) a first lentivirus vector comprising (i) a first promoter, (ii) a second promoter, (iii) a first nucleic acid sequence (e.g., a first polynucleotide) encoding a gRNA operatively coupled to the first promoter, (iv) a second nucleic acid sequence (e.g., a second polynucleotide) encoding a cell surface protein, e.g., a receptor, operatively coupled to the second promoter and (v) a third nucleic acid sequence (e.g., a third polynucleotide) encoding a reporter, e.g., a fluorescent reporter, operatively coupled to the second reporter and (b) a second lentivirus vector comprising (i) a promoter, (ii) a first nucleic acid sequence (e.g., a first polynucleotide) encoding a Cas protein coupled to the promoter and (iii) a second nucleic acid sequence (e.g., a second polynucleotide) encoding a reporter, e.g., a fluorescent reporter, coupled to the promoter. In certain embodiments, the cell surface protein is a receptor. In certain embodiments, the cell surface protein is mThy 1.1.

[0237] In certain embodiments, the first lentivirus vector, e.g., which encodes the gRNA, can be contacted with the plurality of cells at a multiplicity of infection (MOI) of about 0.1 to about 1, e.g., at an MOI of about 0.1 to about 0.3. In certain embodiments, the first lentivirus vector, e.g., which encodes the gRNA, can be contacted with the plurality of cells at a MOI of about 1 or less. In certain embodiments, the first lentivirus vector, e.g., which encodes the gRNA, can be contacted with the plurality of cells at a MOI of about 0.3 or less. As shown in Fig. 15, a MOI of 0.3 or less for the first lentivirus vector, e.g., which encodes the gRNA, results in efficient magnetic enrichment compared to other transduction rates. In certain embodiments, the second lentivirus vector, e.g., which encodes the Cas protein, can be contacted with the plurality of cells at a MOI of about 5 to about 15, e.g., at an MOI of about 8 to about 10.

[0238] In certain embodiments, the plurality of cells is (i) contacted with the first lentivirus vector, e.g., which encodes the gRNA, at an MOI of about 0.1 to about 0.3 and (ii) contacted with the second lentivirus vector, e.g., which encodes the Cas protein, at an MOI of about 8 to about 10.

[0239] In certain embodiments, the plurality of cells is contacted with the first lentivirus vector and the second lentivirus vector at a MOI ratio of about 0.1 : 5 to about 1 : 15 of the first lentivirus vector to the second lentivirus vector. In certain embodiments, the plurality of cells is contacted with the first lentivirus vector and the second lentivirus vector at a MOI ratio of about 0.1:15, about 0.3:15, about 1:15, about 0.1:5, about 0.3:5 or about 1:5 of the first lentivirus vector to the second lentivirus vector.

[0240] 516169085.1 41Attorney Ref. No. 00B206.1710

[0241] GNE Ref. No. P60053-WO In certain embodiments, the method of the present disclosure can further include culturing the plurality of transduced cells, e.g., neurons, for a period of time and under conditions that are sufficient to allow expression of the gRNA, Cas protein and cell surface protein. In certain embodiments, the plurality of transduced cells is cultured for about 2 to about 30 days, e.g., about 2 to about 15 days, as shown in Fig. 11 A. In certain embodiments, the plurality of transduced cells is cultured for about 10 to about 20 days.

[0242] In certain embodiments, a method of the present disclosure can further include enriching for cells that express the gRNA and / or Cas protein from the plurality of transduced cells. In certain embodiments, enriching for cells includes the use of microparticles that are coupled to an agent, e.g., an antibody, that specifically binds to the exogenously expressed cell surface protein. For example, and not by way of limitation, a method of the present disclosure can further include contacting the plurality of transduced cells with magnetic microparticles, e.g., as shown in Fig. 11 A. In certain embodiments, a method of the present disclosure can further include introducing magnetic microparticles into the plurality of cells after lentiviral transduction to enrich for cells expressing an exogenous cell surface protein, e.g., mThyl.l, as shown in Fig. 11 A. In certain embodiments, a method of the present disclosure can further include contacting the plurality of cells after lentiviral transduction with magnetic microparticles to enrich for cells expressing a gRNA by binding the exogenous cell surface protein, e.g., mThyl.l, expressed by one or more lentiviruses disclosed herein. For example, and not by way of limitation, a method of the present disclosure can further include contacting the plurality of cells after lentiviral transduction with magnetic microparticles to enrich for cells expressing an exogenous cell surface protein by the first lentivirus encoding the gRNA. In certain embodiments, the magnetic microparticles are capable of binding to the exogenous cell surface protein directly or indirectly. In certain embodiments, the magnetic microparticles can be any shape, e.g., the magnetic microparticles can have a spherical, non-spherical, oval, oblong, amorphous, circular or cylindrical shape. In certain embodiments, the method includes introducing magnetic microparticles into the plurality of transduced cells and incubating for about 1 minute to about 10 minutes.

[0243] In certain embodiments, a method of the present disclosure further includes washing the magnetic microparticles. In certain embodiments, a method of the present disclosure further includes washing the magnetic microparticles one or more times following introduction of the magnetic microparticles into the plurality of transduced cells. In certain embodiments, the magnetic microparticles are washed at least 2 or more times, at least 3 or more times, at

[0244] 516169085.1 42Attorney Ref. No. 00B206.1710

[0245] GNE Ref. No. P60053-WO least 4 or more times, at least 5 or more times, at least 6 or more times, at least 7 or more times. In certain embodiments, the magnetic microparticles are washed at least 2 or more times.

[0246] In certain embodiments, a method of the present disclosure can include (a) providing a plurality of cells, e.g., neurons, (b) contacting the plurality of cells with a first nucleic acid construct (or a lentivirus vector comprising the first nucleic acid construct) and a second nucleic acid construct (or a lentivirus vector comprising the second nucleic acid construct), where at least one of the nucleic acid constructs comprise a polynucleotide encoding a heterologous surface protein and (c) enriching for a subset of cells that express the gRNA and / or the Cas protein from the plurality of cells using magnetic enrichment, where the magnetic enrichment comprises binding magnetic beads to the heterologous surface protein.

[0247] In certain embodiments, a method of the present disclosure can include (a) providing a plurality of cells, e.g., neurons, (b) contacting the plurality of cells with (i) a first nucleic acid construct (or a lentivirus vector comprising the first nucleic acid construct), where the first nucleic acid construct comprises (1) a first nucleic acid sequence (e.g., a first polynucleotide) encoding a gRNA and (2) a second nucleic acid sequence (e.g., a second polynucleotide) encoding a heterologous cell surface protein, e.g., a receptor, and (ii) a second nucleic acid construct (or a lentivirus vector comprising the second nucleic acid construct), where the second lentivirus vector of the present disclosure comprises (1) a first nucleic acid sequence (e.g., a first polynucleotide) encoding a Cas protein and (2) a second nucleic acid sequence (e.g., a second polynucleotide) encoding a reporter, e.g., a fluorescent reporter, and (c) enriching for a subset of cells that express the gRNA and / or the Cas protein from the plurality of cells using magnetic enrichment, where the magnetic enrichment comprises binding magnetic beads to the heterologous cell surface protein.

[0248] In certain embodiments, a method of the present disclosure can include (a) providing a plurality of cells, e.g., neurons, (b) contacting the plurality of cells with (i) a first nucleic acid construct (or a lentivirus vector comprising the first nucleic acid construct), where the first nucleic acid construct comprises (1) a first nucleic acid sequence (e.g., a first polynucleotide) encoding a gRNA, (2) a second nucleic acid sequence (e.g., a second polynucleotide) encoding a heterologous cell surface protein, e.g., a receptor, and (3) a third nucleic acid sequence (e.g., a third polynucleotide) encoding a reporter, e.g. , a fluorescent reporter, and (ii) a second nucleic acid construct (or a lentivirus vector comprising the second nucleic acid construct), where the second lentivirus vector of the present disclosure comprises (1) a first nucleic acid sequence (e.g, a first polynucleotide) encoding a Cas protein and (2) a second nucleic acid sequence

[0249] 516169085.1 43Attorney Ref. No. 00B206.1710

[0250] GNE Ref. No. P60053-WO (e.g., a second polynucleotide) encoding a reporter, e.g., a fluorescent reporter, and (c) enriching for a subset of cells that express the gRNA and / or the Cas protein from the plurality of cells using magnetic enrichment, where the magnetic enrichment comprises binding magnetic beads to the heterologous cell surface protein.

