WO2022143783A1

WO2022143783A1 - Methods of identifying t-cell modulating genes

Info

Publication number: WO2022143783A1
Application number: PCT/CN2021/142538
Authority: WO
Inventors: Pengfei YUAN; Ming Jin; Yongjian Zhang; Xiaomei Yang; Ling Yang; Meihua SU; Huiming ZONG
Original assignee: Edigene Therapeutics (Beijing) Inc.
Priority date: 2020-12-29
Filing date: 2021-12-29
Publication date: 2022-07-07
Also published as: TW202242139A; CN114874983A

Abstract

Provided are methods of identifying genes that modulate the sensitivity or resistance of T cells (e.g., allogeneic T cells or CAR-T cells (such as allogeneic CAR-T cells) ) to NK cell killing. Also provided are modified T cells (e.g., allogeneic T cells or CAR-T cells (such as allogeneic CAR-T cells)) that are resistant to NK cell killing, and methods and kits for generating thereof.

Description

METHODS OF IDENTIFYING T-CELL MODULATING GENES

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority benefits of International Patent Applications No. PCT/CN2020/140860 filed December 29, 2020, the content of which is incorporated herein by reference in its entirety.

FIELD OF THE PRESENT APPLICATION

The present application relates to methods of identifying genes that modulate the sensitivity or resistance of T cells (e.g., allogeneic T cells or chimeric antigen receptor-expressing T cells (CAR-T cells) , such as allogeneic CAR-T cells) to NK cell killing. Also provided are modified T cells (e.g., allogeneic T cells or CAR-T cells (such as allogeneic CAR-T cells) ) that are resistant to NK cell killing, and methods and kits for generating thereof.

BACKGROUND OF THE PRESENT APPLICATION

Immunotherapeutic approaches, including adoptive T cell therapy (e.g., CAR-T) , are playing an increasingly important role in the treatment of cancer, viral infections, and other pathophysiological autoimmune conditions. Compared to autologous T cell therapy which often requires a long and expensive customized manufacturing process and is not suitable for all patients, allogeneic T cell therapy has become a more appealing approach where T cells are derived from healthy donors and can provide off-the-shelf products suitable for many patients instead of only a single person. One of the major challenges in the allogeneic approach is host rejection in which patient’s immune system (e.g., host T cells, NK cells) will recognize infused non-HLA matched T-cells as foreign and reject them. To overcome this issue, researchers have knocked out Beta-2 microglobulin (B2M) required for human leucocyte antigen (HLA) class I expression in CAR-T cells using the clustered regularly interspaced short palindromic repeats (CRISPR) /Cas9 (CRISPR-associated protein 9) (CRISPR/Cas9) system, to prevents host TCRαβ cells recognizing donor CAR-T cells as foreign via HLA class I (Ren et al., Clin. Cancer Res. 2017; 23: 2255-2266) . However, cells with reduced HLA class I expression are also targeted by NK cells, representing a hurdle to prevent allogeneic T cell rejection (Liu et al. Curr. Res. Transl. Med. 2018; 66: 39-42) .

The activity of NK cells is regulated by a complex interplay of various cell surface inhibitory and activating receptors. Inhibitory receptors include killer immunoglobulin-like receptors (KIRs) and CD94/NKG2A, recognize major histocompatibility complex (MHC) or HLA class I molecules, allow NK cells to recognize autologous cells and prevent them from attacking the host tissue. When no matching MHC class I molecule is present, the inhibition of NK cytotoxicity is released and the balance is shifted towards NK cell activation via activating receptor engagement. During viral infections or malignant transformation, the transformed cells decrease MHC class I antigen expression on cell surface to avoid recognition by T cells. NK cells can recognize such transformed cells as “altered self” whose abnormal level of MHC class I expression results in decreased engagement of KIRs, and increase stimulatory receptor expression to provide effector response and cytotoxic killing of the transformed cells (Nayyar et al. Front Oncol. 2019; 9: 51) .

The CRISPR/Cas9 system enables editing at targeted genomic sites with high efficiency and specificity. One of its extensive applications is to identify functions of coding genes, non-coding RNAs and regulatory elements through high-throughput pooled screening in combination with next generation sequencing ( “NGS” ) analysis. By introducing a pooled single-guide RNA ( “sgRNA” ) or paired-guide RNA ( “pgRNA” ) library into cells expressing Cas9 or catalytically inactive Cas9 (dCas9) fused with effector domains, investigators can perform multifarious genetic screens by generating diverse mutations, large genomic deletions, transcriptional activation or transcriptional repression.

To generate a high-quality cell library of gRNAs for any given pooled CRISPR screen, one must use a low multiplicity of infection ( “MOI” ) during cell library construction to ensure that each cell on average harbors less than one sgRNA or pgRNA to minimize the false-discovery rate (FDR) of the screen. To further reduce the FDR and increase data reproducibility, in-depth coverage of gRNAs and multiple biological replicates are often necessary to obtain hit genes with high statistical significance, resulting in increased workload. Additional difficulties may arise when one performs a large number of genome-wide screens, when cell materials for library construction are limited, or when one conducts more challenging screens (i.e., in vivo screens) for which it is difficult to obtain experimental replicates or control the MOI. The “internal barcodes ( “iBAR” ) methods previously developed by the Applicant (see WO2020125762, the content of which is incorporated herein by reference in its entirety) provide a reliable and highly efficient screening strategy for large-scale target identification in eukaryotic cells, with much lower false-positive and false-negative rates, and allow cell library generation using a high MOI. For example, compared to a conventional CRISPR/Cas screen with a low MOI of 0.3, the iBAR methods can reduce the starting cell numbers for more than 20-fold (e.g., at an MOI of 3) to more than 70-fold (e.g., at an MOI of 10) , while maintaining high efficiency and accuracy. The iBAR system is particularly useful for cell-based screens in which the cells are available in limited quantities, or for in vivo screens in which viral infection to specific cells or tissues is difficult to control at low MOI.

The disclosures of all publications, patents, patent applications and published patent applications referred to herein are hereby incorporated herein by reference in their entirety.

BRIEF SUMMARY OF THE PRESENT APPLICATION

The present invention in one aspect provides a method of identifying a target gene in a T cell (e.g., allogeneic T cell or CAR-T cell (such as allogeneic CAR-T cell) ) that modulates the activity of the T cell, comprising: a) providing a T cell library comprising a plurality of T cells, wherein each of the plurality of T cells has a mutation (e.g., inactivating mutation) at a hit gene ( “hit gene mutation” ) in the genome, wherein the hit gene in at least two of the plurality of T cells are different from each other; b) subjecting the T cell library to treatment with NK cells; c) obtaining a T cell from the T cell library that is sensitive or resistant to the killing of the NK cells; and d) identifying the hit gene in the T cell obtained from step c) , thereby identifying the target gene in the T cell that modulates the activity of the T cell. In some embodiments, the T cell library is generated by subjecting an initial population of T cells to genome-wide gene editing. In some embodiments, T cells in the initial population of T cells express a CAR.

In some embodiments according to any one of the methods described above, the T cell library is generated by contacting an initial population of T cells (e.g., allogeneic T cells or CAR-T cells (such as allogeneic CAR-T cells) ) with i) a single-guide RNA ( “sgRNA” ) library comprising a plurality of sgRNA constructs, wherein each sgRNA construct comprises or encodes an sgRNA, and wherein each sgRNA comprises a guide sequence that is complementary (e.g., at least about any of 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%complementary) to a target site in the hit gene in the genome; and optionally ii) a Cas component comprising a Cas protein or a nucleic acid encoding the Cas protein, under a condition that allows introduction of the sgRNA constructs and the optional Cas component into the initial population of T cells. In some embodiments, the Cas protein is Cas9. In some embodiments, each sgRNA comprises the guide sequence fused to a second sequence, wherein the second sequence comprises a repeat-anti-repeat stem loop that interacts with the Cas9. In some embodiments, the second sequence of each sgRNA further comprises a stem loop 1, a stem loop 2, and/or a stem loop 3. In some embodiments, each sgRNA further comprises an internal barcode (iBAR) sequence ( “sgRNA ^iBAR” ) , wherein each sgRNA ^iBAR is operable with the Cas protein (e.g., Cas9) to modify the hit gene (e.g., cleave the hit gene, or modulate hit gene expression) . In some embodiments, the iBAR sequence of each sgRNA ^iBAR is inserted in the loop region of the repeat-anti-repeat stem loop. In some embodiments, the Cas protein is Cas9, and the iBAR sequence of each sgRNA ^iBAR is inserted in the loop region of the repeat-anti-repeat stem loop. In some embodiments, each sgRNA ^iBAR comprises in the 5’-to-3’ direction a first stem sequence and a second stem sequence, wherein the first stem sequence hybridizes with the second stem sequence to form a double-stranded RNA (dsRNA) region that interacts with the Cas protein, and the iBAR sequence is disposed between the 3’ end of the first stem sequence and the 5’ end of the second stem sequence. In some embodiments, each guide sequence comprises about 17 to about 23 nucleotides. In some embodiments, each iBAR sequence comprises about 1 to about 50 nucleotides (e.g., about 6 nucleotides) . In some embodiments, the sgRNA library comprising a plurality of sgRNA ^iBAR constructs ( “sgRNA ^iBAR library” ) comprises a plurality of sets of sgRNA ^iBAR constructs, wherein each set of sgRNA ^iBAR constructs comprise three or more (e.g., 3, 4, 5, 6, or more) sgRNA ^iBAR constructs each comprising or encoding an sgRNA ^iBAR, wherein the guide sequences for the three or more (e.g., 3, 4, 5, 6, or more) sgRNA ^iBAR constructs are the same, wherein the iBAR sequence for each of the three or more (e.g., 3, 4, 5, 6, or more) sgRNA ^iBAR constructs is different from each other, and wherein the guide sequence of each set of sgRNA ^iBAR constructs is complementary (e.g., at least about any of 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%complementary) to a different target site in the genome. In some embodiments, each set of sgRNA ^iBAR constructs comprises four sgRNA ^iBAR constructs, and the iBAR sequence for each of the four sgRNA ^iBAR constructs is different from each other. In some embodiments, the sgRNA ^iBAR library comprises at least about 100 (e.g., at least about any of 1,000, 10,000, 50,000, or more) sets of sgRNA ^iBAR constructs. In some embodiments, the iBAR sequences for at least two sgRNA ^iBAR constructs among different sets of sgRNA ^iBAR constructs are the same (e.g., the first set and the second set of sgRNA ^iBAR constructs have at least 1, 2, 3, 4, or more shared iBAR sequences among the two sets of sgRNA ^iBAR constructs) . In some embodiments, the iBAR sequences for at least two sets of sgRNA ^iBAR constructs are the same. In some embodiments, the sgRNA library comprising a plurality of sgRNA constructs comprises or encodes sgRNAs with guide sequences complementary (e.g., at least about any of 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%complementary) to target sites of every annotated gene in the genome. In some embodiments, the sgRNA ^iBAR library comprising a plurality of sgRNA ^iBAR constructs comprises or encodes sgRNAs ^iBAR with guide sequences complementary (e.g., at least about any of 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%complementary) to target sites of every annotated gene in the genome. In some embodiments, at least about 95% (e.g., at least about any of 96%, 97%, 98%, 99%, or 100%) of the sgRNA constructs in the sgRNA library (or sgRNA ^iBAR constructs in the sgRNA ^iBAR library) are introduced into the initial population of T cells. In some embodiments, the T cell library has averagely at least about 100-fold (e.g., at least about any of 200-, 500-, 1,000-, 5,000-, or more fold) coverage for each sgRNA ^iBAR. In some embodiments, the T cell library has averagely at least about 400-fold (e.g., at least about any of 600-, 800-, 1,000-, 2,000-, 8,000-, 12,000-, or more fold) coverage for each sgRNA. In some embodiments, the sgRNA library (or sgRNA ^iBAR library) comprises at least about 400 (e.g., at least about any of 600, 1000, 5000, 10,000, 50,000, 100,000, 300,000, 600,000, or more) sgRNA constructs (or sgRNA ^iBAR constructs) . In some embodiments, the sgRNA library (or sgRNA ^iBAR library) comprises at least about 150,000 (e.g., at least about any of 300,000, 600,000, or more) sgRNA constructs (or sgRNA ^iBAR constructs) . In some embodiments, the initial population of T cells express a Cas (e.g., Cas9) protein. In some embodiments, the method further comprises contacting the initial population of T cells or the T cell library with i) an sgRNA construct comprising or encoding an sgRNA which comprises a guide sequence that is complementary (e.g., at least about any of 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%complementary) to a target site in the B2M gene ( “B2M sgRNA” ) ; and optionally ii) a Cas component comprising a Cas protein or a nucleic acid encoding the Cas protein, under a condition that allows introduction of the B2M sgRNA construct and the optional Cas component into the initial population of T cells or the T cell library. In some embodiments, the T cells in the initial population of T cells comprise a B2M mutation (e.g., inactivating B2M mutation) . In some embodiments, each sgRNA construct in the sgRNA library (or each sgRNA ^iBAR construct in the sgRNA ^iBAR library) and/or the B2M sgRNA construct is an RNA. In some embodiments, each sgRNA construct in the sgRNA library (or each sgRNA ^iBAR construct in the sgRNA ^iBAR library) and/or the B2M sgRNA construct is a plasmid. In some embodiments, each sgRNA construct in the sgRNA library (or each sgRNA ^iBAR construct in the sgRNA ^iBAR library) and/or the B2M sgRNA construct is a viral vector, such as a lentiviral vector. In some embodiments, each sgRNA construct in the sgRNA library (or each sgRNA ^iBAR construct in the sgRNA ^iBAR library) and/or the B2M sgRNA construct is a virus, such as a lentivirus. In some embodiments, the sgRNA library (or sgRNA ^iBAR library) and/or the B2M sgRNA construct is contacted with the initial population of T cells at a multiplicity of infection (MOI) of at least about 2, such as 3.

In some embodiments according to any one of the methods described above, the treatment with NK cells comprises: i) an initial treatment step comprising contacting the T cell library with the NK cells; ii) an optional first enrichment step comprising sorting the mixture of treated cells to obtain a first T cell subpopulation that is sensitive or resistant to the killing of the NK cells; iii) an optional first recovery step comprising culturing the first T cell subpopulation; and iv) an optional second treatment step comprising contacting the first T cell subpopulation with the NK cells. In some embodiments, the initial treatment step comprises contacting the T cell library with the NK cells for at least about 48 hours, such as about any of 48 hours, 72 hours, 5 days, or 10 days. In some embodiments, the method comprises a first enrichment step. In some embodiments, the first enrichment step comprises sorting the mixture of treated cells that are B2M-negative (or deficient) and alive, thus obtaining the first T cell subpopulation that is resistant to the killing of the NK cells ( “first alive enrichment” ) . In some embodiments, the first enrichment step comprises sorting the mixture of treated cells that are B2M-negative (or deficient) and dead, thus obtaining the first T cell subpopulation that is sensitive to the killing of the NK cells ( “first dead enrichment” ) . In some embodiments, the method further comprises staining the mixture of treated cells with an anti-B2M antibody before sorting. In some embodiments, the method further comprises staining the mixture of treated cells with propidium iodide (PI) before sorting, wherein PI staining indicates cell death. In some embodiments, the method comprises a first recovery step. In some embodiments, the first recovery step comprises culturing the first T cell subpopulation for at least about 24 hours, such as about 48 hours. In some embodiments, the method comprises a second treatment step. In some embodiments, the second treatment step comprises contacting the first T cell subpopulation with the NK cells for at least about 48 hours, such as 96 hours. In some embodiments, the ratio of the NK cells and the T cells in the T cell library in the initial treatment step is about 0.1: 1 to about 20: 1 (e.g., about 0.3: 1 to about 1: 1, or about 0.5: 1 to about 20: 1) , such as about 0.5: 1 or about 1: 1. In some embodiments, the ratio of the NK cells and the T cells in the first T cell subpopulation in the second treatment step is about 0.1: 1 to about 20: 1 (e.g., about 0.3: 1 to about 1: 1, or about 1: 1 to about 10: 1) , such as about 0.3: 1.

In some embodiments according to any one of the methods described above, obtaining the T cell from the T cell library that is sensitive or resistant to the killing of the NK cells comprises: i) a sorting step comprising sorting the cells obtained from step b) to obtain a second T cell subpopulation that is sensitive or resistant to the killing of the NK cells; and ii) an optional second recovery step comprising culturing the second T cell subpopulation before harvesting the cells. In some embodiments, the sorting step comprises sorting the cells obtained from step b) that are B2M-negative (or deficient) and alive, thus obtaining the second T cell subpopulation that is resistant to the killing of the NK cells ( “harvest alive sorting” ) . In some embodiments, the sorting step comprises sorting the cells obtained from step b) that are B2M-negative (or deficient) and dead, thus obtaining the second T cell subpopulation that is sensitive to the killing of the NK cells ( “harvest dead sorting” ) . In some embodiments, the method further comprises staining the cells obtained from step b) with an anti-B2M antibody before sorting. In some embodiments, the method further comprises staining the cells obtained from step b) with PI before sorting, wherein PI staining indicates cell death. In some embodiments, the method comprises a second recovery step. In some embodiments, the second recovery step comprises culturing the second T cell subpopulation for at least about 24 hours, such as about 48 hours.

In some embodiments according to any one of the methods described above, steps b) and c) comprise: i) an initial treatment step comprising contacting the T cell library with the NK cells for about 72 hours at a ratio of NK cells to T cells of about 0.5: 1; ii) an enrichment step comprising sorting the mixture of treated cells that are B2M-negative (or deficient) and alive, thus obtaining a first T cell subpopulation that is resistant to the killing of the NK cells; iii) a recovery step comprising culturing the first T cell subpopulation for about 48 hours; iv) a second treatment step comprising contacting the first T cell subpopulation post-recovery with the NK cells for about 96 hours at a ratio of NK cells to T cells of about 0.3: 1; and v) a sorting step comprising sorting the final mixture of treated cells that are B2M-negative (or deficient) and alive, thus obtaining a second T cell subpopulation that is resistant to the killing of the NK cells.

In some embodiments according to any one of the methods described above, steps b) and c) comprise: i) a treatment step comprising contacting the T cell library with the NK cells for about 10 days at a ratio of NK cells to T cells of about 0.5: 1; and ii) a sorting step comprising sorting the mixture of treated cells that are B2M-negative (or deficient) and alive, thus obtaining a T cell subpopulation that is resistant to the killing of the NK cells.

In some embodiments according to any one of the methods described above, steps b) and c) comprise: i) a treatment step comprising contacting the T cell library with the NK cells for about 48 hours at a ratio of NK cells to T cells of about 1: 1; ii) a sorting step comprising sorting the mixture of treated cells that are B2M-negative (or deficient) and alive, thus obtaining a T cell subpopulation that is resistant to the killing of the NK cells; and iii) a recovery step comprising culturing the T cell subpopulation for about 48 hours before harvesting the cells.

In some embodiments according to any one of the methods described above, steps b) and c) comprise: i) a treatment step comprising contacting the T cell library with the NK cells for about 48 hours at a ratio of NK cells to T cells of about 1: 1; ii) an enrichment step comprising sorting the mixture of treated cells that are B2M-negative (or deficient) and alive, thus obtaining a first T cell subpopulation that is resistant to the killing of the NK cells; iii) a recovery step comprising culturing the first T cell subpopulation for about 48 hours; and iv) a sorting step comprising sorting the first T cell subpopulation post-recovery that are B2M-negative (or deficient) and alive, thus obtaining a second T cell subpopulation that is resistant to the killing of the NK cells.

In some embodiments according to any one of the methods described above, identifying the hit gene in the T cell obtained from step c) comprises: i) identifying a sequence comprising the hit gene mutation (e.g., inactivating mutation) in the T cell obtained from step c) ; and ii) identifying the hit gene corresponding to the sequence comprising the hit gene mutation (e.g., inactivating mutation) . In some embodiments, identifying the hit gene in the T cell obtained from step c) comprises: i) identifying the sgRNA sequence in the T cell obtained from step c) ; and ii) identifying the hit gene corresponding to the guide sequence of the sgRNA. In some embodiments, the hit gene mutation (e.g., inactivating mutation) or the sgRNA sequence is identified by DNA sequencing or RNA sequencing. In some embodiments, the hit gene mutation (e.g., inactivating mutation) or the sgRNA sequence is identified by next-generation sequencing (NGS) . In some embodiments, identifying the target gene comprises: i) obtaining sequences comprising the hit gene mutations (e.g., inactivating mutations) in the final T cell subpopulation obtained from step c) ; ii) ranking the sequences comprising the hit gene mutations (e.g., inactivating mutations) based on sequence counts; and iii) identifying the hit gene corresponding to a sequence comprising the hit gene mutation (e.g., inactivating mutation) ranked above a predetermined threshold level. In some embodiments, identifying the target gene comprises: i) obtaining sgRNA sequences in the final T cell subpopulation obtained from step c) ; ii) ranking the corresponding guide sequences of the sgRNA sequences based on sequence counts; and iii) identifying the hit gene corresponding to a guide sequence ranked above a predetermined threshold level. In some embodiments, the sgRNA is an sgRNA ^iBAR, and identifying the target gene comprises: i) obtaining sgRNA ^iBAR sequences in the final T cell subpopulation obtained from step c) ; ii) ranking the corresponding guide sequences of the sgRNA ^iBAR sequences based on sequence counts, wherein the ranking comprises adjusting the rank of each guide sequence based on data consistency among the iBAR sequences in the sgRNA ^iBAR sequences corresponding to the guide sequence; and iii) identifying the hit gene corresponding to a guide sequence ranked above a predetermined threshold level. In some embodiments, the method is a positive screening. In some embodiments, the method is a negative screening. In some embodiments, the sequence counts are subject to median ratio normalization followed by mean-variance modeling. In some embodiments, the sgRNA library is an sgRNA ^iBAR library, and the variance of each guide sequence is adjusted based on data consistency among the iBAR sequences in the sgRNA ^iBAR sequences corresponding to the guide sequence. In some embodiments, the sequence counts obtained from the final T cell subpopulation obtained from step c) are compared to corresponding sequence counts obtained from a subpopulation of control T cells to provide fold changes (e.g., actual fold changes, or derivatives of fold changes such as log2 or log 10 fold changes) . In some embodiments, the subpopulation of control T cells are obtained from a same T cell library cultured in the same condition without subjected to treatment with NK cells, and optionally subjected to the same obtaining method in step c) . In some embodiments, the data consistency among the iBAR sequences in the sgRNA ^iBAR sequences corresponding to each guide sequence is determined based on the direction of the fold change of each iBAR sequence, wherein the variance of the guide sequence is increased if the fold changes of the iBAR sequences are in different directions with respect to each other (e.g., increased vs. reduced, increased vs. unchanged, or reduced vs. unchanged) . In some embodiments, the method further comprises culturing a same T cell library under the same condition without subjected to treatment with NK cells, and optionally subjected to the same obtaining method in step c) to obtain a subpopulation of control T cells, wherein the presence of identifying the hit gene corresponding to the sequence comprising the hit gene mutation (e.g., inactivating mutation) or the guide sequence of the sgRNA from the subpopulation of control T cells but absence from the T cell obtained from step c) from the T cell library subjected to treatment with NK cells identifies the hit gene as the target gene.

In some embodiments according to any one of the methods described above, the method comprises subjecting the T cell library from step a) to at least two of the four separate Trials before step d) : (I) Trial I: i) an initial treatment step comprising contacting the T cell library with the NK cells for about 72 hours at a ratio of NK cells to T cells of about 0.5: 1; ii) an enrichment step comprising sorting the mixture of treated cells that are B2M-negative (or deficient) and alive, thus obtaining a first T cell subpopulation that is resistant to the killing of the NK cells; iii) a recovery step comprising culturing the first T cell subpopulation for about 48 hours; iv) a second treatment step comprising contacting the first T cell subpopulation post-recovery with the NK cells for about 96 hours at a ratio of NK cells to T cells of about 0.3: 1; and v) a sorting step comprising sorting the final mixture of treated cells that are B2M-negative (or deficient) and alive, thus obtaining a second T cell subpopulation that is resistant to the killing of the NK cells; (II) Trial II: i) a treatment step comprising contacting the T cell library with the NK cells for about 10 days at a ratio of NK cells to T cells of about 0.5: 1; and ii) a sorting step comprising sorting the mixture of treated cells that are B2M-negative (or deficient) and alive, thus obtaining a T cell subpopulation that is resistant to the killing of the NK cells; (III) Trial III: i) a treatment step comprising contacting the T cell library with the NK cells for about 48 hours at a ratio of NK cells to T cells of about 1: 1; ii) a sorting step comprising sorting the mixture of treated cells that are B2M-negative (or deficient) and alive, thus obtaining a T cell subpopulation that is resistant to the killing of the NK cells; and iii) a recovery step comprising culturing the T cell subpopulation for about 48 hours before harvesting the cells; and (IV) Trial IV: i) a treatment step comprising contacting the T cell library with the NK cells for about 48 hours at a ratio of NK cells to T cells of about 1: 1; ii) an enrichment step comprising sorting the mixture of treated cells that are B2M-negative (or deficient) and alive, thus obtaining a first T cell subpopulation that is resistant to the killing of the NK cells; iii) a recovery step comprising culturing the first T cell subpopulation for about 48 hours; and iv) a sorting step comprising sorting the first T cell subpopulation post-recovery that are B2M-negative (or deficient) and alive, thus obtaining a second T cell subpopulation that is resistant to the killing of the NK cells. In some embodiments, identifying the target gene comprises identifying the hit genes from the at least two of the four separate Trials, wherein: i) the hit genes that are identified as depleted from the final T cell subpopulation (alive) in at least one Trial with FDR ≤ 0.01, or in at least two Trials with FDR ≤ 0.05 (and/or with at least about 2-fold depletion, such as at least about any of 3-, 4-, 5-, 10-, 20-, 50-, 100-fold, or more depletion) are identified as target genes whose mutation (e.g., inactivation) makes the T cells sensitive to NK cell killing; and/or ii) the hit genes that are identified as enriched from the final T cell subpopulation in at least one Trial with FDR ≤ 0.05, or in at least two Trials with FDR ≤ 0.15, (and/or with at least about 2-fold enrichment, such as at least about any of 3-, 4-, 5-, 10-, 20-, 50-, 100-fold, or more enrichment) are identified as target genes whose mutation (e.g., inactivation) makes the T cells resistant to NK cell killing.

In some embodiments according to any one of the methods described above, the method comprises subjecting the T cell library from step a) to at least two separate different treatments with NK cells in step b) , and obtaining the T cells that are sensitive or resistant to the killing of the NK cells from each treatment in step c) . In some embodiments, identifying the target gene comprises identifying the hit genes in the T cells obtained from the at least two separate different treatments with NK cells, wherein: i) the hit genes that are identified as depleted from the final T cell subpopulation resistant to the killing of the NK cells in at least one treatment with NK cells with FDR ≤ 0.01, or in at least two separate different treatments with NK cells with FDR ≤ 0.05 (and/or with at least about 2-fold depletion, such as at least about any of 3-, 4-, 5-, 10-, 20-, 50-, 100-fold, or more depletion) are identified as target genes whose mutation (e.g., inactivation) make the T cells sensitive to NK cell killing; ii) the hit genes that are identified as enriched from the final T cell subpopulation resistant to the killing of the NK cells in at least one treatment with NK cells with FDR ≤ 0.05, or in at least two separate different treatments with NK cells with FDR ≤ 0.15, (and/or with at least about 2-fold enrichment, such as at least about any of 3-, 4-, 5-, 10-, 20-, 50-, 100-fold, or more enrichment) are identified as target genes whose mutation (e.g., inactivation) make the T cells resistant to NK cell killing; iii) the hit genes that are identified as depleted from the final T cell subpopulation sensitive to the killing of the NK cells in at least one treatment with NK cells with FDR ≤ 0.05, or in at least two separate different treatments with NK cells with FDR ≤ 0.15, (and/or with at least about 2-fold depletion, such as at least about any of 3-, 4-, 5-, 10-, 20-, 50-, 100-fold, or more depletion) are identified as target genes whose mutation (e.g., inactivation) make the T cells resistant to NK cell killing; and/or iv) the hit genes that are identified as enriched from the final T cell subpopulation sensitive to the killing of the NK cells in at least one treatment with NK cells with FDR ≤ 0.01, or in at least two separate different treatments with NK cells with FDR ≤ 0.05, (and/or with at least about 2-fold enrichment, such as at least about any of 3-, 4-, 5-, 10-, 20-, 50-, 100-fold, or more enrichment) are identified as target genes whose mutation (e.g., inactivation) make the T cells sensitive to NK cell killing.

In some embodiments according to any one of the methods described above, the method further comprises validating the target gene by: a) modifying a T cell by creating a mutation (e.g., inactivating mutation) in the target gene in the T cell; and b) determining the sensitivity or resistance of the modified T cell to the killing of NK cells. In some embodiments, the method further comprises creating a mutation (e.g., inactivating mutation) in B2M in the T cell.

In another aspect, there is also provided a method of generating a modified T cell (e.g., allogeneic T cell or CAR-T cell (such as allogeneic CAR-T cell) ) , comprising inactivating a target gene identified by any of the methods described above in a host T cell. In some embodiments, the host T cell further comprises a mutation (e.g., inactivating mutation) in B2M. In some embodiments, the host T cell expresses a CAR. In some embodiments, the method further comprises introducing into the host T cell or the modified T cell a nucleic acid encoding a CAR. In some embodiments, the host T cell is allogeneic.

Also provided are modified T cells (e.g., allogeneic T cells or CAR-T cells (such as allogeneic CAR-T cells) ) comprising a mutation (e.g., inactivating mutation) in a target gene, wherein the target gene is selected from the group consisting of TACC2, HES1, STON1, GJD2, LILRB4, PLS1, KLHL24, FANCB, ARNTL, AMY2A, SIX1, USP17L13, PIK3R6, ATP6V1H, TPM3, OR5H14, TFG, SMAD6, PDE4C, SRRM3, LRRC69, KCNJ13, C8orf34, and PACS2. In some embodiments, the modified T cell further comprises a mutation (e.g., inactivating mutation) in B2M. In some embodiments, the target gene is PSCS2. In some embodiments, the modified T cell further expresses a CAR. In some embodiments, the modified T cell is allogeneic.

Further provided are sgRNA (or sgRNA ^iBAR) libraries comprising one or more sgRNA (or sgRNA ^iBAR) constructs, wherein each sgRNA (or sgRNA ^iBAR) construct comprises or encodes an sgRNA (or sgRNA ^iBAR) , and wherein each sgRNA (or sgRNA ^iBAR) comprises a guide sequence that is complementary (e.g., at least about any of 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%complementary) to a target site in a target gene selected from the group consisting of TACC2, HES1, STON1, GJD2, LILRB4, PLS1, KLHL24, FANCB, ARNTL, AMY2A, SIX1, USP17L13, PIK3R6, ATP6V1H, TPM3, OR5H14, TFG, SMAD6, PDE4C, SRRM3, LRRC69, KCNJ13, C8orf34, and PACS2. In some embodiments, the sgRNA (or sgRNA ^iBAR) library further comprises an sgRNA construct comprising or encoding an sgRNA whose guide sequence is complementary (e.g., at least about any of 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%complementary) to a target site in B2M.

Kits and articles of manufacture that are useful for the methods described herein are also provided, such as kits for generating a modified T cell (e.g., allogeneic T cell or CAR-T cell (such as allogeneic CAR-T cell) ) resistant to the killing of NK cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows exemplary procedure for screening genes related to NK cell killing of T cells.

FIG. 2 shows exemplary screening methods for Cas9 ⁺ B2M ^-sgRNA ^iBAR T cell library.

FIGs. 3A-3D show screening results and top-ranking candidates from Trials 3-6, identifying genes conferring resistant phenotype (positive side) or sensitive phenotype (negative side) to NK cell killing after T cell gene knockout. Top ranking genes with FDR≤0.15 are in dark grey above the dotted line.

FIG. 4 shows Venn diagram of top ranking candidates from various screening trials (FDR≤0.15) .

FIG. 5 shows an exemplary target gene identification workflow for Cas9 ⁺ sgRNA ^iBAR T cell library.

DETAILED DESCRIPTION OF THE PRESENT APPLICATION

The present application provides methods of identifying a target gene in a T cell (e.g., allogeneic T cell or CAR-T cell (such as allogeneic CAR-T cell) ) that modulates the activity of the T cell, such as in response to NK cell treatment. The method comprises: a) providing a T cell library comprising a plurality of T cells, wherein each of the plurality of T cells has a mutation (e.g., inactivating mutation) at a hit gene ( “hit gene mutation” ) in the genome, wherein the hit gene in at least two of the plurality of T cells are different from each other; b) subjecting the T cell library to treatment with NK cells; c) obtaining a T cell from the T cell library that is sensitive or resistant to the killing of the NK cells; and d) identifying the hit gene in the T cell, thereby identifying the target gene in the T cell that modulates the activity of the T cell. In some embodiments, the one or more mutations (e.g., inactivating mutations) at one or more hit genes are generated by CRISPR/Cas guide RNAs (e.g., single-guide RNA) or constructs encoding the CRISPR/Cas guide RNAs (e.g., vector such as viral vector, or virus such as lentivirus) , such as sgRNA comprising an iBAR sequence (sgRNA ^iBAR) described herein. Screening assays employing sgRNA ^iBAR molecules, constructs, sets, or libraries described herein provide a reliable and highly efficient screening strategy for large-scale target identification in eukaryotic cells (e.g., T cells) , with much lower false-positive and false-negative rates, and allow cell library generation using a high MOI. Target genes identified herein, especially those whose mutation (e.g., inactivation) renders T cells higher resistance to killing by NK cells, are particularly useful in adoptive T cell therapy (e.g., CAR-T) . For example, allogeneic T cells (e.g., allogeneic CAR-T cells) can be modified to inactivate one or more target genes identified herein to avoid rejecting reactions from host NK cells.