[0251] In certain embodiments, a method of the present disclosure can include contacting multiple plurality of cells with a different composition described herein for each plurality of cells and pooling the multiple plurality of cells together prior to magnetic enrichment. For example, but not by way of limitation, a method of the present disclosure can include (a) providing a first plurality of cells and a second plurality of cells, e.g., neurons; (b) contacting the first plurality of cells with (i) a first nucleic acid construct (or a lentiviral vector comprising the first nucleic acid construct), wherein the first nucleic acid construct comprises (1) a first nucleic acid sequence (e.g., a first polynucleotide) encoding a first gRNA, (2) a second nucleic acid sequence (e.g., a second polynucleotide) encoding a heterologous cell surface protein, e.g., a receptor, and (3) a third nucleic acid sequence (e.g., a third polynucleotide) encoding a reporter, e.g., a fluorescent reporter, and (ii) a second nucleic acid construct (or a lentiviral vector comprising the second nucleic acid construct), wherein the second nucleic acid construct comprises (1) a first nucleic acid sequence (e.g., a first polynucleotide) encoding a Cas protein and (2) a second nucleic acid sequence (e.g., a second polynucleotide) encoding a reporter, e.g., a fluorescent reporter; (c) contacting the second plurality of cells with (i) a first nucleic acid construct (or a lentiviral vector comprising the first nucleic acid construct), wherein the first nucleic acid construct comprises (1) a first nucleic acid sequence (e.g., a first polynucleotide) encoding a second gRNA, (2) a second nucleic acid sequence (e.g., a second polynucleotide) encoding a heterologous cell surface protein, e.g., a receptor, and (3) a third nucleic acid sequence (e.g., a third polynucleotide) encoding a reporter, e.g., a fluorescent reporter, and (ii) a second nucleic acid construct (or a lentiviral vector comprising the second nucleic acid construct), wherein the second nucleic acid construct comprises (1) a first nucleic acid sequence (e.g., a first polynucleotide) encoding a Cas protein and (2) a second nucleic acid sequence (e.g., a second polynucleotide) encoding a reporter, e.g., a fluorescent reporter; (d) pooling the first plurality of cells and the second plurality of cells together to generate a pooled sample, and (e) enriching for a subset of cells that express the gRNA and / or the Cas protein from the pooled sample using magnetic enrichment, wherein the magnetic enrichment comprises binding magnetic beads to the heterologous cell surface protein. In certain embodiments, the heterologous cell surface protein expressed in the first plurality of cells and

[0252] 516169085.1 44Attorney Ref. No. 00B206.1710

[0253] GNE Ref. No. P60053-WO the second plurality of cells is the same. In certain embodiments, the first nucleic construct and the second nucleic construct that is contacted with the first plurality of cells is present in a single composition that is contacted with the first plurality of cells. Alternatively, the first nucleic construct and the second nucleic construct are present in separate compositions and contacted with the first plurality of cells at different time points (e.g., consecutively). In certain embodiments, the first nucleic construct and the second nucleic construct that is contacted with the second plurality of cells is present in a single composition that is contacted with the second plurality of cells. Alternatively, the first nucleic construct and the second nucleic construct are present in separate compositions and contacted with the second plurality of cells at different time points (e.g., consecutively).

[0254] In certain embodiments, a method of the present disclosure further comprises analyzing gene expression of the enriched cells, e.g., enriched cells that express the gRNA and / or Cas protein. For example, but not by way of limitation, a method of the present disclosure can further include performing single cell sequencing of the enriched cells. In certain embodiments, a method of the present disclosure can further include analyzing single-cell mRNA expression data from the enriched cells. For example, but not by way of limitation, a method of the present disclosure can include (a) providing a plurality of cells, e.g., neurons, (b) contacting the plurality of cells with a first nucleic acid construct (or a lentivirus vector comprising the first nucleic acid construct) and a second nucleic acid construct (or a lentivirus vector comprising the second nucleic acid construct), where at least one of the nucleic acid constructs comprise a polynucleotide encoding a heterologous surface protein, (c) enriching for a subset of cells that express the gRNA and / or the Cas protein from the plurality of cells using magnetic enrichment, where the magnetic enrichment comprises binding magnetic beads to the heterologous surface protein and (d) obtaining expression data, e.g., single cell expression data (e.g., single cell mRNA expression data), from the enriched cells. In certain embodiments, a method of the present disclosure can include (a) providing a plurality of cells, e.g., neurons, (b) contacting the plurality of cells with (i) a first nucleic acid construct (or a lentivirus vector comprising the first nucleic acid construct), where the first nucleic acid construct comprises (1) a first nucleic acid sequence (e.g., a first polynucleotide) encoding a gRNA and (2) a second nucleic acid sequence (e.g., a second polynucleotide) encoding a heterologous cell surface protein, e.g., a receptor, and (ii) a second nucleic acid construct (or a lentivirus vector comprising the second nucleic acid construct), where the second lentivirus vector of the present disclosure comprises (1) a first nucleic acid sequence (e.g., a first

[0255] 516169085.1 45Attorney Ref. No. 00B206.1710

[0256] GNE Ref. No. P60053-WO polynucleotide) encoding a Cas protein and (2) a second nucleic acid sequence (e.g., a second polynucleotide) encoding a reporter, e.g., a fluorescent reporter, (c) enriching for a subset of cells that express the gRNA and / or the Cas protein from the plurality of cells using magnetic enrichment, where the magnetic enrichment comprises binding magnetic beads to the heterologous cell surface protein and (d) obtaining expression data, e.g, single cell expression data (e.g., single cell mRNA expression data), from the enriched cells. In certain embodiments, a method of the present disclosure can include (a) providing a plurality of cells, e.g., neurons, (b) contacting the plurality of cells with (i) a first nucleic acid construct (or a lentivirus vector comprising the first nucleic acid construct), where the first nucleic acid construct comprises (1) a first nucleic acid sequence (e.g., a first polynucleotide) encoding a gRNA, (2) a second nucleic acid sequence (e.g., a second polynucleotide) encoding a heterologous cell surface protein, e.g., a receptor, and (3) a third nucleic acid sequence (e.g., a third polynucleotide) encoding a reporter, e.g., a fluorescent reporter, and (ii) a second nucleic acid construct (or a lentivirus vector comprising the second nucleic acid construct), where the second lentivirus vector of the present disclosure comprises (1) a first nucleic acid sequence (e.g., a first polynucleotide) encoding a Cas protein and (2) a second nucleic acid sequence (e.g., a second polynucleotide) encoding a reporter, e.g., a fluorescent reporter, (c) enriching for a subset of cells that express the gRNA and / or the Cas protein from the plurality of cells using magnetic enrichment, where the magnetic enrichment comprises binding magnetic beads to the heterologous cell surface protein and (d) obtaining expression data, e.g., single cell expression data (e.g., single cell mRNA expression data), from the enriched cells. In certain embodiments, the expression data is associated with one genetic perturbation caused by contacting the plurality of cells with the dual lentivirus viruses of step (b). In certain embodiments, obtaining expression data includes the analysis of mRNA expression in single cells and the sequencing of gRNAs present in the single cells, which allows for analyzing the effect of specific gRNAs on mRNA expression. For example, but not by way of limitation, a method of the present disclosure allows for analyzing the effect of about 10 to about 1,000 gRNAs on mRNA expression.

[0257] In certain embodiments, single cell expression data is obtained by partitioning cells of the enriched cell sample into compartments, e.g., emulsion droplets, using a microfluidic device. In certain embodiments, individual particles, e.g., beads, and one or more of the cells or nuclei can be partitioned into emulsion droplets. In certain embodiments, each of the beads is coupled to a polynucleotide comprising a barcode (referred to herein as a “barcode

[0258] 516169085.1 46Attorney Ref. No. 00B206.1710

[0259] GNE Ref. No. P60053-WO polynucleotide”) and a capture sequence e.g., a poly-T sequence), e.g., that binds to the poly-A tails of the mRNAs of the cell, binds to the poly-A tails associated with the gRNA and / or binds to the poly-A tails of the MULTIseq lipid-modified oligonucleotides, and allows for the identification of single cells. In certain embodiments, partitioning the individual particles and the one or more cells of the enriched cell sample into emulsion droplets can comprise bringing the plurality of particles from a first aqueous phase and the cells from a second aqueous phase in contact with another phase, e.g, a non-aqueous phase, that is immiscible with the first and / or second aqueous phase to form emulsion droplets. Alternatively, partitioning the individual particles and the one or more cells or nuclei into emulsion droplets can comprise bringing a first aqueous phase comprising the plurality of particles and the cells in contact with another phase, e.g, a non-aqueous phase, that is immiscible with the first aqueous phase to form emulsion droplets.

[0260] In certain embodiments, the emulsion droplets can be generated by providing the cells and / or nucleic in a first fluid, e.g., a first aqueous phase, and combining the first fluid with a second fluid, e.g., a second aqueous phase or a non-aqueous phase, and shearing the fluids to generate a plurality of emulsion droplets that contain an individual particle and one or more of the cells or nuclei. In certain embodiments, shearing the fluids can be performed using any known method, technique or device for mixing solutions. For example, but not by way of limitation, shearing the fluids can include vortexing, shaking, flicking, stirring and / or pipetting. In certain embodiments, shearing the fluids includes vortexing the fluids to generate emulsion droplets.

[0261] In certain embodiments, the emulsion droplets can be generated using any emulsion droplet generating device. In certain embodiments, the emulsion droplet generating device can be a microfluidic device. For example, but not by way of limitation, droplets can be generated on a Chromium™ platform commercialized by lOx Genomics. In certain embodiments, a first aqueous phase comprising the barcode polynucleotide carrying beads is flowed through a channel segment and a second aqueous phase comprising the cells or nuclei is flowed through a second channel segment towards a channel junction. A partitioning fluid (e.g., oil) is introduced into the channel junction from one or more side channels, and the combined streams are flowed into an outlet channel. Within the channel junction, the two aqueous streams are combined with the partitioning oil, and partitioned into droplets that contain co-partitioned nucleic or cells and beads into emulsion droplets (referred to herein as “GEMs”). In certain embodiments, controlling the flow characteristics of each of the fluids combining at the channel

[0262] 516169085.1 47Attorney Ref. No. 00B206.1710

[0263] GNE Ref. No. P60053-WO junction and controlling the geometry of the channel junction, one can achieve a desired occupancy level of beads, cells (or nuclei) or both, within the droplets that are generated. Additional disclosure regarding microfluidic devices for use in the present disclosure is provided in WO 2017 / 096158 and U.S. Patent No. 11,193,122, the contents of each which are incorporated herein in their entirety.

[0264] In certain embodiments, partitioning the individual particles and the one or more cells can result in the generation of a plurality of emulsion droplets. In certain embodiments, other reagents can be co-partitioned into the emulsion droplets. In certain embodiments, such reagents can be used to incorporate the barcode polynucleotide into the nucleic acids, e.g., mRNAs present in the cell and / or the gRNA present in the cell. For example, but not by way of limitation, the reagents can comprise polymerases, reverse transcriptases, nucleoside triphosphates or NTP analogues, primers, cofactors, endonucleases, lysis reagents, dyes, markers or labels.

[0265] In certain embodiments, the plurality of cells is overloaded onto the microfluidic device for expression analysis. In certain embodiments, at least 40,000 cells are loaded onto one channel of the device for expression analysis. In certain embodiments, at least 50,000 cells are loaded onto one channel of the device for expression analysis. In certain embodiments, at least 60,000 cells are loaded onto one channel of the device for expression analysis. In certain embodiments, at least 70,000 cells are loaded onto one channel of the device for expression analysis. In certain embodiments, at least 80,000 cells are loaded onto one channel of the device for expression analysis. In certain embodiments, at least 90,000 cells are loaded onto one channel of the device for expression analysis. In certain embodiments, at least 100,000 cells are loaded onto one channel of the device for expression analysis. In certain embodiments, the number of cells per emulsion droplet is more than 1, e.g., more than 2, more than 3, more than 4 or more than 5.

[0266] In certain embodiments, a method of the present disclosure can include contacting the plurality of cells with labels, / .< ., oligonucleotides comprising a barcode, prior to loading onto a device for expression analysis. In certain embodiments, the labels are oligonucleotides, e.g., lipid-modified oligonucleotides, e.g., MULTIseq lipid-modified oligonucleotides (Sigma). Non-limiting examples of such lipid-modified oligonucleotides are disclosed in McGinnis et al., Nature Methods 16:619-626 (2019), the contents of which are hereby incorporated in its entirety. In certain embodiments, the labels allow for overloading of the microfluidic device and are used to dissociate singlet and multiplet (e.g., doublet) neurons from the plurality of

[0267] 516169085.1 48Attorney Ref. No. 00B206.1710

[0268] GNE Ref. No. P60053-WO cells. In certain embodiments, the use of the barcode from the MULTIseq lipid-modified oligonucleotides and the barcode from the 10X barcode polynucleotides allow for the identification of RNA transcripts from a single cell even in the presence of overloading of the microfluidic device.

[0269] In certain embodiments, at least 10,000 cells are recovered from one channel of the device for expression analysis. In certain embodiments, at least 20,000 cells are recovered from one channel of the device for expression analysis. In certain embodiments, at least 30,000 cells are recovered from one channel of the device for expression analysis. In certain embodiments, at least 40,000 cells are recovered from one channel of the device for expression analysis. In certain embodiments, at least 50,000 cells are recovered from one channel of the device for expression analysis.

[0270] In certain embodiments, a method of the present disclosure includes (a) providing a plurality of cells, e.g., neurons, (b) contacting the plurality of cells with a first nucleic acid construct (or a lentivirus vector comprising the first nucleic acid construct) and a second nucleic acid construct (or a lentivirus vector comprising the second nucleic acid construct), where at least one of the nucleic acid constructs comprise a polynucleotide encoding a heterologous surface protein, (c) enriching for a subset of cells that express the gRNA and / or the Cas protein from the plurality of cells using magnetic enrichment, where the magnetic enrichment comprises binding magnetic beads to the heterologous surface protein, (d) incorporating one or more barcodes into the nucleic acids (e.g., mRNAs and gRNAs) of the enriched cells and (e) sequencing the barcoded nucleic acids. In certain embodiments, the method further includes analyzing the sequenced barcoded nucleic acids to correlate mRNA expression in single cells to the expression of a specific gRNA, which allows for analyzing the effect of specific gRNAs on mRNA expression.

[0271] In certain embodiments, all or a subset of the barcoded nucleic acids can be sequenced. In certain embodiments, the nucleic acids, e.g. , mRNAs, can be sequenced by a high throughput sequencing method. In certain embodiments, the nucleic acids, e.g, mRNAs, can be sequenced by next-generation sequencing (NGS). For example, but not by way of limitation, sequencing can be performed using the Illumina NGS platform. In certain embodiments, the nucleic acids, e.g, mRNAs, can be sequenced by a pyrosequencing method. In certain embodiments, the nucleic acids, e.g., mRNAs, can be sequenced by Sanger sequencing. In certain embodiments, the nucleic acids, e.g., mRNAs, can be sequenced using nanopore-based sequencing. In certain embodiments, the sequence reads obtained using the methods of the present disclosure can be

[0272] 516169085.1 49Attorney Ref. No. 00B206.1710

[0273] GNE Ref. No. P60053-WO attributed to specific cells based upon the unique sequences included in the barcode polynucleotide coupled to the particles and the barcode from the MULTIseq lipid-modified oligonucleotides.

[0274] In certain embodiments, cells from the enriched cell sample can use the Perturb View method as disclosed in WO 2025 / 137335 and Kudo et al. Nature Biotechnology 43: 1091-1100 (2025), the contents of each of which are incorporated by reference herein. For example, but not by way of limitation, a method of the present disclosure for preparing an enriched cell sample can include the use of a first nucleic acid construct encoding the gRNA comprising a T7 promoter incorporated into a U6 promoter that is operatively coupled to the gRNA for expression, e.g., where the U6 promoter is used for expression of the gRNA in live cells and the T7 promoter drives transcription of the gRNA in fixed cells, e.g., for in situ hybridization or sequencing.

[0275] V. SYSTEMS

[0276] The present disclosure provides systems for performing the methods of the present disclosure. For example, but not by way of limitation, the present disclosure provides systems containing materials and reagents for performing a pooled genomic screen, e.g., a pooled CRISPR screen. In certain embodiments, the present disclosure provides systems that include materials or reagents (e.g., in one or more reservoirs or containers) for performing a method for CRISPR screening cells in a sample. In certain embodiments, the system is an automated system. In certain embodiments, the automated system includes one or more automated pipettes for dispensing materials or reagents onto a sample.

[0277] In certain embodiments, a system of the present disclosure can comprise one or more nucleic acid constructs described herein, e.g., two or more, three or more, four or more or five or more nucleic acid constructs (lentivirus vectors or compositions thereof) in one or more containers. In certain embodiments, a system of the present disclosure can comprise a composition comprising one or more nucleic acid constructs described herein. In certain embodiments, a system of the present disclosure can comprise a vector comprising one or more nucleic acid constructs described herein. For example, but not by way of limitation, a system of the present disclosure can comprise a second nucleic acid construct that comprises one or more nucleic acid sequences encoding a Cas protein, e.g., Cas9, and a fluorescent label under the control of a promoter, e.g., hSynl and / or hPGK, and a first nucleic acid construct comprising one or more nucleic acid sequences encoding a gRNA, a cell surface protein and a

[0278] 516169085.1 50Attorney Ref. No. 00B206.1710

[0279] GNE Ref. No. P60053-WO fluorescent label under the control of a promoter. In certain embodiments, a system of the present disclosure can comprise a second nucleic acid construct that comprises one or more nucleic acid sequences encoding a Cas protein, e.g., Cas9, and a fluorescent label under the control of a promoter, e.g., hSynl and / or hPGK, and a first nucleic acid construct comprising one or more nucleic acid sequences encoding a gRNA, a cell surface protein and a fluorescent label under the control of one or more promoters, e.g., two promoters, as shown in Fig. 14.

[0280] In certain embodiments, a system of the present disclosure can further include magnetic microparticles, e.g., coupled to an agent that specifically binds the cell surface protein, in a container. For example, but not by way of limitation, a system of the present disclosure can further include magnetic microparticles, e.g., coupled to an agent that specifically binds m Thy 1.1.