Thus, the present invention in one aspect provides a method of identifying a target gene in a T cell (e.g., allogeneic T cell or CAR-T cell (such as allogeneic CAR-T cell) ) that modulates the activity of the T cell, comprising: a) providing a T cell library comprising an sgRNA library or an sgRNA ^iBAR library targeting one or more hit genes in the genome (e.g., human whole genome) ; b) subjecting the T cell library to treatment with NK cells; c) obtaining a T cell from the T cell library that is sensitive or resistant to the killing of the NK cells; and d) identifying the hit gene in the T cell, thereby identifying the target gene in the T cell that modulates the activity of the T cell. In some embodiments, the sgRNA library comprises one or a plurality of sgRNA constructs, wherein each sgRNA construct comprises or encodes an sgRNA, and wherein each sgRNA comprises a guide sequence that is complementary (e.g., at least about any of 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%complementary) to a target site in a hit gene in the genome. In some embodiments, the sgRNA ^iBAR library comprises a plurality of sets of sgRNA ^iBAR constructs, wherein each set of sgRNA ^iBAR constructs comprise three or more (e.g., four) sgRNA ^iBAR constructs each comprising or encoding an sgRNA ^iBAR, wherein each sgRNA ^iBAR comprises a guide sequence and an iBAR sequence, wherein the guide sequences for the three or more (e.g., four) sgRNA ^iBAR constructs are the same and are complementary (e.g., at least about any of 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%complementary) to a same target site in the genome, wherein the iBAR sequence for each of the three or more (e.g., four) sgRNA ^iBAR constructs is different from each other, wherein the guide sequence of each set of sgRNA ^iBAR constructs is complementary to a different target site (e.g., different genes, or different sites within the same gene) in the genome, and wherein each sgRNA ^iBAR is operable with a Cas protein (e.g., Cas9) to modify (e.g., cleave or modulate expression) the target site. In some embodiments, the sgRNA library or the sgRNA ^iBAR library is a whole-genome library, i.e., targeting every annotated gene in the genome. In some embodiments, more than one (e.g., 2, 3, 4 or more, such as 2) guide sequence is designed for each hit gene.

Also provided are sgRNA or sgRNA ^iBAR molecules, constructs, sets, or libraries, which are useful for conducting the screening methods described herein. Modified T cells (e.g., allogeneic T cells or CAR-T cells (such as allogeneic CAR-T cells) ) comprising the sgRNA or sgRNA ^iBAR molecules, constructs, sets, or libraries, and methods of generating thereof, are also provided. Further provided are target genes whose mutation (e.g., inactivation such as knock-out) renders T cells higher sensitivity, or higher resistance, to killing by NK cells. sgRNA molecules, constructs, or libraries against target genes whose mutation (e.g., inactivation) renders T cells higher resistance to killing by NK cells, modified T cells (e.g., allogeneic T cells or CAR-T cells (such as allogeneic CAR-T cells) ) comprising thereof, pharmaceutical compositions thereof, and kits, are also provided.

I. Definitions

The present invention will be described with respect to particular embodiments and with reference to certain drawings but the invention is not limited thereto. Any reference signs in the claims shall not be construed as limiting the scope. In the drawings, the size of some of the elements may be exaggerated and not drawn on scale for illustrative purposes. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

As used herein, “internal barcode” or “iBAR” refers to an index inserted into or appended to a molecule, which is useful for tracing the identity and performance of the molecule. The iBAR can be, for example, a short nucleotide sequence inserted in or appended to a guide RNA for a CRISPR/Cas system, as exemplified by the present invention. Multiple iBARs can be used to trace the performance of a single guide RNA sequence within one experiment, thereby providing replicate data for statistical analysis without having to repeat the experiment.

“CRISPR system” or “CRISPR/Cas system” refers collectively to transcripts and other elements involved in the expression and/or directing the activity of CRISPR-associated ( “Cas” ) genes. For example, a CRISPR/Cas system may include sequences encoding a Cas gene, a tracr (trans-activating CRISPR) sequence (e.g., tracrRNA or an active partial tracrRNA) , a tracr-mate sequence (e.g., encompassing a “direct repeat” and a tracrRNA-processed partial direct repeat in an endogenous CRISPR system) , a guide sequence (also referred to as a “spacer” in an endogenous CRISPR system) , and other sequences and transcripts derived from a CRISPR locus.

In the context of formation of a CRISPR complex, “target sequence” refers to a sequence to which a guide sequence is designed to have complementarity, where hybridization between a target sequence and a guide sequence promotes the formation of a CRISPR complex. Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization and promote formation of a CRISPR complex. A target sequence may comprise any polynucleotide, such as DNA or RNA polynucleotides. A CRISPR complex may comprise a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins.

The term “guide sequence” refers to a contiguous sequence of nucleotides in a guide RNA which has partial or complete complementarity to a target sequence in a target polynucleotide and can hybridize to the target sequence by base pairing facilitated by a Cas protein. In a CRISPR/Cas9 system, a target sequence is adjacent to a PAM site. The PAM sequence, and its complementary sequence on the other strand, together constitutes a PAM site.

The terms “single guide RNA, ” “synthetic guide RNA” and “sgRNA” are used interchangeably and refer to a polynucleotide sequence comprising a guide sequence and any other sequence necessary for the function of the sgRNA and/or interaction of the sgRNA with one or more Cas proteins to form a CRISPR complex. In some embodiments, an sgRNA comprises a guide sequence fused to a second sequence comprising a tracr sequence derived from a tracr RNA and a tracr mate sequence derived from a crRNA. A tracr sequence may contain all or part of the sequence from the tracrRNA of a naturally-occurring CRISPR/Cas system. The term “guide sequence” refers to the nucleotide sequence within the guide RNA that specifies the target site and may be used interchangeably with the terms “guide” or “spacer. ” The term “tracr mate sequence” may also be used interchangeably with the term “direct repeat (s) . ” “sgRNA ^iBAR” as used herein refers to a single-guide RNA having an iBAR sequence.

The term “operable with a Cas protein” means that a guide RNA can interact with the Cas protein to form a CRISPR complex.

As used herein the term “wild type” is a term of the art understood by skilled persons and means the typical form of an organism, strain, gene or characteristic as it occurs in nature as distinguished from mutant or variant forms.

As used herein the term “variant” should be taken to mean the exhibition of qualities that have a pattern that deviates from what occurs in nature.

“Complementarity” refers to the ability of a nucleic acid to form hydrogen bond (s) with another nucleic acid sequence by either traditional Watson-Crick base pairing or other non-traditional types. A percent complementarity indicates the percentage of residues in a nucleic acid molecule which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100%complementary) . “Perfectly complementary” means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. “Substantially complementary” as used herein refers to a degree of complementarity that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or more nucleotides, or refers to two nucleic acids that hybridize under stringent conditions.

As used herein, “stringent conditions” for hybridization refer to conditions under which a nucleic acid having complementarity to a target sequence predominantly hybridizes with the target sequence, and substantially does not hybridize to non-target sequences. Stringent conditions are generally sequence-dependent, and vary depending on a number of factors. In general, the longer the sequence, the higher the temperature at which the sequence specifically hybridizes to its target sequence. Non-limiting examples of stringent conditions are described in detail in Tijssen (1993) , Laboratory Techniques In Biochemistry And Molecular Biology-Hybridization With Nucleic Acid Probes Part 1, Second Chapter “Overview of principles of hybridization and the strategy of nucleic acid probe assay” , Elsevier, N.Y.

“Hybridization” refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues. The hydrogen bonding may occur by Watson Crick base pairing, Hoogstein binding, or in any other sequence specific manner. The complex may comprise two strands forming a duplex structure, three or more strands forming a multi stranded complex, a single self-hybridizing strand, or any combination of these. A hybridization reaction may constitute a step in a more extensive process, such as the initiation of PCR, or the cleavage of a polynucleotide by an enzyme. A sequence capable of hybridizing with a given sequence is referred to as the “complement” of the given sequence.

“Construct” as used herein refers to a nucleic acid molecule (e.g., DNA or RNA) , or a vehicle capable of delivering such nucleic acid molecule. For example, when used in the context of an sgRNA, a construct refers to the sgRNA molecule, a nucleic acid molecule (e.g., isolated DNA, or viral vector) encoding the sgRNA, or a vehicle capable of delivering a nucleic acid molecule encoding the sgRNA, such as a lentivirus carrying a nucleic acid molecule encoding the sgRNA. When used in the context of a protein, a construct refers to a nucleic acid molecule comprising a nucleotide sequence that can be transcribed to an RNA or expressed as a protein. A construct may contain necessary regulatory elements operably linked to the nucleotide sequence that allow transcription or expression of the nucleotide sequence when the construct is present in a host cell.

“Operably linked” as used herein means that expression of a gene is under the control of a regulatory element (e.g., a promoter) with which it is spatially connected. A regulatory element may be positioned 5′ (upstream) or 3′ (downstream) to a gene under its control. The distance between the regulatory element (e.g., promoter) and a gene may be approximately the same as the distance between that regulatory element (e.g., promoter) and a gene it naturally controls and from which the regulatory element is derived. As it is known in the art, variation in this distance may be accommodated without loss of function in the regulatory element (e.g., promoter) .

The term “vector” is used to describe a nucleic acid molecule that may be engineered to contain a cloned polynucleotide or polynucleotides that may be propagated in a host cell. Vectors include, but are not limited to, nucleic acid molecules that are single-stranded, double-stranded, or partially double-stranded; nucleic acid molecules that comprise one or more free ends, no free ends (e.g. circular) ; nucleic acid molecules that comprise DNA, RNA, or both; and other varieties of polynucleotides known in the art. One type of vector is a "plasmid, " which refers to a circular double-stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors) . Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as “expression vectors. ” Recombinant expression vectors can comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory elements, which may be selected on basis of the host cells to be used for expression, that is operably linked to the nucleic acid sequence to be expressed.

A “host cell” refers to a cell that may be or has been a recipient of a vector or isolated polynucleotide. Host cells may be prokaryotic cells or eukaryotic cells. In some embodiments, the host cell is a eukaryotic cell that can be cultured in vitro and modified using the methods described herein. The term “cell” includes the primary subject cell and its progeny.

“Multiplicity of infection” or “MOI” are used interchangeably herein to refer to a ratio of agents (e.g., phage, virus, or bacteria) to their infection targets (e.g., cell or organism) . For example, when referring to a group of cells inoculated with viral particles, the multiplicity of infection or MOI is the ratio between the number of viral particles (e.g., viral particles comprising an sgRNA library) and the number of target cells present in a mixture during viral transduction.

A “phenotype” of a cell as used herein refers to an observable characteristic or trait of a cell, such as its morphology, development (e.g., growth, proliferation, differentiation, or death) , biochemical or physiological property, phenology, or behavior. A phenotype may result from expression of genes in a cell, influence from environmental factors, or interactions between the two. In some embodiments, the phenotype is resistance or sensitivity to killing (e.g., by NK cells) . In some embodiments, the phenotype is inhibition of growth or proliferation. In some embodiments, the phenotype is death.

An “isolated” nucleic acid molecule described herein is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the environment in which it was produced. Preferably, the isolated nucleic acid is free of association with all components associated with the production environment. The isolated nucleic acid molecules encoding the polypeptides and antibodies herein is in a form other than in the form or setting in which it is found in nature. Isolated nucleic acid molecules therefore are distinguished from nucleic acid encoding the polypeptides and antibodies herein existing naturally in cells.

Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron (s) .

The term “transfected” or “transformed” or “transduced” as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell (e.g., T cell) . A “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

As used herein, “treatment” or “treating” is an approach for obtaining beneficial or desired results including clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms resulting from the disease, diminishing the extent of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease) , preventing or delaying the spread (e.g., metastasis) of the disease, preventing or delaying the recurrence of the disease, delay or slowing the progression of the disease, ameliorating the disease state, providing a remission (partial or total) of the disease, decreasing the dose of one or more other medications required to treat the disease, delaying the progression of the disease, increasing the quality of life, and/or prolonging survival. Also encompassed by “treatment” is a reduction of pathological consequence of cancer.

As used herein, an “individual” or a “subject” refers to a mammal, including, but not limited to, human, bovine, horse, feline, canine, rodent, or primate. In some embodiments, the individual is a human.

As used herein, the term “autologous” is meant to refer to any material derived from the same individual to whom it is later to be re-introduced into the individual.

“Allogeneic” refers to a graft derived from a different individual of the same species. “Allogeneic T cell” refers to a T cell from a donor having a tissue HLA type that matches the recipient. Typically, matching is performed on the basis of variability at three or more loci of the HLA gene, and a perfect match at these loci is preferred. In some instances allogeneic transplant donors may be related (usually a closely HLA matched sibling) , syngeneic (a monozygotic “identical” twin of the patient) or unrelated (donor who is not related and found to have very close degree of HLA matching) . The HLA genes fall in two categories (Type I and Type II) . In general, mismatches of the Type-I genes (i.e., HLA-A, HLA-B, or HLA-C) increase the risk of graft rejection. A mismatch of an HLA Type II gene (i.e., HLA-DR, or HLA-DQB1) increases the risk of GvHD.

A “patient” as used herein includes any human who is afflicted with a disease (e.g., cancer, or viral infection) . The terms “subject, ” “individual, ” and “patient” are used interchangeably herein. The term “donor subject” or “donor” refers to herein a subject whose cells are being obtained for further in vitro engineering. The donor subject can be a patient that is to be treated with a population of cells generated by the methods described herein (i.e., an autologous donor) , or can be an individual who donates a blood sample (e.g., lymphocyte sample) that, upon generation of the population of cells generated by the methods described herein, will be used to treat a different individual or patient (i.e., an allogeneic donor) . Those subjects who receive the cells that were prepared by the present methods can be referred to as “recipient” or “recipient subject. ”

The term “stimulation” , as used herein, refers to a primary response induced by ligation of a cell surface moiety (e.g., ligand, receptor, or molecule binding to a cell surface moiety) . For example, in the context of receptors, such stimulation entails the ligation of a receptor (e.g., binding of a ligand or molecule to a receptor) and a subsequent signal transduction event. With respect to stimulation of a T cell, such stimulation refers to the ligation of a T cell surface moiety that in one embodiment subsequently induces a signal transduction event, such as binding the TCR/CD3 complex. Further, the stimulation event may activate a cell and upregulate or down-regulate expression or secretion of a molecule, such as down-regulation of TGF-β. Thus, ligation of cell surface moieties, even in the absence of a direct signal transduction event, may result in the reorganization of cytoskeletal structures, or in the coalescing of cell surface moieties, each of which could serve to enhance, modify, or alter subsequent cellular responses.

The term “activation” , as used herein, refers to the state of a cell following sufficient cell surface moiety ligation to induce a noticeable biochemical or morphological change. Within the context of T cells, such activation refers to the state of a T cell that has been sufficiently stimulated to induce cellular proliferation. Activation of a T cell may also induce cytokine production and performance of regulatory or cytolytic effector functions. Within the context of other cells, this term infers either up or down regulation of a particular physico-chemical process. The term “activated T cells” indicates T cells that are currently undergoing cell division, cytokine production, performance of regulatory or cytolytic effector functions, and/or has recently undergone the process of “activation. ”

Where the term “comprising” is used in the present description and claims, it does not exclude other elements or steps.

It is understood that embodiments of the present application described herein include “consisting” and/or “consisting essentially of” embodiments.

Reference to “about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X” .

As used herein, reference to “not” a value or parameter generally means and describes “other than” a value or parameter. For example, the method is not used to treat cancer of type X means the method is used to treat cancer of types other than X.

The term “about X-Y” used herein has the same meaning as “about X to about Y. ”

For the recitation of numeric ranges of nucleotides herein, each intervening number therebetween, is explicitly contemplated. For example, for the range of 19-2 lnt, the number 20nt is contemplated in addition to 19nt and 2 lnt, and for the range of MOI, each intervening number therebetween, whether it is integral or decimal, is explicitly contemplated.

As used herein and in the appended claims, the singular forms “a, ” “or, ” and “the” include plural referents unless the context clearly dictates otherwise.

II. Methods of identifying target genes that modulate the sensitivity or resistance of T cells to the killing by NK cells

The present application provides methods of identifying a target gene in a T cell (e.g., allogeneic T cell or CAR-T cell (such as allogeneic CAR-T cell) ) that modulates the activity of the T cell, such as in response to NK cell treatment. In some embodiments, there is provided a method of identifying a target gene in a T cell that modulates the activity of the T cell, comprising: a) providing a T cell library comprising a plurality of T cells, wherein each of the plurality of T cells has a mutation (e.g., inactivating mutation) at a hit gene in the genome, wherein the hit gene in at least two of the plurality of T cells are different from each other; b) subjecting the T cell library to treatment with NK cells; c) obtaining a T cell from the T cell library that is sensitive or resistant to the killing of the NK cells; and d) identifying the hit gene in the T cell, thereby identifying the target gene in the T cell that modulates the activity of the T cell. In some embodiments, the T cell library is generated by subjecting an initial population of T cells to genome-wide gene editing. In some embodiments, the T cell library is generated by contacting an initial population of T cells (e.g., allogeneic T cells, or CAR-T cells (such as allogeneic CAR-T cells) ) with i) an sgRNA library comprising a plurality of sgRNA constructs, wherein each sgRNA construct comprises or encodes an sgRNA, and wherein each sgRNA comprises a guide sequence that is complementary (e.g., at least about any of 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%complementary) to a target site in the hit gene in the genome; and optionally ii) a Cas component comprising a Cas protein or a nucleic acid encoding the Cas protein, under a condition that allows introduction of the sgRNA constructs and the optional Cas component into the initial population of T cells. In some embodiments, the Cas protein is Cas9. In some embodiments, each sgRNA comprises the guide sequence fused to a second sequence, wherein the second sequence comprises a repeat-anti-repeat stem loop that interacts with the Cas9. In some embodiments, the second sequence of each sgRNA further comprises a stem loop 1, a stem loop 2, and/or a stem loop 3. In some embodiments, each sgRNA further comprises an iBAR sequence ( “sgRNA ^iBAR” ) , wherein each sgRNA ^iBAR is operable with the Cas protein to modify (e.g., cleave or modulate expression) the hit gene. In some embodiments, the iBAR sequence of each sgRNA ^iBAR is inserted in the loop region of the repeat-anti-repeat stem loop. In some embodiments, each sgRNA ^iBAR comprises in the 5’-to-3’ direction a first stem sequence and a second stem sequence, wherein the first stem sequence hybridizes with the second stem sequence to form a double-stranded RNA (dsRNA) region that interacts with the Cas protein, and wherein the iBAR sequence is disposed between the 3’ end of the first stem sequence and the 5’ end of the second stem sequence. In some embodiments, each guide sequence comprises about 17 to about 23 nucleotides.