[0281] Non-limiting examples of suitable containers for use in a system of the present disclosure comprise bottles, test tubes, vials and microtiter plates. The containers can be formed from a variety of materials such as glass or plastic.

[0282] In certain embodiments, the system can comprise other materials or reagents desirable from a commercial and user standpoint, including other buffers and diluents.

[0283] In certain embodiments, the components of the system are provided in predetermined ratios, with the relative amounts of the various reagents suitably varied to obtain the desired sensitivity and throughput of the disclosed methods.

[0284] VI. KITS

[0285] The present disclosure provides kits for performing the methods of the present disclosure. For example, but not by way of limitation, the present disclosure provides kits containing materials and reagents for performing a pooled genomic screen, e.g., a pooled CRISPR screen.

[0286] In certain embodiments, a kit of the present disclosure can comprise one or more nucleic acid constructs described herein, e.g., two or more, three or more, four or more or five or more nucleic acid constructs (lentivirus vectors or compositions thereof) in one or more containers. In certain embodiments, a kit of the present disclosure can comprise a composition comprising one or more nucleic acid constructs described herein. In certain embodiments, a kit of the present disclosure can comprise a vector comprising one or more nucleic acid constructs described herein. For example, but not by way of limitation, a kit of the present disclosure can comprise a second nucleic acid construct that comprises one or more nucleic acid sequences

[0287] 516169085.1 51Attorney Ref. No. 00B206.1710

[0288] GNE Ref. No. P60053-WO encoding a Cas protein, e.g., Cas9, and a fluorescent label under the control of a promoter, e.g., hSynl and / or hPGK, and a first nucleic acid construct comprising one or more nucleic acid sequences encoding a gRNA, a cell surface protein and a fluorescent label under the control of one or more promoters.

[0289] In certain embodiments, a kit of the present disclosure can further include magnetic microparticles, e.g., coupled to an agent that specifically binds the cell surface protein, in a container. For example, but not by way of limitation, a kit of the present disclosure can further include magnetic microparticles, e.g., coupled to an agent that specifically binds mThyl.l.

[0290] Non-limiting examples of suitable containers for use in a kit of the present disclosure comprise bottles, test tubes, vials and microtiter plates. The containers can be formed from a variety of materials such as glass or plastic.

[0291] In certain embodiments, the kit can comprise other materials or reagents desirable from a commercial and user standpoint, including other buffers and diluents.

[0292] In certain embodiments, the components of the kit are provided in predetermined ratios, with the relative amounts of the various reagents suitably varied to obtain the desired sensitivity and throughput of the disclosed methods.

[0293] VII. EXEMPLARY EMBODIMENTS

[0294] A. The present disclosure provides a method for performing a genomic screen, comprising:

[0295] a) providing a plurality of cells;

[0296] b) contacting the plurality of cells with (a) a first lentivirus vector comprising (i) a first nucleic acid sequence encoding a gRNA and (ii) a second nucleic acid sequence encoding a cell surface protein, and (b) a second lentivirus vector comprising (i) a first nucleic acid sequence encoding a Cas protein to obtain a plurality of transduced cells; and

[0297] c) contacting the plurality of transduced cells with magnetic microparticles coupled to an agent that binds to the cell surface protein to obtain an enriched population of cells that express the cell surface protein and the gRNA.

[0298] Al. The method of A further comprising culturing the transduced cells for a duration sufficient for expression of the gRNA, the cell surface protein and / or the Cas protein.

[0299] A2. The method of A or Al further comprising contacting the enriched population of cells with one or more polynucleotides comprising a barcode to label each cell with a barcode. A3. The method of any one of A-A2 further comprising analyzing single-cell RNA expression data from the enriched population of cells.

[0300] 516169085.1 52Attorney Ref. No. 00B206.1710

[0301] GNE Ref. No. P60053-WO A4. The method of A3, wherein analyzing single-cell RNA expression data from the enriched population of cells comprises sequencing the RNAs of single cells obtained from the enriched population of cells.

[0302] A5. The method of A4 further correlating a change in expression level of one or more RNAs to the gRNA expressed in a single cell of the enriched population of cells.

[0303] A6. The method of any one of A-A5, wherein at least one of the first lentivirus vector and the second lentivirus vector further comprises a nucleic acid sequence encoding a reporter. A7. The method of any one of A-A6, wherein the first lentivirus vector further comprises (iii) a third nucleic acid sequence encoding a reporter.

[0304] A8. The method of any one of A-A7, wherein the second lentivirus vector further comprises (ii) a second nucleic acid sequence encoding a reporter.

[0305] A9. The method of any one of A-A8, wherein the cell surface protein is a receptor.

[0306] A10. The method of A9, wherein the receptor is mThy 1.1.

[0307] All. The method of any one of A-A10, wherein the agent is an antibody that specifically binds to the cell surface protein.

[0308] A12. The method of any one of A-All, wherein the expression of the cell surface protein and / or the Cas protein are controlled by a promoter that enables expression of the cell surface protein and / or the Cas protein in the plurality of cells.

[0309] A13. The method of any one of A-A12, wherein the expression of the cell surface protein is controlled by a promoter that enables expression of the cell surface protein in the plurality of cells.

[0310] A14. The method of any one of A-A13, wherein the expression of the Cas protein is controlled by a promoter that enables expression of the Cas protein in the plurality of cells. A15. The method of any one of A12-A14, wherein the promoter is selected from the group consisting of an EF-la, an Synl and an PGK promoter.

[0311] Al 6. The method of Al 5, wherein the promoter is the EF-la promoter.

[0312] Al 7. The method of Al 5, wherein the promoter is the Synl promoter.

[0313] Al 8. The method of Al 5, wherein the promoter is the PGK promoter.

[0314] Al 9. The method of A-A18, wherein the expression of the gRNA is controlled by a first promoter and the expression of the cell surface protein is controlled by a second promoter. A20. The method of Al 9, wherein the first promoter is a U6 promoter, a T7 promoter or a combination thereof.

[0315] A21. The method of A20, wherein the first promoter is a U6 promoter.

[0316] 516169085.1 53Attorney Ref. No. 00B206.1710

[0317] GNE Ref. No. P60053-WO A22. The method of A20, wherein the first promoter is a T7 promoter.

[0318] A23. The method of A20, wherein the first promoter is a combination of a U6 promoter and a T7 promoter.

[0319] A24. The method of any one of A19-A23, wherein the second promoter is an EF-la promoter, an Synl promoter or an PGK promoter.

[0320] A25. The method of A24, wherein the second promoter is an EF-la promoter.

[0321] A26. The method of A24, wherein the second promoter is an Synl promoter.

[0322] A27. The method of A24, wherein the second promoter is an PGK promoter.

[0323] A28. The method of any one of A-A27, wherein the plurality of cells is contacted with the first lentivirus vector and the second lentivirus vector simultaneously.

[0324] A29. The method of any one of A-A28, wherein the plurality of cells is contacted with the first lentivirus vector at an MOI of 0.3 or less.

[0325] A30. The method of any one of A-A29, wherein the plurality of cells comprises cells of the nervous system or cancer cells.

[0326] A31. The method of A30, wherein the plurality of cells comprises cells of the nervous system.

[0327] A32. The method of any one of A- A31, wherein the plurality of cells comprises neurons. A33. The method of any one of A-A31, wherein the plurality of cells comprises microglia. A34. The method of A30, wherein the plurality of cells comprises cancer cells.

[0328] A35. The method of any one of A-A34, wherein the plurality of cells comprises from about 10 to about 1,000,000,000 cells.

[0329] A36. The method of any one of A- A35, wherein contacting the plurality of cells with a first lentivirus vector comprises contacting the plurality of cells with a plurality of first lentivirus vectors, wherein each first lentivirus vector encodes a gRNA targeting a different target gene. A37. The method of A36, wherein the plurality of first lentivirus vectors comprises from about 10 to about 100,000 first lentivirus vectors.

[0330] A38. The method of A36 or A37, wherein the plurality of first lentivirus vectors comprises from about 10 to about 1,000 first lentivirus vectors.

[0331] B. The present disclosure provides a composition for use in performing a genomic screen, comprising:

[0332] a) a first nucleic acid construct comprising (i) a first nucleic acid sequence encoding a gRNA and (ii) a second nucleic acid sequence encoding a cell surface protein; and

[0333] 516169085.1 54Attorney Ref. No. 00B206.1710

[0334] GNE Ref. No. P60053-WO b) a second nucleic acid construct comprising (i) a first nucleic acid sequence encoding a Cas protein.

[0335] Bl. The composition of B, wherein at least one of the first nucleic acid construct and the second nucleic acid construct further comprises a nucleic acid sequence encoding a reporter. B2. The composition of B or Bl, wherein the first nucleic acid construct comprises (i) a first nucleic acid sequence encoding a gRNA, (ii) a second nucleic acid sequence encoding a cell surface protein and (iii) a third nucleic acid sequence encoding a reporter.

[0336] B3. The composition of any one of B-B2, wherein the second nucleic acid construct comprises (i) a first nucleic acid sequence encoding a Cas protein and (ii) a second nucleic acid sequence encoding a reporter.

[0337] C. The present disclosure provides a composition for use in performing a genomic screen, comprising:

[0338] a) a first lentivirus vector comprising (i) a first nucleic acid sequence encoding a gRNA and (ii) a second nucleic acid sequence encoding a cell surface protein; and

[0339] b) a second lentivirus vector comprising (i) a first nucleic acid sequence encoding a Cas protein.

[0340] Cl . The composition of C, wherein at least one of the first lentivirus vector and the second lentivirus vector further comprises a nucleic acid sequence encoding a reporter.

[0341] C2. The composition of C or Cl, wherein the first lentivirus vector comprises (i) a first nucleic acid sequence encoding a gRNA, (ii) a second nucleic acid sequence encoding a cell surface protein and (iii) a third nucleic acid sequence encoding a reporter.

[0342] C3. The composition of any one of C-C2, wherein the second lentivirus vector comprises (i) a first nucleic acid sequence encoding a Cas protein and (ii) a second nucleic acid sequence encoding a reporter.

[0343] C4. The composition of any one of B-C3, wherein the first nucleic acid sequence encoding the gRNA is coupled to a first promoter and the second nucleic acid sequence encoding the cell surface protein is coupled to a second promoter.

[0344] C5. The composition of C4, wherein the first promoter is a U6 promoter, a T7 promoter or a combination thereof.

[0345] C6. The composition of C4 or C5, wherein the second promoter is an EF-la promoter, an Synl promoter or an PGK promoter.

[0346] C7. The composition of any one of B-C6, wherein the first nucleic acid sequence encoding the Cas protein is coupled to a promoter.

[0347] 516169085.1 55Attorney Ref. No. 00B206.1710

[0348] GNE Ref. No. P60053-WO C8. The composition of C7, wherein the promoter is selected from the group consisting of an EF-la, an Synl and an PGK promoter.

[0349] C9. The composition of any one of B-C8, wherein the cell surface protein is a receptor. CIO. The composition of C9, wherein the receptor is mThy 1.1.

[0350] Cl 1. The composition of any one of B-C10, wherein the Cas protein is a Cas9 protein. D. A kit for use in performing a genomic screen of any one of A-A38.

[0351] E. A system for use in performing a genomic screen of any one of A- A38.

[0352] F. A kit comprising a composition of any one of B-Cl 1.

[0353] G. A system comprising a composition of any one of B-Cl 1.

[0354] H. The present disclosure provides a nucleic acid construct comprising (i) a first nucleic acid sequence encoding a gRNA and (ii) a second nucleic acid sequence encoding a cell surface protein.

[0355] Hl . The nucleic acid construct of H, wherein the nucleic acid construct further comprises a nucleic acid sequence encoding a reporter.

[0356] H2. The nucleic acid construct of H or Hl, wherein the nucleic acid construct comprises (i) a first nucleic acid sequence encoding a gRNA, (ii) a second nucleic acid sequence encoding a cell surface protein and (iii) a third nucleic acid sequence encoding a reporter.

[0357] H3. The nucleic acid construct of any one of H-H2, wherein the nucleic acid sequence encoding the gRNA is coupled to a first promoter and the second nucleic acid sequence encoding the cell surface protein is coupled to a second promoter.

[0358] H4. The nucleic acid construct of H3, wherein the first promoter is a U6 promoter, a T7 promoter or a combination thereof.

[0359] H5. The nucleic acid construct of H3 or H4, wherein the second promoter is an EF-la promoter, an Synl promoter or an PGK promoter.

[0360] H6. The nucleic acid construct of any one of H-H5, wherein the cell surface protein is a receptor.

[0361] H7. The nucleic acid construct of H6, wherein the receptor is mThy 1.1.

[0362] H8. A lentivirus vector comprising the nucleic acid construct of any one of H-H7.

[0363] I. The present disclosure provides a nucleic acid construct comprising (i) a first nucleic acid sequence encoding a Cas protein and (ii) a second nucleic acid sequence encoding a reporter.

[0364] II. The nucleic acid construct of I, wherein the first nucleic acid sequence encoding the Cas protein is coupled to a promoter.

[0365] 516169085.1 56Attorney Ref. No. 00B206.1710

[0366] GNE Ref. No. P60053-WO 12. The nucleic acid construct of I and II, wherein the promoter is selected from the group consisting of an EF-la, an Synl and an PGK promoter.

[0367] 13. The nucleic acid construct of any one of 11-13, wherein the second nucleic acid sequence encoding the reporter is coupled to the promoter.

[0368] 14. The nucleic acid construct of any one of I-I3, wherein the Cas protein is a Cas9 protein.

[0369] 15. A lentivirus vector comprising the nucleic acid construct of any one of I-I4.

[0370] J. A kit comprising a nucleic acid construct or a lentivirus vector of any one of H-I4. K. A system comprising a nucleic acid construct or a lentivirus vector of any one of H-I4.

[0371] EXAMPLES

[0372] The presently disclosed subject matter will be better understood by reference to the following examples, which are provided as exemplary of the presently disclosed subject matter, and not by way of limitation.

[0373] Example 1: Double Lentivirus Transduction

[0374] This example discloses a lentiviral toolbox enabling pooled CRISPR screening in all wild-type cells, including iPSC-derived neurons. This toolbox includes double lentiviral infection with Cas9 and gRNA-expressing viruses that are compatible with pooled CRISPR-knock-out (“CRISPRko”) screening. These tools allow greater temporal control over the timing of the perturbation, and they enable pooled CRISPR screening in any cell type amenable to lentiviral infections. It also streamlines the process of screen enablement by eliminating the need to create and test multiple Cas9-expressing cell types within or across cell lines, as the same virus can be applied to multiple cell types at once. Finally, both the Cas9 and the gRNA are stably integrated into the cells post-transduction, which may enable additional, future perturbations in the same cells if desired, which is not possible with transient Cas9 transfection. Non-limiting examples of lentivirus vector expressing a gRNA or Cas 9 are shown in Figs.

[0375] 13A-13B and 14

[0376] Methods.

[0377] Cell Culture:

[0378] HEK 293T cells were obtained from ATCC and maintained in DMEM supplemented with 10% Fetal Bovine Serum (FBS; v / v), 100 units / ml penicillin, 100 pg / ml streptomycin and IX GlutaMAX.

[0379] After thawing, iNeurons were plated at 50- 100k cells per cm2on a poly-D-lysine and

[0380] 516169085.1 57Attorney Ref. No. 00B206.1710

[0381] GNE Ref. No. P60053-WO iMatrix-coated culture vessel of a suitable format, e.g., a 6 well-plate, a 96-well plate, a T225 flask or a multilayer Celldisc. On day 8, cells were transduced by adding appropriate volumes of lentiviral supernatant overnight to achieve an MOI of < 0.3. On DIV21, cells were collected for 10X loading.

[0382] All cells were cultured at 37°C and 5% CO2 in a humidified incubator.

[0383] Lentiviral transduction:

[0384] HEK293T cells:

[0385] HEK293T cells were seeded into 6-well plates at a density of 1 million cells per well. The following day, cells were transfected with the expression plasmid, delta8.9 and pCMV-VSV-G at a molar ratio of (1:2.3:0.2) using Lipofectamine 3000 (ThermoFisher Scientific L3000015). Opti-MEM (Gibco 31985062) was exchanged to culture media after 4 hours. Viral supernatant was harvested 2 days after transfection and filtered through a 0.45 pm filter. For infection of BMDMs, viral supernatant was further concentrated by adding Lenti-X concentrator (Takara 31231) and centrifuging at 4°C for Ihr. Lentivirus titer was quantified before the screens using CellTiter-Glo kit (Promega G7570).

[0386] iNeurons:

[0387] Lentiviruses were packaged in LentiX-HEK293T cells. Lentiviral Cas9 and lentiviral gRNA were simultaneously delivered, at an MOI of 8-10 and 0.1-0.3, respectively, to the desired culture anytime from DIV1 onwards. See, e.g., Figs.3A and 4A-B. Cells were cultured for 7-14 days before assessing KO efficiency. The high MOI for Cas9 was aimed at transducing as many cells in the culture as possible. This number depends both on the cell type and on the number of cells in the culture.

[0388] FACS Sortins. Sorting was performed on a Sony MA900 Cell Sorter. Sorting was conducted using 130-micron nozzles with a standard nozzle and sort speed. A normal sorting mode was employed to maximize recovery during sorting. As part of the 4-part sort-gating strategy, (1) neurons were isolated from debris (SSC-A v. FSC-A), (2) singlets were isolated from multiplets (FSC-H vs. FSC-A), (3) mCherry+ expressing neurons were isolated (FL3-A), and finally (4) mTurq2+ expressing neurons were isolated. See Fig. 3B.