In some embodiments, the T cell library is generated by contacting an initial population of T cells (e.g., allogeneic T cells or CAR-T cells (such as allogeneic CAR-T cells) ) with i) an sgRNA ^iBAR library comprising a plurality of sets of sgRNA ^iBAR constructs, wherein each set of sgRNA ^iBAR constructs comprise three or more (e.g., four) sgRNA ^iBAR constructs each comprising or encoding an sgRNA ^iBAR, wherein each sgRNA ^iBAR comprises a guide sequence and an iBAR sequence, wherein the guide sequences for the three or more (e.g., four) sgRNA ^iBAR constructs are the same and are complementary (e.g., at least about any of 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%complementary) to a same target site in the genome, wherein the iBAR sequence for each of the three or more (e.g., four) sgRNA ^iBAR constructs is different from each other, wherein the guide sequence of each set of sgRNA ^iBAR constructs is complementary to a different target site (e.g., different hit genes, or different sites within the same hit gene) in the genome, and wherein each sgRNA ^iBAR is operable with a Cas9 protein to modify the target site; and optionally ii) a Cas9 component comprising a Cas9 protein or a nucleic acid encoding the Cas9 protein, under a condition that allows introduction of the sgRNA ^iBAR constructs and the optional Cas component into the initial population of T cells. In some embodiments, the T cell library is generated by contacting an initial population of T cells with i) an sgRNA ^iBAR library comprising a plurality of sets of sgRNA ^iBAR constructs, wherein each set of sgRNA ^iBAR constructs comprise three or more (e.g., four) sgRNA ^iBAR constructs each comprising or encoding an sgRNA ^iBAR, wherein each sgRNA ^iBAR comprises a guide sequence, a second sequence, and an iBAR sequence, wherein the guide sequences for the three or more (e.g., four) sgRNA ^iBAR constructs are the same and are complementary (e.g., at least about any of 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%complementary) to a same target site in the genome, wherein the iBAR sequence for each of the three or more (e.g., four) sgRNA ^iBAR constructs is different from each other, wherein the guide sequence is fused to a second sequence, wherein the second sequence comprises a repeat-anti-repeat stem loop that interacts with a Cas9 protein, wherein the iBAR sequence is inserted in the loop region of the repeat-anti-repeat stem loop, wherein the guide sequence of each set of sgRNA ^iBAR constructs is complementary to a different target site in the genome, and wherein each sgRNA ^iBAR is operable with the Cas9 protein to modify the target site; and optionally ii) a Cas9 component comprising a Cas9 protein or a nucleic acid encoding the Cas9 protein, under a condition that allows introduction of the sgRNA ^iBAR constructs and the optional Cas9 component into the initial population of T cells. In some embodiments, each iBAR sequence comprises about 1 to about 50 nucleotides. In some embodiments, each set of sgRNA ^iBAR constructs comprises four sgRNA ^iBAR constructs, and wherein the iBAR sequence for each of the four sgRNA ^iBAR constructs is different from each other. In some embodiments, the sgRNA ^iBAR library comprises at least about 100 sets of sgRNA ^iBAR constructs. In some embodiments, the iBAR sequences for at least two sgRNA ^iBAR constructs among different sets of sgRNA ^iBAR constructs are the same (e.g., the first set and the second set of sgRNA ^iBAR constructs have at least 1, 2, 3, 4, or more shared iBAR sequences among the two sets of sgRNA ^iBAR constructs) . In some embodiments, the iBAR sequences for at least two sets of sgRNA ^iBAR constructs are the same. In some embodiments, the sgRNA ^iBAR library is contacted with the initial population of T cells at an MOI of more than about 2 (e.g., at least about 3, 5, or 10) . In some embodiments, the sgRNA ^iBAR library comprising a plurality of sgRNA ^iBAR constructs comprises or encodes sgRNA ^iBAR with guide sequences complementary to target sites of every annotated gene in the genome. In some embodiments, at least about 95% (e.g., at least about any of 96%, 97%, 98%, 99%, or more) , such as at least about 99%, of the sgRNA ^iBAR constructs in the sgRNAs ^iBAR library are introduced into the initial population of T cells. In some embodiments, the T cell library has averagely at least about 100-fold (e.g., at least about any of 200-, 400-, 500-, 1,000-, or more fold) coverage for each sgRNA ^iBAR. In some embodiments, the T cell library has averagely at least about 400-fold (e.g., at least about any of 800-, 1000-, 2000-, 4000-, or more fold) coverage for each set of sgRNA ^iBAR. In some embodiments, the T cell library has averagely at least about 100-fold (e.g., at least about any of 200-, 400-, 500-, 1,000-, or more fold) coverage for the sgRNAs ^iBAR library. In some embodiments, the T cell library has averagely at least about 800-fold (e.g., at least about any of 1200-, 1600-, 2000-, 3000-, 4000-, 10,000-, or more fold) coverage for each hit gene. In some embodiments, the T cell library further comprises a B2M mutation (e.g., inactivating B2M mutation) . In some embodiments, the B2M mutation (e.g., inactivating B2M mutation) is generated by contacting the T cell library or the initial population of T cells for generating the T cell library with a B2M sgRNA construct (e.g., a viral vector or a virus) that comprises or encodes a B2M sgRNA, which comprises a guide sequence that is complementary (e.g., at least about any of 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%complementary) to a target site in the B2M gene. In some embodiments, the B2M sgRNA construct is contacted with the T cell library or the initial population of T cells for generating the T cell library at an MOI of more than about 2 (e.g., at least about 3, 5 or 10) .

Screening methods using sgRNAs ^iBAR libraries described herein in some embodiments can improve target identification and data reproducibility by statistical analysis and reduce false discovery rate (FDR) . In conventional CRISPR/Cas-based screening methods using a pooled sgRNA library, a high-quality cell library expressing gRNAs are generated using a low MOI during cell library construction to ensure that each cell harbors on average less than one sgRNA or paired guide RNA ( “pgRNA” ) . Because the sgRNA molecules in a library are randomly integrated in the transfected cells, a sufficiently low MOI ensures that each cell expresses a single sgRNA, thereby minimizing the FDR of the screen. To further reduce FDR and increase data reproducibility, in-depth coverage of gRNAs and multiple biological replicates are often necessary to obtain hit genes with high statistical significance. The conventional screen methods face difficulties when a large number of genome-wide screens are needed, when cell materials for library construction are limited, or when one conducts more challenging screens (i.e., in vivo screen) for which it is difficult to arrange the experimental replications or control the MOI. The screening methods using sgRNA ^iBAR libraries described herein overcome the difficulties by including an iBAR sequence in each sgRNA, which enables collection of internal replicates within each sgRNA set having the same guide sequence but different iBAR sequences. Such iBAR method can reduce experimental noise. For example, an iBAR with four nucleotides for each sgRNA, as demonstrated in WO2020125762, can provide sufficient internal replicates to evaluate data consistency among different sgRNA ^iBAR constructs targeting the same genomic locus. The high level of consistency between the two independent experiments in WO2020125762 indicates that one experimental replicate is sufficient for CRISPR/Cas screens using the iBAR method. Because library coverage is significantly increased with a high MOI during viral transduction of host cells, the cell number in the initial cell population could be reduced more than 20-fold to reach the same library coverage, as demonstrated in the constructed genome-wide human library in WO2020125762. By the same token, workload for each genome-wide screen using sgRNA ^iBAR can be reduced proportionally. Using sgRNAs with different iBAR sequences, one could then trace the performance of each guide sequence multiple times within the same experiment by counting both the guide sequence and the corresponding iBAR nucleotide sequences, thereby drastically reducing FDR, and increasing efficiency and liability. Transduction efficiency and library coverage could be further increased, a high viral titer is used during the viral transduction step, for example, with MOI ＞1 (e.g., MOI＞1.5, MOI ＞2, MOI ＞2.5, MOI ＞3, MOI ＞3.5, MOI ＞4, MOI ＞4.5, MOI ＞5, MOI ＞5.5, MOI ＞6, MOI ＞6.5, MOI ＞7, MOI ＞7.5, MOI ＞8, MOI ＞8.5, MOI ＞9, MOI ＞9.5 or MOI ＞10; such as, MOI is about any of 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10) .

In some embodiments, there is provided a method of identifying a target gene in a T cell (e.g., allogeneic T cell or CAR-T cell (such as allogeneic CAR-T cell) ) that modulates the activity of the T cell, comprising: a) providing a T cell library comprising an sgRNAs ^iBAR library described herein targeting one or more hit genes in the genome; b) subjecting the T cell library to treatment with NK cells; c) obtaining a T cell from the T cell library that is sensitive or resistant to the killing of the NK cells; and d) identifying the hit gene in the T cell, thereby identifying the target gene in the T cell that modulates the activity of the T cell. In some embodiments, there is provided a method of identifying a target gene in a T cell that modulates the activity of the T cell, comprising: a) providing a T cell library comprising an sgRNAs ^iBAR library described herein targeting one or more hit genes in the genome; b) subjecting the T cell library to treatment with NK cells; c) obtaining a T cell from the T cell library that is sensitive or resistant to the killing of the NK cells; and d) identifying the hit gene in the T cell, thereby identifying the target gene in the T cell that modulates the activity of the T cell; wherein the treatment with NK cells comprises: i) an initial treatment step comprising contacting the T cell library with the NK cells; ii) an optional first enrichment step comprising sorting the mixture of treated cells to obtain a first T cell subpopulation that is sensitive or resistant to the killing of the NK cells; iii) an optional first recovery step comprising culturing the first T cell subpopulation; and iv) an optional second treatment step comprising contacting the first T cell subpopulation with the NK cells; and/or wherein obtaining the T cell from the T cell library that is sensitive or resistant to the killing of the NK cells comprises: i) a sorting step comprising sorting the cells obtained from step b) to obtain a second T cell subpopulation that is sensitive or resistant to the killing of the NK cells; and ii) an optional second recovery step comprising culturing the second T cell subpopulation before harvesting the cells. In some embodiments, the sgRNAs ^iBAR library targets every annotated gene in the genome (i.e., the sgRNAs ^iBAR library is a whole-genome sgRNAs ^iBAR library) . In some embodiments, the T cell library has averagely at least about 100-fold (e.g., at least about 400-fold) coverage for the whole-genome sgRNAs ^iBAR library. In some embodiments, identifying the hit gene in the T cell obtained from step c) comprises: i) identifying the sgRNAs ^iBAR sequence in the T cell obtained from step c) ; and ii) identifying the hit gene corresponding to the guide sequence of the sgRNAs ^iBAR. In some embodiments, identifying the target gene comprises: i) obtaining sgRNA ^iBAR sequences in the final T cell subpopulation obtained from step c) ; ii) ranking the corresponding guide sequences of the sgRNA ^iBAR sequences based on sequence counts, wherein the ranking comprises adjusting the rank of each guide sequence based on data consistency among the iBAR sequences in the sgRNA ^iBAR sequences corresponding to the guide sequence; and iii) identifying the hit gene corresponding to a guide sequence ranked above a predetermined threshold level. In some embodiments, the method is a positive screening. In some embodiments, the method is a negative screening. In some embodiments, the sequence counts obtained from the final T cell subpopulation obtained from step c) are compared to corresponding sequence counts obtained from a subpopulation of control T cells to provide fold changes (e.g., actual fold changes, or derivatives of fold changes such as log2 or log 10 fold changes) . In some embodiments, the subpopulation of control T cells are obtained from a same T cell library cultured in the same condition without subjected to treatment with NK cells, and optionally subjected to the same obtaining method in step c) . In some embodiments, the T cell library further comprises a B2M mutation (e.g., inactivating B2M mutation) . In some embodiments, the B2M mutation (e.g., inactivating B2M mutation) is generated by contacting the T cell library or the initial population of T cells for generating the T cell library with a B2M sgRNA construct (e.g., a viral vector or a virus) described herein. In some embodiments, subjecting the T cell library to treatment with NK cells in step b) comprises growing the T cell library in the presence of the NK cells.

In some embodiments, there is provided a method of identifying a target gene in a T cell (e.g., allogeneic T cell or CAR-T cell (such as allogeneic CAR-T cell) ) that modulates the activity of the T cell, comprising: a) providing a T cell library comprising a plurality of T cells, wherein each of the plurality of T cells has a mutation (e.g., inactivating mutation) at a hit gene in the genome, wherein the hit gene in at least two of the plurality of T cells are different from each other; b) subjecting the T cell library to treatment with NK cells; c) obtaining a T cell (or a post-treatment T cell population) from the T cell library that is sensitive or resistant to the killing of the NK cells; and d) identifying the target gene based on the difference between profiles of the hit gene mutations in the obtained T cell (or the post-treatment T cell population) from step c) and a control T cell (or a control T cell population) . In some embodiments, the control T cell (or control T cell population) are obtained from a same T cell library cultured in the same condition without subjected to treatment with NK cells, and optionally subjected to the same obtaining method in step c) . In some embodiments, subjecting the T cell library to treatment with NK cells comprises growing the T cell library in the presence of the NK cells. In some embodiments, the profiles of the hit gene mutations in the obtained T cell (or post-treatment T cell population) from step c) and the control T cell (or control T cell population) are identified by next generation sequencing. In some embodiments, the T cell library is subjected to two or more (e.g., 2, 3, 4, or more) separate different treatments with NK cells and/or obtaining methods in steps b) and c) , and the target genes are identified based on difference between profiles from each treatment. In some embodiments, the sequence counts comprising the hit gene mutations are subject to median ratio normalization followed by mean-variance modeling. In some embodiments, the method comprises comparing sequence counts comprising the hit gene mutations obtained from the obtained T cell (or post-treatment T cell population) from step c) with sequence counts comprising the hit gene mutations obtained from the control T cell (or control T cell population) , wherein i) the hit genes whose corresponding mutations are identified as depleted in the obtained T cell (or post-treatment T cell population) from step c) resistant to the killing of the NK cells in at least one treatment with NK cells with FDR ≤ 0.01 or in at least two separate different treatments with NK cells with FDR ≤ 0.05 (and/or with at least about 2-fold depletion, such as at least about any of 3-, 4-, 5-, 10-, 20-, 50-, 100-fold, or more depletion) , are identified as target genes whose mutations make the T cells sensitive to NK cell killing; ii) the hit genes whose corresponding mutations are identified as enriched in the obtained T cell (or post-treatment T cell population) from step c) resistant to the killing of the NK cells in at least one treatment with NK cells with FDR ≤ 0.05, or in at least two separate different treatments with NK cells with FDR ≤ 0.15 (and/or with at least about 2-fold enrichment, such as at least about any of 3-, 4-, 5-, 10-, 20-, 50-, 100-fold, or more enrichment) , are identified as target genes whose mutations make the T cells resistant to NK cell killing; iii) the hit genes whose corresponding mutations are identified as depleted in the obtained T cell (or post-treatment T cell population) from step c) sensitive to the killing of the NK cells in at least one treatment with NK cells with FDR ≤ 0.05, or in at least two separate different treatments with NK cells with FDR ≤ 0.15 (and/or with at least about 2-fold depletion, such as at least about any of 3-, 4-, 5-, 10-, 20-, 50-, 100-fold, or more depletion) , are identified as target genes whose mutations make the T cells resistant to NK cell killing; and/or iv) the hit genes whose corresponding mutations are identified as enriched in the obtained T cell (or post-treatment T cell population) from step c) sensitive to the killing of the NK cells in at least one treatment with NK cells with FDR ≤ 0.01, or in at least two separate different treatments with NK cells with FDR ≤ 0.05 (and/or with at least about 2-fold enrichment, such as at least about any of 3-, 4-, 5-, 10-, 20-, 50-, 100-fold, or more enrichment) , are identified as target genes whose mutation make the T cells sensitive to NK cell killing.