[0389] Assessment of editing efficiency. For cell surface gene targets, cells were washed with PBS, incubated in a blocking buffer of 10% normal donkey serum in PBS for half an hour, then exposed to fluorophore-conjugated antibodies (e.g., Alexa Fluor 488, PE / Cy7) for Ih followed by a PBS wash. For cytoplasmic gene targets, cells were fixed briefly with 4% paraformaldehyde in PBS with 10% sucrose and permeabilized in 0.5% Triton-X in PBS for

[0390] 516169085.1 58Attorney Ref. No. 00B206.1710

[0391] GNE Ref. No. P60053-WO 15 minutes. After washing, the cells were incubated in blocking buffer for 30 minutes and exposed to fluorophore-conjugated antibodies (e.g., Alexa Fluor 488, PE / Cy7) for Ih followed by a PBS wash. Antibody signal was measured by flow cytometry (Sony, MA900 MultiApplication Cell Sorter) gated on mTurq2+ / mCherry+ cells based on forward and side scatter.

[0392] Results:

[0393] To test the editing capability of the double lentivirus system (Fig. 1), lentiviral gRNA targeting Target Gene 1 (“Tl”) were simultaneously delivered to A549 cells. Fig.2 illustrates the editing efficiency of double lentivirus transduction in A549 cells. A Tl knock-out of 95.6% was observed when measured by flow cytometry (Fig. 2).

[0394] An experimental approach to assay the editing of double lentivirus efficiency in iNeurons is illustrated in Fig.3A. iNeurons were transduced with the double lentivirus system on DIV1 and were sorted and analyzed by FACS on DIV21. A single-cell sorting mode was employed to deposit cells accurately during sorting. As part of the 4-part sort-gating strategy, (1) neurons were isolated from debris (SSC-A v. FSC-A), (2) singlets were isolated from multiplets (FSC-H vs. FSC-A), (3) mCherry+ expressing neurons were isolated (FL3-A), and finally (4) mTurq2+ expressing neurons were isolated (Fig.3B). Knock-out of gene targets T2 and T3 were >80% and >90%, respectively (Fig. 3C). The ability of the double lentivirus system to efficiently edit iNeurons was further assessed using two iNeuron types, / .< ., iNeuron type 1 and iNeuron type 2 (Figs. 4A-4B). iNeuron type 1 and iNeuron type 2 express different neurotransmitters and are functionally and molecularly distinct. The double lentivirus system achieved 87.8% knock-out of T4 in iNeuron type 1 (Fig. 4A) and 60% knock-out of T4 in iNeuron type 2 using the same guide (Fig. 4B).

[0395] A significant percentage of DIV21 iNeurons showed a loss of the target protein and expression of the Cas9 reporter (mCherry) but did not express the gRNA reporter (mTurq) (Fig. 5A). To limit the silencing of the gRNA reporter by transduced cells, iNeurons were transduced atDIVl, DIV7, andDIV14 (Fig.5B) and collected on DIV21. Transducing atDIV7 and DIV14 vastly reduced the percentage of edited mTurq-cells, with transduction at DIV7 showing the greatest reduction (2.13% silenced cells) (Fig. 5B). Editing efficiency for transduction at DIV7 and DIV14 was also assessed (Fig. 5C). Transduction at DIV7 showed 54% editing efficiency, significantly more effective than the 28% editing efficiency observed for transduction at DIV14 (Fig. 5C).

[0396] Next, lentiviral vectors with Cas9 under the control of three different promoters (Efl a, hPGK, and hSynl) were assessed for expression efficiency. The greatest expression was

[0397] 516169085.1 59Attorney Ref. No. 00B206.1710

[0398] GNE Ref. No. P60053-WO observed with hPGK (60%), followed by hSynl (46%), and then Efla (30%) (Fig. 6).

[0399] Next, to scale the disclosed double lentivirus system, the delivery of multiple lentiviral gRNAs to iNeurons was assessed. iNeurons were transduced with a pool of 197 control gRNAs (NTCs) (Fig.7A). Fig.7B illustrates an example distribution of NTC counts for a single guide, showing that cells display a bimodal distribution of guide counts. By assigning cells with 2+ NTC counts for a single guide, guides can be detected in transduced cells with a high signal-to-noise ratio. Using the same gRNA pool and assigning cells with 2+ NTC counts, 386 singlets containing 171 distinct guides were isolated (Figs. 7C-7D).

[0400] To evaluate the scalability and data quality of a pooled CRISPR screen in iNeurons, a perturb-seq pilot targeting 150 genes was performed, transducing on DIV7 and enriching for guide-expressing cells via FACS on DIV21. This 150 gene panel was highly curated and included a large fraction of positive controls for which a strong transcriptional response to KO, such as proteasomal and ribosomal genes, was anticipated. iNeurons from the perturb-seq pilot were projected onto the reference iNeuron atlas (Fig. 8A). The distribution of 8,893 guide-assigned iNeurons (~30 cells per guide) across iNeuron subtypes (red / orange, PRKCE subtypes; green / blue, RMST subtypes) was observed (Fig.8B). 78 gRNAs targeting 49 distinct genes are shown to affect their target gene, with red and blue indicating up- and downregulation relative to control (Fig. 8C). The regulatory matrix shows the impact of knocking out 50 genes (columns) on 865 affected genes (rows), with red and blue indicating up- and downregulation of affected genes in perturbed cells relative to control (Fig. 8D). Horizontal and vertical black lines delineate co-regulated gene programs (Fig. 8E; covariance colored by purple / green) and their regulatory modules (Fig.8F; covariance colored by blue and red), respectively. Genes are clustered and color-coded by module (Fig. 8F) or by program (Fig. 8E). Fig. 8G illustrates a schematic of functional gene networks, color-coded by cluster as in Figs. 8D-8F. A selected subset of genes in each module or program are listed as examples. Blue point arrows and red blunt arrows denote activation and inhibition respectively. Fig. 8H illustrates the experimental schematic and amounts of cells involved.

[0401] Example 2: Enrichment of Genetically Perturbed Cells

[0402] This example illustrates two enrichment methods, as an alternative to FACS, to enable scaling of the disclosed systems and methods. The speed and throughput of FACS makes scaling difficult. Thus, (1) nuclei isolation and FACS enrichment and (2) magnetic enrichment were assessed. Magnetic enrichment of guide-expressing human iPSC-derived neurons was

[0403] 516169085.1 60Attorney Ref. No. 00B206.1710

[0404] GNE Ref. No. P60053-WO achieved through the overexpression of a mouse cell surface receptor. This advancement leverages either synthetic or biologically relevant cell surface receptors and offers a significantly faster and higher throughput approach to FACS, in addition to allowing a more even distribution of iNeuron cell types.

[0405] Methods:

[0406] Dissociation ofDIV21 Cells.

[0407] Papain. A vial of papain (Worthington PAP2) was resuspended with 10 mL of HBSS with no Ca and Mg. A vial of DNase I (Worthington D2) resuspended in 500 pL of HBSS with no Ca and Mg was added to the vial of resuspended papain. The enzymatic solution was warmed for 10 minutes at 37°C.

[0408] Neurobasal inhibitor: 1 mL of 10% fetal bovine serum, 1 mL of ovomucoid inhibitor (Worthington OI-BSA, resuspended in 32 mL of HBSS with no Ca or Mg), and 8 mL of Neurobasal (Thermofisher 21103049) was combined and warmed for 10 minutes at 37°C.

[0409] Cell media was removed and the cells were incubated with warm 1 : 1 papain:HBSS with no Ca and Mg at 37°C for 5 minutes. After gently replacing the enzymatic solution with the Neurobasal inhibitor solution, the flask was gently swirled to lift the iNeurons. The cells are pelleted at 300g for 5 minutes at room temperature and then resuspended in 3% BSA in HBSS with no Ca or Mg.

[0410] Enrichment of mThy 1.1 -expressing cells (guide-positive cells) using Miltenyi.

[0411] Following resuspension of cells in 3% BSA in HBSS with no Ca or Mg, cells were incubated with 10 pL / mL of CD90.1 MicroBeads (Miltenyi 130-121-273) for 10 minutes. In the meantime, LD columns (Miltenyi 130-042-901) were rinsed with 2 mL of cold MACS buffer (PBS, pH 7.2, 0.5% BSA, and 2 mM EDTA) to prepare for loading. Cells were brought up to 500 pL of volume, filtered through a 100 pM Easy Strainer, then applied to the column. When the column reservoir was emptied, the column was washed twice with 1 mL MACS buffer. Once emptied, the column was flushed with 3 mL of 1% BSA in HBSS with no Ca or Mg using the provided plunger. Cells were spun down at 400g for 5 minutes and resuspended in either 1% BSA in Neurobasal minus phenol red or Neurobasal alone if following with MULTIseq barcoding.

[0412] Magnetic Enrichment of mThy 1.1 -expressing cells (guide-positive cells) usingEasySep.