In some embodiments, there is provided a method of identifying a target gene in a T cell (e.g., allogeneic T cell or CAR-T cell (such as allogeneic CAR-T cell) ) that modulates the activity of the T cell, comprising: a) providing a T cell library comprising a plurality of T cells, wherein each of the plurality of T cells has a mutation (e.g., inactivating mutation) at a hit gene in the genome, wherein the hit gene in at least two of the plurality of T cells are different from each other, wherein the T cell library is generated by contacting an initial population of T cells with i) an sgRNA library comprising a plurality of sgRNA constructs, wherein each sgRNA construct comprises or encodes an sgRNA, and wherein each sgRNA comprises a guide sequence that is complementary to a target site in a corresponding hit gene; and ii) a Cas component comprising a Cas protein (e.g., Cas9) or a nucleic acid encoding the Cas protein, under a condition that allows introduction of the sgRNA constructs and the Cas component into the initial population of T cells and generation of the mutations at the hit genes; b) subjecting the T cell library to treatment with NK cells; c) obtaining a T cell (or a post-treatment T cell population) from the T cell library that is sensitive or resistant to the killing of the NK cells; and d) identifying the target gene based on the difference between profiles of sgRNAs or hit gene mutations in the obtained T cell (or the post-treatment T cell population) from step c) and a control T cell (or a control T cell population) . In some embodiments, the sgRNA library and the Cas component are introduced into the initial population of T cells sequentially. In some embodiments, the control T cell (or control T cell population) are obtained from a same T cell library cultured in the same condition without subjected to treatment with NK cells, and optionally subjected to the same obtaining method in step c) . In some embodiments, subjecting the T cell library to treatment with NK cells comprises growing the T cell library in the presence of the NK cells. In some embodiments, the profiles of sgRNAs or hit gene mutations in the obtained T cell (or post-treatment T cell population) from step c) and the control T cell (or control T cell population) are identified by next generation sequencing. In some embodiments, the T cell library is subjected to two or more (e.g., 2, 3, 4, or more) separate different treatments with NK cells and/or obtaining methods in steps b) and c) , and the target genes are identified based on difference between profiles from each treatment. In some embodiments, the sequence counts comprising the sgRNAs or hit gene mutations are subject to median ratio normalization followed by mean-variance modeling. In some embodiments, the method comprises comparing sequence counts comprising the sgRNAs or hit gene mutations obtained from the obtained T cell (or post-treatment T cell population) from step c) with sequence counts comprising the sgRNAs or hit gene mutations obtained from the control T cell (or control T cell population) , wherein i) the hit genes whose corresponding sgRNA guide sequences or mutations are identified as depleted in the obtained T cell (or post-treatment T cell population) from step c) resistant to the killing of the NK cells in at least one treatment with NK cells with FDR ≤ 0.01, or in at least two separate different treatments with NK cells with FDR ≤ 0.05 (and/or with at least about 2-fold depletion, such as at least about any of 3-, 4-, 5-, 10-, 20-, 50-, 100-fold, or more depletion) , are identified as target genes whose mutations make the T cells sensitive to NK cell killing; ii) the hit genes whose corresponding sgRNA guide sequences or mutations are identified as enriched in the obtained T cell (or post-treatment T cell population) from step c) resistant to the killing of the NK cells in at least one treatment with NK cells with FDR ≤ 0.05, or in at least two separate different treatments with NK cells with FDR ≤ 0.15 (and/or with at least about 2-fold enrichment, such as at least about any of 3-, 4-, 5-, 10-, 20-, 50-, 100-fold, or more enrichment) , are identified as target genes whose mutations make the T cells resistant to NK cell killing; iii) the hit genes whose corresponding sgRNA guide sequences or mutations are identified as depleted in the obtained T cell (or post-treatment T cell population) from step c) sensitive to the killing of the NK cells in at least one treatment with NK cells with FDR ≤ 0.05, or in at least two separate different treatments with NK cells with FDR ≤ 0.15 (and/or with at least about 2-fold depletion, such as at least about any of 3-, 4-, 5-, 10-, 20-, 50-, 100-fold, or more depletion) , are identified as target genes whose mutations make the T cells resistant to NK cell killing; and/or iv) the hit genes whose corresponding sgRNA guide sequences or mutations are identified as enriched in the obtained T cell (or post-treatment T cell population) from step c) sensitive to the killing of the NK cells in at least one treatment with NK cells with FDR ≤ 0.01, or in at least two separate different treatments with NK cells with FDR ≤ 0.05 (and/or with at least about 2-fold enrichment, such as at least about any of 3-, 4-, 5-, 10-, 20-, 50-, 100-fold, or more enrichment) , are identified as target genes whose mutation make the T cells sensitive to NK cell killing.

In some embodiments, there is provided a method of identifying a target gene in a T cell (e.g., allogeneic T cell or CAR-T cell (such as allogeneic CAR-T cell) ) that modulates the activity of the T cell, comprising: a) providing a T cell library comprising a plurality oft cells, wherein each of the plurality of T cells has a mutation (e.g., inactivating mutation) at a hit gene in the genome, wherein the hit gene in at least two of the plurality of T cells are different from each other, wherein the T cell library is generated by contacting an initial population of T cells with i) an sgRNA ^iBAR library comprising a plurality of sets of sgRNA ^iBAR constructs, wherein each set of sgRNA ^iBAR constructs comprise three or more (e.g., 4) sgRNA ^iBAR constructs each comprising or encoding an sgRNA ^iBAR, wherein each sgRNA ^iBAR comprises a guide sequence that is complementary to a target site in a corresponding hit gene, wherein the guide sequences for the three or more (e.g., 4) sgRNA ^iBAR constructs are the same, wherein the iBAR sequence for each of the three or more (e.g., 4) sgRNA ^iBAR constructs is different from each other, and wherein the guide sequence of each set of sgRNA ^iBAR constructs is complementary to a different target site in the hit gene; and ii) a Cas component comprising a Cas protein (e.g., Cas9) or a nucleic acid encoding the Cas protein, under a condition that allows introduction of the sgRNA ^iBAR constructs and the Cas component into the initial population of T cells and generation of the mutations at the hit genes; b) subjecting the T cell library to treatment with NK cells; c) obtaining a T cell (or a post-treatment T cell population) from the T cell library that is sensitive or resistant to the killing of the NK cells; and d) identifying the target gene based on the difference between profiles of sgRNAs or hit gene mutations in the obtained T cell (or the post-treatment T cell population) from step c) and a control T cell (or a control T cell population) . In some embodiments, the sgRNA ^iBAR library and the Cas component are introduced into the initial population of T cells sequentially. In some embodiments, the iBAR sequence of each sgRNA ^iBAR is inserted in the loop region of the repeat-anti-repeat stem loop. In some embodiments, the control T cell (or control T cell population) are obtained from a same T cell library cultured in the same condition without subjected to treatment with NK cells, and optionally subjected to the same obtaining method in step c) . In some embodiments, subjecting the T cell library to treatment with NK cells comprises growing the T cell library in the presence of the NK cells. In some embodiments, the profiles of sgRNA ^iBAR or hit gene mutations in the obtained T cell (or post-treatment T cell population) from step c) and the control T cell (or control T cell population) are identified by next generation sequencing. In some embodiments, the T cell library is subjected to two or more (e.g., 2, 3, 4, or more) separate different treatments with NK cells and/or obtaining methods in steps b) and c) , and the target genes are identified based on difference between profiles from each treatment. In some embodiments, the sequence counts comprising the sgRNAs ^iBAR or hit gene mutations are subject to median ratio normalization followed by mean-variance modeling. In some embodiments, the variance of each guide sequence is adjusted based on data consistency among the iBAR sequences in the sgRNA ^iBAR sequences corresponding to the guide sequence. In some embodiments, the data consistency among the iBAR sequences in the sgRNA ^iBAR sequences corresponding to each guide sequence is determined based on the direction of the fold change of each iBAR sequence, wherein the variance of the guide sequence is increased if the fold changes of the iBAR sequences are in different directions with respect to each other. In some embodiments, the method comprises comparing sequence counts comprising the sgRNAs ^iBAR or hit gene mutations obtained from the obtained T cell (or post-treatment T cell population) from step c) with sequence counts comprising the sgRNAs ^iBAR or hit gene mutations obtained from the control T cell (or control T cell population) , wherein i) the hit genes whose corresponding sgRNAs ^iBAR guide sequences or mutations are identified as depleted in the obtained T cell (or post-treatment T cell population) from step c) resistant to the killing of the NK cells in at least one treatment with NK cells with FDR ≤ 0.01, or in at least two separate different treatments with NK cells with FDR ≤ 0.05 (and/or with at least about 2-fold depletion, such as at least about any of 3-, 4-, 5-, 10-, 20-, 50-, 100-fold, or more depletion) , are identified as target genes whose mutations make the T cells sensitive to NK cell killing; ii) the hit genes whose corresponding sgRNAs ^iBAR guide sequences or mutations are identified as enriched in the obtained T cell (or post-treatment T cell population) from step c) resistant to the killing of the NK cells in at least one treatment with NK cells with FDR ≤ 0.05, or in at least two separate different treatments with NK cells with FDR ≤ 0.15 (and/or with at least about 2-fold enrichment, such as at least about any of 3-, 4-, 5-, 10-, 20-, 50-, 100-fold, or more enrichment) , are identified as target genes whose mutations make the T cells resistant to NK cell killing; iii) the hit genes whose corresponding sgRNAs ^iBAR guide sequences or mutations are identified as depleted in the obtained T cell (or post-treatment T cell population) from step c) sensitive to the killing of the NK cells in at least one treatment with NK cells with FDR ≤ 0.05, or in at least two separate different treatments with NK cells with FDR ≤ 0.15 (and/or with at least about 2-fold depletion, such as at least about any of 3-, 4-, 5-, 10-, 20-, 50-, 100-fold, or more depletion) , are identified as target genes whose mutations make the T cells resistant to NK cell killing; and/or iv) the hit genes whose corresponding sgRNAs ^iBAR guide sequences or mutations are identified as enriched in the obtained T cell (or post-treatment T cell population) from step c) sensitive to the killing of the NK cells in at least one treatment with NK cells with FDR ≤ 0.01, or in at least two separate different treatments with NK cells with FDR ≤0.05 (and/or with at least about 2-fold enrichment, such as at least about any of 3-, 4-, 5-, 10-, 20-, 50-, 100-fold, or more enrichment) , are identified as target genes whose mutation make the T cells sensitive to NK cell killing.

In some embodiments, there is provided a method of identifying a target gene in a T cell (e.g., allogeneic T cell or CAR-T cell (such as allogeneic CAR-T cell) ) that modulates the activity of the T cell, comprising: a) providing a T cell library comprising an sgRNAs ^iBAR library described herein targeting one or more hit genes in the genome; b) subjecting the T cell library to treatment with NK cells; c) obtaining a T cell from the T cell library that is resistant to the killing of the NK cells; and d) identifying the hit gene in the T cell obtained from step c) , thereby identifying the target gene in the T cell that modulates the activity of the T cell; wherein steps b) and c) comprise: i) an initial treatment step comprising contacting the T cell library with the NK cells for about 72 hours at a ratio of NK cells to T cells of about 0.5∶1; ii) an enrichment step comprising sorting the mixture of treated cells that are T cells (e.g., B2M-negative (or deficient) or CD3+) and alive, thus obtaining a first T cell subpopulation that is resistant to the killing of the NK cells; iii) a recovery step comprising culturing the first T cell subpopulation for about 48 hours; iv) a second treatment step comprising contacting the first T cell subpopulation post-recovery with the NK cells for about 96 hours at a ratio of NK cells to T cells of about 0.3∶1; and v) a sorting step comprising sorting the final mixture of treated cells that are T cells (e.g., B2M-negative (or deficient) or CD3+) and alive, thus obtaining a second T cell subpopulation that is resistant to the killing of the NK cells. In some embodiments, identifying the hit gene in the T cell obtained from step c) comprises: i) identifying the sgRNAs ^iBAR sequence in the T cell obtained from step c) ; and ii) identifying the hit gene corresponding to the guide sequence of the sgRNAs ^iBAR. In some embodiments, there is provided a method of identifying a target gene in a T cell that modulates the activity of the T cell, comprising: a) providing a T cell library comprising an sgRNAs ^iBAR library described herein targeting one or more hit genes in the genome; b) subjecting the T cell library to treatment with NK cells; c) obtaining a T cell from the T cell library that is resistant to the killing of the NK cells; wherein steps b) and c) comprise: i) an initial treatment step comprising contacting the T cell library with the NK cells for about 72 hours at a ratio of NK cells to T cells of about 0.5∶1; ii) an enrichment step comprising sorting the mixture of treated cells that are T cells (e.g., B2M-negative (or deficient) or CD3+) and alive, thus obtaining a first T cell subpopulation that is resistant to the killing of the NK cells; iii) a recovery step comprising culturing the first T cell subpopulation for about 48 hours; iv) a second treatment step comprising contacting the first T cell subpopulation post-recovery with the NK cells for about 96 hours at a ratio of NK cells to T cells of about 0.3∶1; and v) a sorting step comprising sorting the final mixture of treated cells that are T cells (e.g., B2M-negative (or deficient) or CD3+) and alive, thus obtaining a second T cell subpopulation that is resistant to the killing of the NK cells; and d) identifying the target gene in the T cell that modulates the activity of the T cell, wherein identifying the target gene comprises: i) obtaining sgRNA ^iBAR sequences in the final T cell subpopulation obtained from step c) ; ii) ranking the corresponding guide sequences of the sgRNA ^iBAR sequences based on sequence counts, wherein the ranking comprises adjusting the rank of each guide sequence based on data consistency among the iBAR sequences in the sgRNA ^iBAR sequences corresponding to the guide sequence; and iii) identifying the hit gene corresponding to a guide sequence ranked above a predetermined threshold level. In some embodiments, the sgRNAs ^iBAR library targets every annotated gene in the genome. In some embodiments, the T cell library has averagely at least about 100-fold (e.g., at least about 400-fold) coverage for the whole-genome sgRNAs ^iBAR library. In some embodiments, the method is a positive screening. In some embodiments, the method is a negative screening. In some embodiments, the sequence counts are subject to median ratio normalization followed by mean-variance modeling. In some embodiments, the variance of each guide sequence is adjusted based on data consistency among the iBAR sequences in the sgRNA ^iBAR sequences corresponding to the guide sequence. In some embodiments, the data consistency among the iBAR sequences in the sgRNA ^iBAR sequences corresponding to each guide sequence is determined based on the direction of the fold change of each iBAR sequence, wherein the variance of the guide sequence is increased if the fold changes of the iBAR sequences are in different directions with respect to each other (e.g., increased vs. reduced, increased vs. unchanged, or reduced vs. unchanged) . In some embodiments, the sequence counts obtained from the final T cell subpopulation obtained from step c) are compared to corresponding sequence counts obtained from a subpopulation of control T cells to provide fold changes (e.g., actual fold changes, or derivatives of fold changes such as log2 or log 10 fold changes) . In some embodiments, the subpopulation of control T cells are obtained from a same T cell library cultured in the same condition without subjected to treatment with NK cells, and optionally subjected to the same obtaining method in step c) . In some embodiments, the T cell library further comprises a B2M mutation (e.g., inactivating B2M mutation) . In some embodiments, the B2M mutation (e.g., inactivating B2M mutation) is generated by contacting the T cell library or the initial population of T cells for generating the T cell library with a B2M sgRNA construct (e.g., a viral vector or a virus) described herein. In some embodiments, subjecting the T cell library to treatment with NK cells in step b) comprises growing the T cell library in the presence of the NK cells.

In some embodiments, there is provided a method of identifying a target gene in a T cell (e.g., allogeneic T cell or CAR-T cell (such as allogeneic CAR-T cell) ) that modulates the activity of the T cell, comprising: a) providing a T cell library comprising an sgRNAs ^iBAR library described herein targeting one or more hit genes in the genome; b) subjecting the T cell library to treatment with NK cells; c) obtaining a T cell from the T cell library that is resistant to the killing of the NK cells; and d) identifying the hit gene in the T cell obtained from step c) , thereby identifying the target gene in the T cell that modulates the activity of the T cell; wherein steps b) and c) comprise: i) a treatment step comprising contacting the T cell library with the NK cells for about 10 days at a ratio of NK cells to T cells of about 0.5∶1; and ii) a sorting step comprising sorting the mixture of treated cells that are T cells (e.g., B2M-negative (or deficient) or CD3+) and alive, thus obtaining a T cell subpopulation that is resistant to the killing of the NK cells. In some embodiments, identifying the hit gene in the T cell obtained from step c) comprises: i) identifying the sgRNAs ^iBAR sequence in the T cell obtained from step c) ; and ii) identifying the hit gene corresponding to the guide sequence of the sgRNAs ^iBAR. In some embodiments, there is provided a method of identifying a target gene in a T cell that modulates the activity of the T cell, comprising: a) providing a T cell library comprising an sgRNAs ^iBAR library described herein targeting one or more hit genes in the genome; b) subjecting the T cell library to treatment with NK cells; c) obtaining a T cell from the T cell library that is resistant to the killing of the NK cells; wherein steps b) and c) comprise: i) a treatment step comprising contacting the T cell library with the NK cells for about 10 days at a ratio of NK cells to T cells of about 0.5∶1; and ii) a sorting step comprising sorting the mixture of treated cells that are T cells (e.g., B2M-negative (or deficient) or CD3+) and alive, thus obtaining a T cell subpopulation that is resistant to the killing of the NK cells; and d) identifying the target gene in the T cell that modulates the activity of the T cell, wherein identifying the target gene comprises: i) obtaining sgRNA ^iBAR sequences in the final T cell subpopulation obtained from step c) ; ii) ranking the corresponding guide sequences of the sgRNA ^iBAR sequences based on sequence counts, wherein the ranking comprises adjusting the rank of each guide sequence based on data consistency among the iBAR sequences in the sgRNA ^iBAR sequences corresponding to the guide sequence; and iii) identifying the hit gene corresponding to a guide sequence ranked above a predetermined threshold level. In some embodiments, the sgRNAs ^iBAR library targets every annotated gene in the genome. In some embodiments, the T cell library has averagely at least about 100-fold (e.g., at least about 400-fold) coverage for the whole-genome sgRNAs ^iBAR library. In some embodiments, the method is a positive screening. In some embodiments, the method is a negative screening. In some embodiments, the sequence counts are subject to median ratio normalization followed by mean- variance modeling. In some embodiments, the variance of each guide sequence is adjusted based on data consistency among the iBAR sequences in the sgRNA ^iBAR sequences corresponding to the guide sequence. In some embodiments, the data consistency among the iBAR sequences in the sgRNA ^iBAR sequences corresponding to each guide sequence is determined based on the direction of the fold change of each iBAR sequence, wherein the variance of the guide sequence is increased if the fold changes of the iBAR sequences are in different directions with respect to each other (e.g., increased vs. reduced, increased vs. unchanged, or reduced vs. unchanged) . In some embodiments, the sequence counts obtained from the final T cell subpopulation obtained from step c) are compared to corresponding sequence counts obtained from a subpopulation of control T cells to provide fold changes (e.g., actual fold changes, or derivatives of fold changes such as log2 or log 10 fold changes) . In some embodiments, the subpopulation of control T cells are obtained from a same T cell library cultured in the same condition without subjected to treatment with NK cells, and optionally subjected to the same obtaining method in step c) . In some embodiments, the T cell library further comprises a B2M mutation (e.g., inactivating B2M mutation) . In some embodiments, the B2M mutation (e.g., inactivating B2M mutation) is generated by contacting the T cell library or the initial population of T cells for generating the T cell library with a B2M sgRNA construct (e.g., a viral vector or a virus) described herein. In some embodiments, subjecting the T cell library to treatment with NK cells in step b) comprises growing the T cell library in the presence of the NK cells.