[0413] After resuspending the pellet of iNeurons in 3% BSA in HBSS with no Ca and Mg, cells were incubated with 25 pL / mL of EasySep Mouse CD90.1 positive selection cocktail for 3 minutes (StemCell Technology cat no 18958). Samples were then incubated with 25 pL / mL

[0414] 516169085.1 61Attorney Ref. No. 00B206.1710

[0415] GNE Ref. No. P60053-WO of EasySep RapidSheres for 3 minutes. After bringing the volume up to 10 mL, tubes were placed into the EasySep magnet and incubated for 3 minutes before the supernatant was discarded. Rinsing was repeated 2 more times before the final pellet was resuspended with 1% BSA and spun down at 300g for 5 minutes. Cells were finally resuspended in either 1% BSA in Neurobasal minus phenol red or Neurobasal alone if following with MULTIseq barcoding.

[0416] Results:

[0417] Fig. 9A illustrates the experimental schematic for nuclei isolation and FACS enrichment. iNeurons were transduced one week after thaw with a guide lentivirus. At DIV21, iNeurons were dissociated and frozen down before lysing with TST buffer. The subsequent nuclei retain expression of H2B-GFP, enabling FACS-based enrichment of transduced neurons. gRNA counts versus cells (left) and reads (right) across different states was observed (Fig. 9B) A degree of bias was observed in the distribution of guide-assigned nuclei across iNeuron subtypes (PRKCE subtypes as red / orange; RMST subtypes as green / blue) (Fig. 9C).

[0418] Fig. 10A illustrates the experimental schematic for magnetic enrichment. iNeurons were transduced one week after thaw with a guide lentivirus expressing GFP2xNLS and m Thy 1.1. mThy 1.1 -positive cells were isolated via commercially available magnetic beadbased positive selection kits. Using GFP to measure mThy 1.1 expression, 56% of cells were mThy 1.1 -positive after magnetic enrichment, compared to 23% of mThy 1.1 -positive cells before magnetic enrichment (Fig. 10B). Fig. 10C shows the proportion of guide-assigned cells and their distribution across iNeuron subtypes, with -40% of cells assigned to a single guide. Less bias was observed in the subtypes recovered compared to FACS (Figs 10C-D; Fig. 9C), better reflecting the intrinsic heterogeneity of iNeurons. In addition to less bias, magnetic enrichment scales significantly more efficiently than FACS (10X greater recovery of enriched cells over significantly reduced experimental time) (Table 1).

[0419] Table 1. Scaling of Magnetic Enrichment versus FACS Enrichment

[0420] # cells # cells transduced # cells in # cells transduced # mTurq2+ cells seeded (-30%) suspension (-30%) post-FACS

[0421] 63M 18.9M 15M 4.5M 109,340 (2%) (over a full day of sorting)

[0422]

[0423] 516169085.1 62Attorney Ref. No. 00B206.1710

[0424] GNE Ref. No. P60053-WO

[0425] # cells # cells transduced # cells in # cells transduced # GFP2xNLS+ cells seeded (-30%) suspension (-30%) post- magnetic enrichment 122.5M 44. IM 26M 9.36M 1.8M (19%)

[0426] (-20 min)

[0427] 145M 60.3M 32.2M I3.4M 4.9M (37%)

[0428] (-20 min)

[0429]

[0430] Next, two enrichment technologies were compared. Fig. 11A illustrates the experimental schematic for magnetic enrichment. Cells remained intact through the enrichment protocol using either Miltenyi and StemCell Tech (Easy Sep) magnetic beads (Fig. 11B).

[0431] However, gRNA counts were enriched in cells isolated using EasySep beads compared to Miltenyi (Figs. 11C-E), and a higher proportion of guide-assigned cells were observed in cell and nuclei samples using EasySep compared to Miltenyi (Fig. HF and Fig. 11G).

[0432] Further experiments were performed to determine the optimal transduction rate and cell concentration to maximize magnetic enrichment efficiency. The experiments were performed as described in Example 1 using the StemCell EasySep kit with 25 pl / mL of beads per sample except that various MOIs and iNeuron cell concentrations were tested. MOIs ranging from 0.1 to 0.4 were analyzed. The percent GFP (% GFP) was used as a proxy for the initial MOI prior to enrichment, e.g., a 20% GFP+ population of iNeurons indicates that a MOI was used for transduction. Efficiency (as shown in Fig. 15 and Fig. 16) was determined by analyzing the fold change in the proportion of transduced cells following enrichment. As shown in Fig. 15, the most efficient magnetic enrichment was observed with a transduction rate MOI of less than 0.3, which resulted in the greatest fold change in the number of GFP+ cells. In addition, efficient magnetic enrichment was observed when iNeuron populations containing between 1 to 3 million iNeurons are transduced (Fig. 16), which resulted in the greatest fold change in the number of GFP+ cells.

[0433] Example 3 — Scaling Through MULTI sei / Barcodins:

[0434] This example illustrates MULTIseq barcoding and demultiplexing to further improve the scale of the presently disclosed systems and methods. Traditional 10X channels enable the recovery of at most 20,000 cells, with a multiplet rate of 8%. For a genome-wide screen aiming to ultimately recover 10-30 guide-assigned cells per perturbation, this would require loading over 300 10X channels. Overloading 10X channels (loading more and subsequently recovering

[0435] 516169085.1 63Attorney Ref. No. 00B206.1710

[0436] GNE Ref. No. P60053-WO more than the standard amount of cells) is thus critical for scaling perturb-seq studies, enabling both significant savings in both cost, labor, and time and making the experiment more feasible. A major caveat of overloading was the high multiplet rate. Existing algorithms are frequently unable to accurately distinguish multiplets from singlets, which would impair the ability to accurately assign perturbations to single cells. To overcome this obstacle, there are several methods to hash, or barcode, cells with an oligonucleotide prior to loading. These barcodes can then be used to demultiplex cells and accurately assign singlets and doublets. Antibody-based hashing methods take longer compared to lipid-modified oligonucleotides (MULTIseq), which can compromise the health of delicate cells such as iNeurons. However, MULTIseq has not previously been implemented in iNeurons.

[0437] Here, it is shown that MULTIseq labeling can be performed on dissociated neurons, and that the barcodes can be demultiplexed downstream of 10X to accurately identify singlets from overloaded channels.

[0438] Methods:

[0439] Preparation of MULTIseq barcode plate. Barcode plates were ordered from Integrated DNA Technologies. The anchor stock and barcode lipid modified oligonucleotides (Sigma) were each diluted 1 : 10 to 5 pM. 5 pL of working anchor solution and 5 pL of barcode working solution was added to each well. The co-anchor stock was diluted 1 :20 to 2.5 pM. 0 pL of coanchor working solution was distributed to a separate set of wells, 1 per barcode. Plates can be prepared beforehand and stored at -20°C.

[0440] Label cells. Cells (suspended in cold HBSS with no BSA) were distributed across the wells of a protein loBind 2mL Deepwell 96-well plate, aliquoting 80 pL of cells per well to a maximum of 500,000 cells per well. 10 pL of anchor: barcode solution was transferred to 80 pL of cells. The solution was pipetted gently ~10 times to mix well and incubated for 5 minutes on ice. 10 pL of co-anchor solution was added per well. The solution was pipetted gently ~10 times to mix well and incubated for 5 minutes on ice. To prevent barcode swapping between cells, all subsequent reagents must be kept ice-cold during labeling. The labeling was quenched by adding 1 mL of 3% BSA in Neurobasal media to each well. The plate was spun at 400g for 5 minutes at 4°C. The media was gently removed, and each pellet was resuspended in 2% BSA in Neurobasal media. Each well was pooled into 25 mL of ice-cold 2% BSA in Neurobasal media. The samples were centrifuged at 400g for 5 minutes to collect labeled cells and resuspended in 200 pL of 1% BSA in Neurobasal media. If necessary, the cells were filtered through a 100 pM filter before loading on a 10X chip.

[0441] 516169085.1 64Attorney Ref. No. 00B206.1710

[0442] GNE Ref. No. P60053-WO The distribution of recovered doublets and singlets across 3 10X v4 channels overloaded with 60-70k cells is shown in Fig. 12A. UMAP barcode space (color-coded as in Fig. 12A) shows the localization of unknowns and doublets in intermediate spaces between barcode clusters and the localization of singlets at the barcode clusters (Fig. 12B). This localization allows for the barcodes to be demultiplexed downstream of 10X to accurately identify singlets from overloaded channels. Finally, Fig. 12C shows the expression of MULTIseq barcodes across demultiplexed cells (rows correspond to individual barcodes, columns correspond to barcode assignments), showing that MULTIseq barcoding enables accurate singlet recovery from overloaded 10X channels.

[0443] Example 4 — High Throughput Magnetic Enrichment

[0444] Additional experiments were performed to scale up the number of cells that can undergo magnetic enrichment. In brief, iPSC cells that express dCas9-Zim3 were transduced with the lentivirus vector shown in Fig. 146 days after initiation of differentiation (DIV6). The cells were transduced with the vector at a transduction rate of about 40% (MOI of 0.4). As shown in Fig- 14, the lentivirus vector comprises (i) a nucleotide sequence encoding a gRNA under the control of a promoter that includes a T7 promoter incorporated into a U6 promoter (referred to as a “U6 / T7” promoter) and (ii) a nucleotide sequence encoding Thy 1.1 and (iii) a nucleotide sequence encoding a fluorescent reporter, e.g., BFP-2XNLS, where the nucleotide sequences encoding Thy 1.1 and the reporter were under the control of a full-length EF-la promoter. The resulting transduced iNeurons were magnetically enriched for cells expressing mThyl.l at DIV11 while replating them for a DIV21 readout. The 130 million cells were split into 16 subsamples so that magnetic enrichment was performed with the optimal cell concentration. 8 magnets were used at once to enable their simultaneous enrichment over two separate, sequential experimental rounds. Magnetic enrichment using the StemCell EasySep kit using 25 pl / mL of beads per subsample was successfully performed on the 130 million iNeurons (-40% transduction rate), which resulted in the collection of 75 million cells post-enrichment, where the transduced cells now representing -51% of the total population. These data show that a 10-fold increase in scale for magnetic enrichment was successful.

[0445] Although the presently disclosed subject matter and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the present disclosure. Moreover, the

[0446] 516169085.1 65Attorney Ref. No. 00B206.1710

[0447] GNE Ref. No. P60053-WO scope of the present application is not intended to be limited to the particular embodiments of the process, machine, manufacture, and composition of matter, means, methods and steps described in the specification. Accordingly, the appended claims are intended to comprise within their scope such processes, machines, manufacture, compositions of matter, means, methods or steps.

[0448] Various patents, patent applications, publications, product descriptions and protocols are cited throughout this application, the disclosure of which are incorporated herein by reference in their entireties for all purposes.

[0449] 516169085.1 66

Claims

1. Attorney Ref. No. 00B206.1710GNE Ref. No. P60053-WO WHAT IS CLAIMED IS:

1. A method for performing a genomic screen, comprising:a) providing a plurality of cells;b) contacting the plurality of cells with (a) a first lentivirus vector comprising (i) a first nucleic acid sequence encoding a gRNA and (ii) a second nucleic acid sequence encoding a cell surface protein, and (b) a second lentivirus vector comprising (i) a first nucleic acid sequence encoding a Cas protein to obtain a plurality of transduced cells; and c) contacting the plurality of transduced cells with magnetic microparticles coupled to an agent that binds to the cell surface protein to obtain an enriched population of cells that express the cell surface protein and the gRNA.

2. The method of claim 1 further comprising culturing the transduced cells for a duration sufficient for expression of the gRNA, the cell surface protein and / or the Cas protein.

3. The method of claim 1 or 2 further comprising contacting the enriched population of cells with one or more polynucleotides comprising a barcode to label each cell with a barcode.

4. The method of any one of claims 1-3 further comprising analyzing single-cell RNA expression data from the enriched population of cells.

5. The method of claim 4, wherein analyzing single-cell RNA expression data from the enriched population of cells comprises sequencing the RNAs of single cells obtained from the enriched population of cells.

6. The method of claim 5 further correlating a change in expression level of one or more RNAs to the gRNA expressed in a single cell of the enriched population of cells.

7. The method of any one of claims 1-6, wherein at least one of the first lentivirus vector and the second lentivirus vector further comprises a nucleic acid sequence encoding a reporter.

8. The method of any one of claims 1-7, wherein the first lentivirus vector further comprises (iii) a third nucleic acid sequence encoding a reporter.

9. The method of any one of claims 1-8, wherein the second lentivirus vector further comprises (ii) a second nucleic acid sequence encoding a reporter.

10. The method of any one of claims 1-9, wherein the cell surface protein is a receptor.

11. The method of claim 10, wherein the receptor is mThy 1.1.

12. The method of any one of claims 1-10, wherein the agent is an antibody that specifically binds to the cell surface protein.516169085.1 67Attorney Ref. No. 00B206.1710GNE Ref. No. P60053-WO 13. The method of any one of claims 1-12, wherein the expression of the cell surface protein and / or the Cas protein are controlled by a promoter that enables expression of the cell surface protein and / or the Cas protein in the plurality of cells.

14. The method of claim 13, wherein the promoter is selected from the group consisting of an EF-la, an Synl and an PGK promoter.

15. The method of any one of claims 1-14, wherein the expression of the gRNA is controlled by a first promoter and the expression of the cell surface protein is controlled by a second promoter.

16. The method of claim 15, wherein the first promoter is a U6 promoter, a T7 promoter or a combination thereof.

17. The method of claim 15 or 16, wherein the second promoter is an EF-la promoter, an Synl promoter or an PGK promoter.

18. The method of any one of claims 1-17, wherein the plurality of cells is contacted with the first lentivirus vector and the second lentivirus vector simultaneously.

19. The method of any one of claims 1-18, wherein the plurality of cells is contacted with the first lentivirus vector at an MOI of 0.3 or less.

20. The method of any one of claims 1-19, wherein the plurality of cells comprises cells of the nervous system or cancer cells.

21. The method of any one of claims 1-20, wherein the plurality of cells comprises neurons.

22. The method of any one of claims 1-21, wherein the plurality of cells comprises microglia.

23. The method of any one of claims 1-22, wherein the plurality of cells comprises from about 10 to about 1,000,000,000 cells.

24. The method of any one of claims 1-23, wherein contacting the plurality of cells with a first lentivirus vector comprises contacting the plurality of cells with a plurality of first lentivirus vectors, wherein each first lentivirus vector encodes a gRNA targeting a different target gene.

25. The method of claim 24, wherein the plurality of first lentivirus vectors comprises from about 10 to about 1,000 first lentivirus vectors.

26. A composition for use in performing a genomic screen, comprising:a) a first nucleic acid construct comprising (i) a first nucleic acid sequence encoding a gRNA and (ii) a second nucleic acid sequence encoding a cell surface protein; and516169085.1 68Attorney Ref. No. 00B206.1710GNE Ref. No. P60053-WO b) a second nucleic acid construct comprising (i) a first nucleic acid sequence encoding a Cas protein.

27. The composition of claim 26, wherein at least one of the first nucleic acid construct and the second nucleic acid construct further comprises a nucleic acid sequence encoding a reporter.

28. The composition of claim 27, wherein the first nucleic acid construct comprises (i) a first nucleic acid sequence encoding a gRNA, (ii) a second nucleic acid sequence encoding a cell surface protein and (iii) a third nucleic acid sequence encoding a reporter.

29. The composition of claim 27 or 28, wherein the second nucleic acid construct comprises (i) a first nucleic acid sequence encoding a Cas protein and (ii) a second nucleic acid sequence encoding a reporter.

30. A composition for use in performing a genomic screen, comprising:a) a first lentivirus vector comprising (i) a first nucleic acid sequence encoding a gRNA and (ii) a second nucleic acid sequence encoding a cell surface protein; and b) a second lentivirus vector comprising (i) a first nucleic acid sequence encoding a Cas protein.

31. The composition of claim 30, wherein at least one of the first lentivirus vector and the second lentivirus vector further comprises a nucleic acid sequence encoding a reporter.

32. The composition of claim 31, wherein the first lentivirus vector comprises (i) a first nucleic acid sequence encoding a gRNA, (ii) a second nucleic acid sequence encoding a cell surface protein and (iii) a third nucleic acid sequence encoding a reporter.

33. The composition of claim 31 or 32, wherein the second lentivirus vector comprises (i) a first nucleic acid sequence encoding a Cas protein and (ii) a second nucleic acid sequence encoding a reporter.

34. The composition of any one of claims 26-33, wherein the first nucleic acid sequence encoding the gRNA is coupled to a first promoter and the second nucleic acid sequence encoding the cell surface protein is coupled to a second promoter.

35. The composition of claim 34, wherein the first promoter is a U6 promoter, a T7 promoter or a combination thereof.

36. The composition of claim 34 or 35, wherein the second promoter is an EF-la promoter, an Synl promoter or an PGK promoter.

37. The composition of any one of claims 26-36, wherein the first nucleic acid sequence encoding the Cas protein is coupled to a promoter.516169085.1 69Attorney Ref. No. 00B206.1710GNE Ref. No. P60053-WO 38. The composition of claim 37, wherein the promoter is selected from the group consisting of an EF-la, an Synl and an PGK promoter.

39. The composition of any one of claims 26-38, wherein the cell surface protein is a receptor.

40. The composition of claim 39, wherein the receptor is mThyl.l.

41. The composition of any one of claims 26-40, wherein the Cas protein is a Cas9 protein.

42. A kit for use in performing a genomic screen of any one of claims 1-25.

43. A system for use in performing a genomic screen of any one of claims 1-25.

44. A kit comprising a composition of any one of claims 26-41.

45. A system comprising a composition of any one of claims 26-41.516169085.1 70

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