In some embodiments, there is provided a method of identifying a target gene in a T cell (e.g., allogeneic T cell or CAR-T cell (such as allogeneic CAR-T cell) ) that modulates the activity of the T cell, comprising: a) providing a T cell library comprising an sgRNAs ^iBAR library described herein targeting one or more hit genes in the genome; b) subjecting the T cell library to treatment with NK cells; c) obtaining a T cell from the T cell library that is resistant to the killing of the NK cells; and d) identifying the hit gene in the T cell obtained from step c) , thereby identifying the target gene in the T cell that modulates the activity of the T cell; wherein steps b) and c) comprise: i) a treatment step comprising contacting the T cell library with the NK cells for about 48 hours at a ratio of NK cells to T cells of about 1∶1; ii) a sorting step comprising sorting the mixture of treated cells that are T cells (e.g., B2M-negative (or deficient) or CD3+) and alive, thus obtaining a T cell subpopulation that is resistant to the killing of the NK cells; and iii) a recovery step comprising culturing the T cell subpopulation for about 48 hours before harvesting the cells. In some embodiments, identifying the hit gene in the T cell obtained from step c) comprises: i) identifying the sgRNAs ^iBAR sequence in the T cell obtained from step c) ; and ii) identifying the hit gene corresponding to the guide sequence of the sgRNAs ^iBAR. In some embodiments, there is provided a method of identifying a target gene in a T cell that modulates the activity of the T cell, comprising: a) providing a T cell library comprising an sgRNAs ^iBAR library described herein targeting one or more hit genes in the genome; b) subjecting the T cell library to treatment with NK cells; c) obtaining a T cell from the T cell library that is resistant to the killing of the NK cells; wherein steps b) and c) comprise: i) a treatment step comprising contacting the T cell library with the NK cells for about 48 hours at a ratio of NK cells to T cells of about 1∶1; ii) a sorting step comprising sorting the mixture of treated cells that are T cells (e.g., B2M-negative (or deficient) or CD3+) and alive, thus obtaining a T cell subpopulation that is resistant to the killing of the NK cells; and iii) a recovery step comprising culturing the T cell subpopulation for about 48 hours before harvesting the cells; and d) identifying the target gene in the T cell that modulates the activity of the T cell, wherein identifying the target gene comprises: i) obtaining sgRNA ^iBAR sequences in the final T cell subpopulation obtained from step c) ; ii) ranking the corresponding guide sequences of the sgRNA ^iBAR sequences based on sequence counts, wherein the ranking comprises adjusting the rank of each guide sequence based on data consistency among the iBAR sequences in the sgRNA ^iBAR sequences corresponding to the guide sequence; and iii) identifying the hit gene corresponding to a guide sequence ranked above a predetermined threshold level. In some embodiments, the sgRNAs ^iBAR library targets every annotated gene in the genome. In some embodiments, the T cell library has averagely at least about 100-fold (e.g., at least about 400-fold) coverage for the whole-genome sgRNAs ^iBAR library. In some embodiments, the method is a positive screening. In some embodiments, the method is a negative screening. In some embodiments, the sequence counts are subject to median ratio normalization followed by mean-variance modeling. In some embodiments, the variance of each guide sequence is adjusted based on data consistency among the iBAR sequences in the sgRNA ^iBAR sequences corresponding to the guide sequence. In some embodiments, the data consistency among the iBAR sequences in the sgRNA ^iBAR sequences corresponding to each guide sequence is determined based on the direction of the fold change of each iBAR sequence, wherein the variance of the guide sequence is increased if the fold changes of the iBAR sequences are in different directions with respect to each other (e.g., increased vs. reduced, increased vs. unchanged, or reduced vs. unchanged) . In some embodiments, the sequence counts obtained from the final T cell subpopulation obtained from step c) are compared to corresponding sequence counts obtained from a subpopulation of control T cells to provide fold changes (e.g., actual fold changes, or derivatives of fold changes such as log2 or log 10 fold changes) . In some embodiments, the subpopulation of control T cells are obtained from a same T cell library cultured in the same condition without subjected to treatment with NK cells, and optionally subjected to the same obtaining method in step c) . In some embodiments, the T cell library further comprises a B2M mutation (e.g., inactivating B2M mutation) . In some embodiments, the B2M mutation (e.g., inactivating B2M mutation) is generated by contacting the T cell library or the initial population of T cells for generating the T cell library with a B2M sgRNA construct (e.g., a viral vector or a virus) described herein. In some embodiments, subjecting the T cell library to treatment with NK cells in step b) comprises growing the T cell library in the presence of the NK cells.

In some embodiments, there is provided a method of identifying a target gene in a T cell (e.g., allogeneic T cell or CAR-T cell (such as allogeneic CAR-T cell) ) that modulates the activity of the T cell, comprising: a) providing a T cell library comprising an sgRNAs ^iBAR library described herein targeting one or more hit genes in the genome; b) subjecting the T cell library to treatment with NK cells; c) obtaining a T cell from the T cell library that is resistant to the killing of the NK cells; and d) identifying the hit gene in the T cell obtained from step c) , thereby identifying the target gene in the T cell that modulates the activity of the T cell; wherein steps b) and c) comprise: i) a treatment step comprising contacting the T cell library with the NK cells for about 48 hours at a ratio of NK cells to T cells of about 1∶1; ii) an enrichment step comprising sorting the mixture of treated cells that are T cells (e.g., B2M-negative (or deficient) or CD3+) and alive, thus obtaining a first T cell subpopulation that is resistant to the killing of the NK cells; iii) a recovery step comprising culturing the first T cell subpopulation for about 48 hours; and iv) a sorting step comprising sorting the first T cell subpopulation post-recovery that are T cells (e.g., B2M-negative (or deficient) or CD3+) and alive, thus obtaining a second T cell subpopulation that is resistant to the killing of the NK cells. In some embodiments, identifying the hit gene in the T cell obtained from step c) comprises: i) identifying the sgRNAs ^iBAR sequence in the T cell obtained from step c) ; and ii) identifying the hit gene corresponding to the guide sequence of the sgRNAs ^iBAR. In some embodiments, there is provided a method of identifying a target gene in a T cell that modulates the activity of the T cell, comprising: a) providing a T cell library comprising an sgRNAs ^iBAR library described herein targeting one or more hit genes in the genome; b) subjecting the T cell library to treatment with NK cells; c) obtaining a T cell from the T cell library that is resistant to the killing of the NK cells; wherein steps b) and c) comprise: i) a treatment step comprising contacting the T cell library with the NK cells for about 48 hours at a ratio of NK cells to T cells of about 1∶1; ii) an enrichment step comprising sorting the mixture of treated cells that are T cells (e.g., B2M-negative (or deficient) or CD3+) and alive, thus obtaining a first T cell subpopulation that is resistant to the killing of the NK cells; iii) a recovery step comprising culturing the first T cell subpopulation for about 48 hours; and iv) a sorting step comprising sorting the first T cell subpopulation post-recovery that are T cells (e.g., B2M-negative (or deficient) or CD3+) and alive, thus obtaining a second T cell subpopulation that is resistant to the killing of the NK cells; and d) identifying the target gene in the T cell that modulates the activity of the T cell, wherein identifying the target gene comprises: i) obtaining sgRNA ^iBAR sequences in the final T cell subpopulation obtained from step c) ; ii) ranking the corresponding guide sequences of the sgRNA ^iBAR sequences based on sequence counts, wherein the ranking comprises adjusting the rank of each guide sequence based on data consistency among the iBAR sequences in the sgRNA ^iBAR sequences corresponding to the guide sequence; and iii) identifying the hit gene corresponding to a guide sequence ranked above a predetermined threshold level. In some embodiments, the sgRNAs ^iBAR library targets every annotated gene in the genome. In some embodiments, the T cell library has averagely at least about 100-fold (at least about 400-fold) coverage for the whole-genome sgRNAs ^iBAR library. In some embodiments, the method is a positive screening. In some embodiments, the method is a negative screening. In some embodiments, the sequence counts are subject to median ratio normalization followed by mean-variance modeling. In some embodiments, the variance of each guide sequence is adjusted based on data consistency among the iBAR sequences in the sgRNA ^iBAR sequences corresponding to the guide sequence. In some embodiments, the data consistency among the iBAR sequences in the sgRNA ^iBAR sequences corresponding to each guide sequence is determined based on the direction of the fold change of each iBAR sequence, wherein the variance of the guide sequence is increased if the fold changes of the iBAR sequences are in different directions with respect to each other (e.g., increased vs. reduced, increased vs. unchanged, or reduced vs. unchanged) . In some embodiments, the sequence counts obtained from the final T cell subpopulation obtained from step c) are compared to corresponding sequence counts obtained from a subpopulation of control T cells to provide fold changes (e.g., actual fold changes, or derivatives of fold changes such as log2 or log 10 fold changes) . In some embodiments, the subpopulation of control T cells are obtained from a same T cell library cultured in the same condition without subjected to treatment with NK cells, and optionally subjected to the same obtaining method in step c) . In some embodiments, the T cell library further comprises a B2M mutation (e.g., inactivating B2M mutation) . In some embodiments, the B2M mutation (e.g., inactivating B2M mutation) is generated by contacting the T cell library or the initial population of T cells for generating the T cell library with a B2M sgRNA construct (e.g., a viral vector or a virus) described herein. In some embodiments, subjecting the T cell library to treatment with NK cells in step b) comprises growing the T cell library in the presence of the NK cells.

In some embodiments, there is provided a method of identifying a target gene in a T cell (e.g., allogeneic T cell or CAR-T cell (such as allogeneic CAR-T cell) ) that modulates the activity of the T cell, comprising: a) providing a T cell library comprising an sgRNAs ^iBAR library described herein targeting one or more hit genes in the genome; b) subjecting the T cell library to at least two separate different treatments with NK cells described herein; c) obtaining a T cell (or a T cell subpopulation) from the T cell library that is sensitive or resistant to the killing of the NK cells from each treatment in step b) ; and d) identifying the target gene in the T cell that modulates the activity of the T cell; wherein identifying the target gene comprises: i) obtaining sgRNA ^iBAR sequences in the T cell (or a T cell subpopulation) obtained in step c) for each NK cell treatment; ii) ranking the corresponding guide sequences of the sgRNA ^iBAR sequences based on sequence counts for each NK cell treatment, wherein the ranking comprises adjusting the rank of each guide sequence based on data consistency among the iBAR sequences in the sgRNA ^iBAR sequences corresponding to the guide sequence; and iii) identifying the hit gene corresponding to a guide sequence ranked above a predetermined threshold level for each NK cell treatment; wherein (1) the hit genes that are identified as depleted from the final T cell subpopulation (from step c) resistant to the killing of the NK cells in at least one treatment with NK cells with FDR ≤0.01, or in at least two separate different treatments with NK cells with FDR ≤ 0.05 (and/or with at least about 2-fold depletion, such as at least about any of 3-, 4-, 5-, 10-, 20-, 50-, 100-fold, or more depletion) are identified as target genes whose mutation (e.g., inactivation) make the T cells sensitive to NK cell killing; (2) the hit genes that are identified as enriched from the final T cell subpopulation (from step c) resistant to the killing of the NK cells in at least one treatment with NK cells with FDR ≤ 0.05, or in at least two separate different treatments with NK cells with FDR ≤ 0.15 (and/or with at least about 2-fold enrichment, such as at least about any of 3-, 4-, 5-, 10-, 20-, 50-, 100-fold, or more enrichment) , are identified as target genes whose mutation (e.g., inactivation) make the T cells resistant to NK cell killing; (3) the hit genes that are identified as depleted from the final T cell subpopulation (from step c) sensitive to the killing of the NK cells in at least one treatment with NK cells with FDR ≤ 0.05, or in at least two separate different treatments with NK cells with FDR ≤ 0.15 (and/or with at least about 2-fold depletion, such as at least about any of 3-, 4-, 5-, 10-, 20-, 50-, 100-fold, or more depletion) , are identified as target genes whose mutation (e.g., inactivation) make the T cells resistant to NK cell killing; and/or; (4) the hit genes that are identified as enriched from the final T cell subpopulation (from step c) sensitive to the killing of the NK cells in at least one treatment with NK cells with FDR ≤0.01, or in at least two separate different treatments with NK cells with FDR ≤ 0.05 (and/or with at least about 2-fold enrichment, such as at least about any of 3-, 4-, 5-, 10-, 20-, 50-, 100-fold, or more enrichment) are identified as target genes whose mutation (e.g., inactivation) make the T cells sensitive to NK cell killing. In some embodiments, the sgRNAs ^iBAR library targets every annotated gene in the genome. In some embodiments, the T cell library has averagely at least about 100-fold (at least about 400-fold) coverage for the whole-genome sgRNAs ^iBAR library. In some embodiments, the method is a positive screening. In some embodiments, the method is a negative screening. In some embodiments, the sequence counts are subject to median ratio normalization followed by mean-variance modeling. In some embodiments, the variance of each guide sequence is adjusted based on data consistency among the iBAR sequences in the sgRNA ^iBAR sequences corresponding to the guide sequence. In some embodiments, the data consistency among the iBAR sequences in the sgRNA ^iBAR sequences corresponding to each guide sequence is determined based on the direction of the fold change of each iBAR sequence, wherein the variance of the guide sequence is increased if the fold changes of the iBAR sequences are in different directions with respect to each other (e.g., increased vs. reduced, increased vs. unchanged, or reduced vs. unchanged) . In some embodiments, the sequence counts obtained from the final T cell subpopulation obtained from step c) for each NK cell treatment are compared to corresponding sequence counts obtained from a subpopulation of control T cells to provide fold changes (e.g., actual fold changes, or derivatives of fold changes such as log2 or log 10 fold changes) . In some embodiments, the subpopulation of control T cells are obtained from a same T cell library cultured in the same condition without subjected to corresponding treatment with NK cells in step b) , and optionally subjected to the same corresponding obtaining method in step c) . In some embodiments, the T cell library further comprises a B2M mutation (e.g., inactivating B2M mutation) . In some embodiments, the B2M mutation (e.g., inactivating B2M mutation) is generated by contacting the T cell library or the initial population of T cells for generating the T cell library with a B2M sgRNA construct (e.g., a viral vector or a virus) described herein. In some embodiments, subjecting the T cell library to treatment with NK cells in step b) comprises growing the T cell library in the presence of the NK cells.

In some embodiments, there is provided a method of identifying a target gene in a T cell (e.g., allogeneic T cell or CAR-T cell (such as allogeneic CAR-T cell) ) that modulates the activity of the T cell, comprising: a) providing a T cell library comprising an sgRNAs ^iBAR library described herein targeting one or more hit genes in the genome; b-c) subjecting the T cell library to at least two of four separate Trials of NK cell treatment (e.g., killing) , thus obtaining a T cell from the T cell library that is resistant to the killing of the NK cells from each Trial; and d) identifying the hit gene in the T cell obtained from each Trial of step b-c) , thereby identifying the target gene in the T cell that modulates the activity of the T cell; wherein the four Trials are: (I) Trial I: i) an initial treatment step comprising contacting the T cell library with the NK cells for about 72 hours at a ratio of NK cells to T cells of about 0.5∶1; ii) an enrichment step comprising sorting the mixture of treated cells that are B2M-negative (or deficient) and alive, thus obtaining a first T cell subpopulation that is resistant to the killing of the NK cells; iii) a recovery step comprising culturing the first T cell subpopulation for about 48 hours; iv) a second treatment step comprising contacting the first T cell subpopulation post-recovery with the NK cells for about 96 hours at a ratio of NK cells to T cells of about 0.3∶1; and v) a sorting step comprising sorting the final mixture of treated cells that are B2M-negative (or deficient) and alive, thus obtaining a second T cell subpopulation that is resistant to the killing of the NK cells; (II) Trial II: i) a treatment step comprising contacting the T cell library with the NK cells for about 10 days at a ratio of NK cells to T cells of about 0.5∶1; and ii) a sorting step comprising sorting the mixture of treated cells that are T cells (e.g., B2M-negative (or deficient) , or CD3+) and alive, thus obtaining a T cell subpopulation that is resistant to the killing of the NK cells; (III) Trial III: i) a treatment step comprising contacting the T cell library with the NK cells for about 48 hours at a ratio of NK cells to T cells of about 1∶1; ii) a sorting step comprising sorting the mixture of treated cells that are T cells (e.g., B2M-negative (or deficient) , or CD3+) and alive, thus obtaining a T cell subpopulation that is resistant to the killing of the NK cells; and iii) a recovery step comprising culturing the T cell subpopulation for about 48 hours before harvesting the cells; and (IV) Trial IV: i) a treatment step comprising contacting the T cell library with the NK cells for about 48 hours at a ratio of NK cells to T cells of about 1∶1; ii) an enrichment step comprising sorting the mixture of treated cells that are T cells (e.g., B2M-negative (or deficient) , or CD3+) and alive, thus obtaining a first T cell subpopulation that is resistant to the killing of the NK cells; iii) a recovery step comprising culturing the first T cell subpopulation for about 48 hours; and iv) a sorting step comprising sorting the first T cell subpopulation post-recovery that are T cells (e.g., B2M-negative (or deficient) , or CD3+) and alive, thus obtaining a second T cell subpopulation that is resistant to the killing of the NK cells. In some embodiments, identifying the hit gene in the T cell obtained from step c) comprises: i) identifying the sgRNAs ^iBAR sequence in the T cell obtained from step c) ; and ii) identifying the hit gene corresponding to the guide sequence of the sgRNAs ^iBAR. In some embodiments, identifying the target gene comprises identifying the hit genes from the at least two of the four separate Trials, wherein: i) the hit genes that are identified as depleted from the final T cell subpopulation in at least one Trial with FDR ≤0.01 or in at least two Trials with FDR ≤ 0.05 (e.g., FDR ≤ any of 0.04, 0.03, 0.02, 0.01, 0.005, 0.001, or less) (and/or with at least about 2-fold depletion, such as at least about any of 3-, 4-, 5-, 10-, 20-, 50-, 100-fold, or more depletion) are identified as target genes whose mutation (e.g., inactivation) makes the T cells sensitive to NK cell killing; and/or; ii) the hit genes that are identified as enriched from the final T cell subpopulation in at least one Trial with FDR ≤ 0.05, or in at least two Trials with FDR ≤ 0.15 (e.g., FDR ≤ any of 0.14, 0.13, 0.12, 0.11, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, 0.01, or less) (and/or with at least about 2-fold enrichment, such as at least about any of 3-, 4-, 5-, 10-, 20-, 50-, 100-fold, or more enrichment) , are identified as target genes whose mutation (e.g., inactivation) makes the T cells resistant to NK cell killing. In some embodiments, the sgRNAs ^iBAR library targets every annotated gene in the genome. In some embodiments, the T cell library has averagely at least about 100-fold (e.g., at least about 400-fold) coverage for the whole-genome sgRNAs ^iBAR library. In some embodiments, the method is a positive screening. In some embodiments, the method is a negative screening. In some embodiments, the sequence counts are subject to median ratio normalization followed by mean-variance modeling. In some embodiments, the variance of each guide sequence is adjusted based on data consistency among the iBAR sequences in the sgRNA ^iBAR sequences corresponding to the guide sequence. In some embodiments, the data consistency among the iBAR sequences in the sgRNA ^iBAR sequences corresponding to each guide sequence is determined based on the direction of the fold change of each iBAR sequence, wherein the variance of the guide sequence is increased if the fold changes of the iBAR sequences are in different directions with respect to each other (e.g., increased vs. reduced, increased vs. unchanged, or reduced vs. unchanged) . In some embodiments, the sequence counts obtained from the final T cell subpopulation obtained from each Trial of step b-c) are compared to corresponding sequence counts obtained from a subpopulation of control T cells to provide fold changes (e.g., actual fold changes, or derivatives of fold changes such as log2 or log 10 fold changes) . In some embodiments, the subpopulation of control T cells are obtained from a same T cell library cultured in the same condition without subjected to treatment with NK cells in corresponding Trial of step b-c) , and optionally subjected to the same obtaining method in corresponding Trial of step b-c) . In some embodiments, the T cell library further comprises a B2M mutation (e.g., inactivating B2M mutation) . In some embodiments, the B2M mutation (e.g., inactivating B2M mutation) is generated by contacting the T cell library or the initial population of T cells for generating the T cell library with a B2M sgRNA construct (e.g., a viral vector or a virus) described herein. In some embodiments, subjecting the T cell library to treatment with NK cells in step b-c) comprises growing the T cell library in the presence of the NK cells.

In some embodiments, there is provided a method of identifying a target gene in a T cell (e.g., allogeneic T cell or CAR-T cell (such as allogeneic CAR-T cell) ) that modulates the activity of the T cell, comprising: a) providing a T cell library comprising an sgRNAs ^iBAR library described herein targeting one or more hit genes in the genome; b-c) subjecting the T cell library to at least two of four separate Trials of NK cell treatment (e.g., killing) , thus obtaining a T cell from the T cell library that is resistant to the killing of the NK cells from each Trial; wherein the four Trials are: (I) Trial I: i) an initial treatment step comprising contacting the T cell library with the NK cells for about 72 hours at a ratio of NK cells to T cells of about 0.5∶1; ii) an enrichment step comprising sorting the mixture of treated cells that are B2M-negative (or deficient) and alive, thus obtaining a first T cell subpopulation that is resistant to the killing of the NK cells; iii) a recovery step comprising culturing the first T cell subpopulation for about 48 hours; iv) a second treatment step comprising contacting the first T cell subpopulation post-recovery with the NK cells for about 96 hours at a ratio of NK cells to T cells of about 0.3∶1; and v) a sorting step comprising sorting the final mixture of treated cells that are B2M-negative (or deficient) and alive, thus obtaining a second T cell subpopulation that is resistant to the killing of the NK cells; (II) Trial II: i) a treatment step comprising contacting the T cell library with the NK cells for about 10 days at a ratio of NK cells to T cells of about 0.5∶1; and ii) a sorting step comprising sorting the mixture of treated cells that are T cells (e.g., B2M-negative (or deficient) , or CD3+) and alive, thus obtaining a T cell subpopulation that is resistant to the killing of the NK cells; (III) Trial III: i) a treatment step comprising contacting the T cell library with the NK cells for about 48 hours at a ratio of NK cells to T cells of about 1∶1; ii) a sorting step comprising sorting the mixture of treated cells that are T cells (e.g., B2M-negative (or deficient) , or CD3+) and alive, thus obtaining a T cell subpopulation that is resistant to the killing of the NK cells; and iii) a recovery step comprising culturing the T cell subpopulation for about 48 hours before harvesting the cells; and (IV) Trial IV: i) a treatment step comprising contacting the T cell library with the NK cells for about 48 hours at a ratio of NK cells to T cells of about 1∶1; ii) an enrichment step comprising sorting the mixture of treated cells that are T cells (e.g., B2M-negative (or deficient) , or CD3+) and alive, thus obtaining a first T cell subpopulation that is resistant to the killing of the NK cells; iii) a recovery step comprising culturing the first T cell subpopulation for about 48 hours; and iv) a sorting step comprising sorting the first T cell subpopulation post-recovery that are T cells (e.g., B2M-negative (or deficient) , or CD3+) and alive, thus obtaining a second T cell subpopulation that is resistant to the killing of the NK cells; and d) identifying the target gene in the T cell that modulates the activity of the T cell, wherein identifying the target gene comprises: i) obtaining sgRNA ^iBAR sequences in the final T cell subpopulation obtained from each Trial of step b-c) ; ii) ranking the corresponding guide sequences of the sgRNA ^iBAR sequences based on sequence counts for each Trial, wherein the ranking comprises adjusting the rank of each guide sequence based on data consistency among the iBAR sequences in the sgRNA ^iBAR sequences corresponding to the guide sequence; and iii) identifying the hit gene corresponding to a guide sequence ranked above a predetermined threshold level for each Trial; wherein (1) the hit genes that are identified as depleted from the final T cell subpopulation in at least one Trial with FDR ≤ 0.01 in at least two Trials with FDR ≤ 0.05 (e.g., FDR ≤ any of 0.04, 0.03, 0.02, 0.01, 0.005, 0.001, or less) (and/or with at least about 2-fold depletion, such as at least about any of 3-, 4-, 5-, 10-, 20-, 50-, 100-fold, or more depletion) are identified as target genes whose mutation (e.g., inactivation) makes the T cells sensitive to NK cell killing; and/or; (2) the hit genes that are identified as enriched from the final T cell subpopulation in at least one Trial with FDR ≤ 0.05, or in at least two Trials with FDR ≤ 0.15 (e.g., FDR ≤ any of 0.14, 0.13, 0.12, 0.11, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, 0.01, or less) (and/or with at least about 2-fold enrichment, such as at least about any of 3-, 4-, 5-, 10-, 20-, 50-, 100-fold, or more enrichment) , are identified as target genes whose mutation (e.g., inactivation) makes the T cells resistant to NK cell killing. In some embodiments, the sgRNAs ^iBAR library targets every annotated gene in the genome. In some embodiments, the T cell library has averagely at least about 100-fold (e.g., at least about 400-fold) coverage for the whole-genome sgRNAs ^iBAR library. In some embodiments, the method is a positive screening. In some embodiments, the method is a negative screening. In some embodiments, the sequence counts are subject to median ratio normalization followed by mean-variance modeling. In some embodiments, the variance of each guide sequence is adjusted based on data consistency among the iBAR sequences in the sgRNA ^iBAR sequences corresponding to the guide sequence. In some embodiments, the data consistency among the iBAR sequences in the sgRNA ^iBAR sequences corresponding to each guide sequence is determined based on the direction of the fold change of each iBAR sequence, wherein the variance of the guide sequence is increased if the fold changes of the iBAR sequences are in different directions with respect to each other (e.g., increased vs. reduced, increased vs. unchanged, or reduced vs. unchanged) . In some embodiments, the sequence counts obtained from the final T cell subpopulation obtained from each Trial of step b-c) are compared to corresponding sequence counts obtained from a subpopulation of control T cells to provide fold changes (e.g., actual fold changes, or derivatives of fold changes such as log2 or log 10 fold changes) . In some embodiments, the subpopulation of control T cells are obtained from a same T cell library cultured in the same condition without subjected to treatment with NK cells in corresponding Trial of step b-c) , and optionally subjected to the same obtaining method in corresponding Trial of step b-c) . In some embodiments, the T cell library further comprises a B2M mutation (e.g., inactivating B2M mutation) . In some embodiments, the B2M mutation (e.g., inactivating B2M mutation) is generated by contacting the T cell library or the initial population of T cells for generating the T cell library with a B2M sgRNA construct (e.g., a viral vector or a virus) described herein. In some embodiments, subjecting the T cell library to treatment with NK cells in step b-c) comprises growing the T cell library in the presence of the NK cells.

In some embodiments, any of the identification methods described herein further comprise validating the target gene by: a) modifying a T cell (e.g., allogeneic T cell or CAR-T cell (such as allogeneic CAR-T cell) ) by creating a mutation (e.g., inactivating mutation) in the target gene in the T cell; b) determining the sensitivity or resistance of the modified T cell to the killing of NK cells. In some embodiments, the method further comprising creating a mutation (e.g., inactivating mutation) in B2M in the T cell.

Further provided are modified T cells (e.g., modified allogeneic T cell or modified CAR-T cells (such as modified allogeneic CAR-T cells) ) ) obtained by inactivating one or more target genes identified by any of the methods described herein.

Single-guide RNA (sgRNA) library and sgRNA ^iBAR library

In some embodiments, the present invention uses CRISPR/Cas guide RNAs (e.g., single-guide RNA) and constructs encoding the CRISPR/Cas guide RNAs to generate mutations (e.g., inactivating mutations) in one or more hit genes in the genome. In some embodiments, the mutations are generated by cleaving the hit gene (e.g., with CRISPR/Cas9) . In some embodiments, the mutations are generated by modulating (e.g., repressing or reducing) the expression of the hit gene (e.g., with CRISPR/dCas fused to a repressor domain) .

In some embodiments, there is provided an sgRNA library comprising one or a plurality of (e.g., 1, 2, 3, 4, 5, 10, 100, 1,000, 10,000, 20,000, or more) sgRNA constructs, wherein each sgRNA construct (e.g., lentivirus or lentiviral vector encoding the sgRNA) comprises or encodes an sgRNA, and wherein each sgRNA comprises a guide sequence that is complementary (e.g., at least about any of 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%complementary) to a target site in a corresponding hit gene. In some embodiments, the sgRNA library comprises a plurality of (e.g., 2, 3, 4, 5, 10, 100, 1,000, 10,000, 20,000, or more) sgRNA constructs, wherein at least two hit genes that the guide sequences are complementary to are different from each other. In some embodiments, the sgRNA construct comprises (or consists of) an sgRNA. In some embodiments, the sgRNA construct encodes an sgRNA. In some embodiments, the sgRNA construct is a plasmid that encodes the sgRNA. In some embodiments, the sgRNA construct is a viral vector (e.g., lentiviral vector) encoding the sgRNA. In some embodiments, the sgRNA construct is a virus (e.g., lentivirus) encoding the sgRNA. In some embodiments, each sgRNA comprises the guide sequence fused to a second sequence, wherein the second sequence comprises a repeat-anti-repeat stem loop that interacts with a Cas protein (e.g., Cas9) . In some embodiments, the second sequence of each sgRNA further comprises a stem loop 1, a stem loop 2, and/or a stem loop 3. In some embodiments, each guide sequence comprises about 17 to about 23 nucleotides. In some embodiments, the sgRNA library comprises at least about 100 sgRNAs constructs, such as at least about any of 200, 300, 400, 1,000, 1,600, 4,000, 10,000, 15,000, 16,000, 19,000, 20,000, 38,000, 50,000, 100,000, 150,000, 155,000, 200,000, or more sgRNA constructs. In some embodiments, the sgRNA library comprising a plurality of sgRNA constructs comprises or encodes sgRNAs with guide sequences complementary to target sites of every annotated gene in the genome (hereinafter also referred to as “whole-genome sgRNA library” ) . In some embodiments, the sgRNA library comprises at least two sgRNA constructs comprising or encoding sgRNAs with guide sequences complementary to at least two different target sites of the same hit gene, i.e., the sgRNA library has averagely at least two-fold coverage for that hit gene. In some embodiments, the sgRNA library comprises at least two (e.g., 2, 3, 4, 5, or more) sgRNA constructs comprising or encoding sgRNAs with guide sequences complementary to at least two different target sites within the same hit gene for every annotated gene in the genome, i.e., the sgRNA library has averagely at least two-fold coverage for the whole genome. In some embodiments, the sgRNA library further comprises one or a plurality of (e.g., 1, 2, 3, 4, 5, 10, 100, 1,000, 2,000, 10,000, or more) “negative control sgRNA constructs” , wherein each negative control sgRNA construct (e.g., lentivirus or lentiviral vector encoding the negative control sgRNA) comprises or encodes a negative control sgRNA, and wherein each negative control sgRNA comprises a guide sequence that is complementary to an irrelevant sequence that is not in the genome, is complementary to a control gene (e.g., known to respond the same or similar between test and control groups after gene inactivation) , or is complementary to a sequence not associated with any annotated gene in the genome. In some embodiments, the sgRNA library further comprises negative control sgRNA constructs in the amount of about 3%to about 30%of the number of hit gene sgRNA constructs in the sgRNA library. In some embodiments, the sgRNA library further comprises about 1,000 negative control sgRNA constructs.

In some embodiments, the sgRNA further comprises an internal barcode (iBAR) sequence (such sgRNA is hereinafter referred to as “sgRNA ^iBAR” ) . In some embodiments, the iBAR is positioned in the sgRNA such that the resulting sgRNA ^iBAR is operable with a Cas protein (e.g., Cas9) to modify (e.g., cleave or modulate expression) the hit gene complementary to the guide sequence of the sgRNA ^iBAR. Thus in some embodiments, the sgRNA library described herein is an sgRNA ^iBAR library. In some embodiments, the sgRNA ^iBAR library comprises one or a plurality of (e.g., 1, 2, 3, 4, 5, 10, 100, 1,000, 20,000, 10,000, or more) sgRNA ^iBAR constructs, wherein each sgRNA ^iBAR construct comprises or encodes an sgRNA ^iBAR, wherein each sgRNA ^iBAR comprises a guide sequence and an iBAR sequence, and wherein each guide sequence is complementary (e.g., at least about any of 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%complementary) to a target site in a corresponding hit gene in the genome. In some embodiments, the sgRNA ^iBAR library comprises a plurality of (e.g., 2, 3, 4, 5, 10, 100, 1,000, 10,000, or more) sgRNA ^iBAR constructs, wherein at least two hit genes that the guide sequences are complementary to are different from each other. In some embodiments, each sgRNA (or sgRNA ^iBAR) comprises the guide sequence fused to a second sequence, wherein the second sequence comprises a repeat-anti-repeat stem loop that interacts with the Cas protein (e.g., Cas9) . In some embodiments, the second sequence of each sgRNA (or sgRNA ^iBAR) further comprises a stem loop 1, a stem loop 2, and/or a stem loop 3. In some embodiments, the iBAR sequence of each sgRNA ^iBAR is inserted in the loop region of the repeat-anti-repeat stem loop. In some embodiments, each sgRNA ^iBAR comprises in the 5'-to-3' direction a first stem sequence and a second stem sequence, wherein the first stem sequence hybridizes with the second stem sequence to form a double-stranded RNA (dsRNA) region that interacts with the Cas protein, and wherein the iBAR sequence is disposed between the 3' end of the first stem sequence and the 5' end of the second stem sequence. In some embodiments, each sgRNA ^iBAR comprises from 5'-to-3': a guide sequence, a repeat-anti-repeat stem loop with iBAR sequence inserted in the loop region, a stem loop 1, a stem loop 2, and a stem loop 3. In some embodiments, there is provided an sgRNA ^iBAR library comprising a plurality of sets of sgRNA ^iBAR constructs, wherein each set of sgRNA ^iBAR constructs comprise three or more (e.g., 3, 4, 5, or more, such as 4) sgRNA ^iBAR constructs (e.g., lentivirus or lentiviral vector encoding the sgRNAs ^iBAR) each comprising or encoding an sgRNA ^iBAR, wherein each sgRNA ^iBAR comprises a guide sequence and an iBAR sequence, wherein the guide sequences for the three or more sgRNA ^iBAR constructs are the same, wherein the iBAR sequence for each of the three or more sgRNA ^iBAR constructs is different from each other, and wherein the guide sequence of each set of sgRNA ^iBAR constructs is complementary (e.g., at least about any of 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%complementary) to a different target site in a corresponding hit gene (e.g., different hit genes, or different sites within the same hit gene) in the genome. In some embodiments, each set of sgRNA ^iBAR constructs comprises four sgRNA ^iBAR constructs, and wherein the iBAR sequence for each of the four sgRNA ^iBAR constructs is different from each other. Hence in some embodiments, there is provided an sgRNA ^iBAR library comprising a plurality of sets of sgRNA ^iBAR constructs, wherein each set of sgRNA ^iBAR constructs comprise four sgRNA ^iBAR constructs each comprising or encoding an sgRNA ^iBAR, wherein each sgRNA ^iBAR comprises a guide sequence and an iBAR sequence, wherein the guide sequences for the four sgRNA ^iBAR constructs are the same, wherein the iBAR sequence for each of the four sgRNA ^iBAR constructs is different from each other, and wherein the guide sequence of each set of sgRNA ^iBAR constructs is complementary (e.g., at least about any of 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%complementary) to a different target site in a corresponding hit gene (e.g., different hit genes, or different sites within the same hit gene) in the genome. In some embodiments, the sgRNA ^iBAR library comprises at least about 100 (e.g., at least about any of 200, 400, 1,000, 1,200, 1,600, 4,000, 10,000, 15,000, 19,000, 20,000, 38,000, 50,000, 100,000, 150,000, 155,000, 200,000, or more) sets of sgRNA ^iBAR constructs. In some embodiments, the iBAR sequences for at least two sgRNA ^iBAR constructs among different sets of sgRNA ^iBAR constructs are the same (e.g., the first set and the second set of sgRNA ^iBAR constructs have at least 1, 2, 3, 4, or more shared iBAR sequences among the two sets of sgRNA ^iBAR constructs) . In some embodiments, the iBAR sequences for at least two sets of sgRNA ^iBAR constructs are the same. In some embodiments, the sgRNA ^iBAR library comprising a plurality of sets sgRNA ^iBAR constructs comprises or encodes sgRNAs ^iBAR with guide sequences complementary to target sites of every annotated gene in the genome (hereinafter also referred to as “whole-genome sgRNA ^iBAR library” ) . In some embodiments, the sgRNA ^iBAR library comprises at least two (e.g., 2, 3, 4, 5, or more) sets sgRNA ^iBAR constructs comprising or encoding sgRNAs ^iBAR with guide sequences complementary to at least two (e.g., 2, 3, 4, 5, or more, such as 2) different target sites of the same hit gene, i.e., the sgRNA ^iBAR library has averagely at least two-fold coverage for that hit gene. In some embodiments, for each hit gene, the sgRNA ^iBAR library comprises 2 sets sgRNA ^iBAR constructs comprising or encoding sgRNAs ^iBAR with guide sequences complementary to 2 different target sites of the same hit gene. In some embodiments, the sgRNA ^iBAR library comprises at least two (e.g., 2, 3, 4, 5, or more) sets sgRNA ^iBAR constructs comprising or encoding sgRNAs ^iBAR with guide sequences complementary to at least two (e.g., 2, 3, 4, 5, or more, such as 2) different target sites within the same hit gene for every annotated gene in the genome, i.e., the sgRNA ^iBAR library has averagely at least two-fold coverage for the whole genome. In some embodiments, each guide sequence comprises about 17 to about 23 nucleotides. In some embodiments, each iBAR sequence comprises about 1 to about 50 (e.g., about 6) nucleotides. In some embodiments, the sgRNA ^iBAR construct comprises (or consists of) an sgRNA ^iBAR. In some embodiments, the sgRNA ^iBAR construct encodes an sgRNA ^iBAR. In some embodiments, the sgRNA ^iBAR construct is a plasmid that encodes the sgRNA ^iBAR. In some embodiments, the sgRNA ^iBAR construct is a viral vector (e.g., lentiviral vector) encoding the sgRNA ^iBAR. In some embodiments, the sgRNA ^iBAR construct is a virus (e.g., lentivirus) encoding the sgRNA ^iBAR. Different sgRNA ^iBAR constructs of a set having different iBAR sequences can be used in a single gene-editing and screening experiment to provide replicate data. In some embodiments, the sgRNA ^iBAR library further comprises one or a plurality of sets of “negative control sgRNA ^iBAR constructs” , wherein each set of negative control sgRNA ^iBAR constructs comprise three or more (e.g., 3, 4, 5, or more, such as 4) negative control sgRNA ^iBAR constructs (e.g., lentivirus or lentiviral vector encoding the negative control sgRNAs ^iBAR) each comprising or encoding a negative control sgRNA ^iBAR, wherein each negative control sgRNA ^iBAR comprises a guide sequence and an iBAR sequence, wherein the guide sequences for the three or more negative control sgRNA ^iBAR constructs are the same, wherein the iBAR sequence for each of the three or more negative control sgRNA ^iBAR constructs is different from each other, and wherein the guide sequence of each set of negative control sgRNA ^iBAR constructs is complementary to a target site not associated with any annotated gene in the genome, is complementary to a control gene (e.g., known to respond the same or similar between test and control groups after gene inactivation) , or is complementary to an irrelevant sequence that is not in the genome. In some embodiments, the sgRNA ^iBAR library further comprises negative control sgRNA ^iBAR constructs in the amount of about 3%to about 30%of the number of hit gene sgRNA ^iBAR constructs in the sgRNA ^iBAR library. In some embodiments, the sgRNA ^iBAR library further comprises about 1,000 negative control sgRNA ^iBAR constructs.

In some embodiments, there is provided an sgRNA library (e.g., sgRNA ^iBAR library) comprising one or more sgRNA constructs (e.g., sgRNA ^iBAR constructs) , wherein each sgRNA construct (e.g., lentivirus or lentiviral vector encoding the sgRNA) comprises or encodes an sgRNA (e.g., sgRNA ^iBAR) , and wherein each sgRNA (e.g., sgRNA ^iBAR) comprises a guide sequence that is complementary (e.g., at least about any of 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%complementary) to a target site in a target gene selected from the group consisting of TACC2, HES1, STON1, GJD2, LILRB4, PLS1, KLHL24, FANCB, ARNTL, AMY2A, SIX1, USP17L13, PIK3R6, ATP6V1H, TPM3, OR5H14, TFG, SMAD6, PDE4C, SRRM3, LRRC69, KCNJ13, C8orf34, and PACS2. In some embodiments, the sgRNA library further comprises an sgRNA construct (e.g., lentivirus or lentiviral vector encoding the sgRNA) comprising or encoding an sgRNA whose guide sequence is complementary to a target site in B2M.

In some embodiments, there is provided an sgRNA ^iBAR library comprising a plurality of sets of sgRNA ^iBAR constructs, wherein each set of sgRNA ^iBAR constructs comprise three or more (e.g., 3, 4, 5, or more, such as 4) sgRNA ^iBAR constructs (e.g., lentiviruses or lentiviral vectors encoding the sgRNAs ^iBAR) each comprising or encoding an sgRNA ^iBAR, wherein each sgRNA ^iBAR comprises a guide sequence and an iBAR sequence, wherein the guide sequences for the three or more sgRNA ^iBAR constructs are the same, wherein the iBAR sequence for each of the three or more sgRNA ^iBAR constructs is different from each other, wherein the guide sequence of each set of sgRNA ^iBAR constructs is complementary (e.g., at least about any of 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%complementary) to a different target site in a corresponding hit gene (e.g., different hit genes, or different sites within the same hit gene) in the genome, and wherein each sgRNA ^iBAR is operable with a Cas9 protein to modify the target site. In some embodiments, there is provided an sgRNA ^iBAR library comprising a plurality of sets of sgRNA ^iBAR constructs, wherein each set of sgRNA ^iBAR constructs comprise four sgRNA ^iBAR constructs each comprising or encoding an sgRNA ^iBAR, wherein each sgRNA ^iBAR comprises a guide sequence and an iBAR sequence, wherein the guide sequences for the four sgRNA ^iBAR constructs are the same, wherein the iBAR sequence for each of the four sgRNA ^iBAR constructs is different from each other, wherein the guide sequence of each set of sgRNA ^iBAR constructs is complementary (e.g., at least about any of 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%complementary) to a different target site in a corresponding hit gene (e.g., different hit genes, or different sites within the same hit gene) in the genome, and wherein each sgRNA ^iBAR is operable with a Cas9 protein to modify the target site. In some embodiments, each sgRNA ^iBAR sequence comprises a guide sequence fused to a second sequence, wherein the second sequence comprises a repeat-anti-repeat stem loop that interacts with the Cas9. In some embodiments, the second sequence of each sgRNA ^iBAR sequence further comprises a stem loop 1, stem loop 2, and/or stem loop 3. In some embodiments, the iBAR sequence is inserted in the loop region of the repeat-anti-repeat stem loop, and/or the loop region of the stem loop 1, stem loop 2, or stem loop 3. In some embodiments, each iBAR sequence comprises about 1-50 (e.g., about 6) nucleotides. In some embodiments, each sgRNA ^iBAR construct is an RNA, a plasmid, a viral vector (e.g., lentiviral vector) , or a virus (e.g., lentivirus) . In some embodiments, the sgRNA ^iBAR library comprises at least about 100 (e.g., at least about any of 200, 400, 1,000, 1,200, 1,600, 4,000, 10,000, 15,000, 19,000, 20,000, 38,000, 50,000, 100,000, 150,000, 155,000, 200,000, or more) sets of sgRNA ^iBAR constructs. In some embodiments, the iBAR sequences for at least two sgRNA ^iBAR constructs among different sets of sgRNA ^iBAR constructs are the same (e.g., the first set and the second set of sgRNA ^iBAR constructs have at least 1, 2, 3, 4, or more shared iBAR sequences among the two sets of sgRNA ^iBAR constructs) . In some embodiments, the iBAR sequences for at least two sets of sgRNA ^iBAR constructs are the same. In some embodiments, the sgRNA ^iBAR library comprising a plurality of sets sgRNA ^iBAR constructs comprises or encodes sgRNAs ^iBAR with guide sequences complementary to target sites of every annotated gene in the genome. In some embodiments, the sgRNA ^iBAR library comprises at least two (e.g., 2, 3, 4, 5, or more) sets sgRNA ^iBAR constructs comprising or encoding sgRNAs ^iBAR with guide sequences complementary to at least two (e.g., 2, 3, 4, 5, or more, such as 2) different target sites within the same hit gene for every annotated gene in the genome. In some embodiments, each guide sequence comprises about 17 to about 23 nucleotides.

In some embodiments, there is provided an sgRNA ^iBAR library comprising a plurality of sets of sgRNA ^iBAR constructs, wherein each set of sgRNA ^iBAR constructs comprise three or more (e.g., 3, 4, 5, or more, such as 4) sgRNA ^iBAR constructs each comprising or encoding an sgRNA ^iBAR, wherein each sgRNA ^iBAR comprises a guide sequence, a second sequence, and an iBAR sequence, wherein the guide sequences for the three or more sgRNA ^iBAR constructs are the same, wherein the iBAR sequence for each of the three or more sgRNA ^iBAR constructs is different from each other, wherein the guide sequence is fused to the second sequence, wherein the second sequence comprises a repeat-anti-repeat stem loop that interacts with a Cas9 protein, wherein the iBAR sequence is inserted in the loop region of the repeat-anti-repeat stem loop, wherein the guide sequence of each set of sgRNA ^iBAR constructs is complementary (e.g., at least about any of 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%complementary) to a different target site of a corresponding hit gene (e.g., different hit genes, or different sites within the same hit gene) in the genome, and wherein each sgRNA ^iBAR is operable with the Cas9 protein to modify the target site. In some embodiments, there is provided an sgRNAiB AR library comprising a plurality of sets of sgRNA ^iBAR constructs, wherein each set of sgRNA ^iBAR constructs comprise four sgRNA ^iBAR constructs each comprising or encoding an sgRNA ^iBAR, wherein each sgRNA ^iBAR comprises a guide sequence, a second sequence, and an iBAR sequence, wherein the guide sequences for the four sgRNA ^iBAR constructs are the same, wherein the iBAR sequence for each of the four sgRNA ^iBAR constructs is different from each other, wherein the guide sequence is fused to the second sequence, wherein the second sequence comprises a repeat-anti-repeat stem loop that interacts with a Cas9 protein, wherein the iBAR sequence is inserted in the loop region of the repeat-anti-repeat stem loop, wherein the guide sequence of each set of sgRNA ^iBAR constructs is complementary (e.g., at least about any of 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%complementary) to a different target site of a corresponding hit gene (e.g., different hit genes, or different sites within the same hit gene) in the genome, and wherein each sgRNA ^iBAR is operable with the Cas9 protein to modify the target site. In some embodiments, the second sequence of each sgRNA ^iBAR sequence further comprises a stem loop 1, stem loop 2, and/or stem loop 3, e.g., fused to the 3' end of the repeat-anti-repeat stem loop sequence. In some embodiments, each iBAR sequence comprises about 1-50 (e.g., 6) nucleotides. In some embodiments, each sgRNA ^iBAR construct is an RNA, a plasmid, a viral vector (e.g., lentiviral vector) , or a virus (e.g., lentivirus) . In some embodiments, the sgRNA ^iBAR library comprises at least about 100 (e.g., at least about any of 200, 400, 1,000, 1,200, 1,600, 4,000, 10,000, 15,000, 19,000, 20,000, 38,000, 50,000, 100,000, 150,000, 155,000, 200,000, or more) sets of sgRNA ^iBAR constructs. In some embodiments, the iBAR sequences for at least two sgRNA ^iBAR constructs among different sets of sgRNA ^iBAR constructs are the same (e.g., the first set and the second set of sgRNA ^iBAR constructs have at least 1, 2, 3, 4, or more shared iBAR sequences among the two sets of sgRNA ^iBAR constructs) . In some embodiments, the iBAR sequences for at least two sets of sgRNA ^iBAR constructs are the same. In some embodiments, the sgRNA ^iBAR library comprising a plurality of sets sgRNA ^iBAR constructs comprises or encodes sgRNAs ^iBAR with guide sequences complementary to target sites of every annotated gene in the genome. In some embodiments, the sgRNA ^iBAR library comprises at least two (e.g., 2, 3, 4, 5, or more) sets sgRNA ^iBAR constructs comprising or encoding sgRNAs ^iBAR with guide sequences complementary to at least two (e.g., 2, 3, 4, 5, or more, such as 2) different target sites within the same hit gene for every annotated gene in the genome. In some embodiments, each guide sequence comprises about 17 to about 23 nucleotides.

In some embodiments, there is provided an sgRNA ^iBAR construct comprising a guide sequence targeting (e.g., at least about any of 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%complementary to) a target site in a corresponding hit gene in the genome, and a guide hairpin coding sequence for a Repeat: Anti-Repeat Duplex and a tetraloop, wherein an iBAR is embedded in the tetraloop serving as internal replicates. In some embodiments, the iBAR comprises a 1 nucleotide ( “nt” ) -50nt (e.g., 1nt-40nt, 1nt-30nt, 1nt-25nt, 2nt-20nt, 3nt-18nt, 3nt-16nt, 3nt-14nt, 3nt-12nt, 3nt-10nt, 3nt-9nt, 4nt-8nt, 5nt-7nt; preferably, 3nt, 4nt, 5nt, 6nt, 7nt) sequence consisting of A, T, C, and G nucleotides. In some embodiments, the guide sequence is about any of 17-23, 18-22, or 19-21 nucleotides in length, and the hairpin sequence once transcribed can be bound to a Cas nuclease (e.g., Cas9) . In some embodiments, the sgRNA ^iBAR construct further comprises a sequence coding for stem loop 1, stem loop 2 and/or stem loop 3. In some embodiments, each sgRNA ^iBAR construct is an RNA, a plasmid, a viral vector (e.g., lentiviral vector) , or a virus (e.g., lentivirus) .

Also provided are sgRNA molecules encoded by any one of the sgRNA constructs or libraries described herein. Also provided are sgRNA ^iBAR molecules encoded by any one of the sgRNA ^iBAR constructs, sets, or libraries described herein. Compositions and kits comprising any one of the sgRNA or sgRNA ^iBAR constructs, molecules, sets, or libraries are further provided.

In some embodiments, there is provided isolated T cells (e.g., allogeneic T cells, or CAR-T cells (such as allogeneic CAR-T cells) ) comprising any one of the sgRNA or sgRNA ^iBAR constructs, molecules, sets, or libraries described herein. In some embodiments, there is provided a T cell library wherein each T cell comprises one or more sgRNA constructs from an sgRNA library described herein, or one or more sgRNA ^iBAR constructs from an sgRNA ^iBAR library described herein. In some embodiments, the T cell library comprises an sgRNA library or an sgRNA ^iBAR library described herein targeting every annotated gene in the genome. In some embodiments, the host cell comprises or expresses one or more components of the CRISPR/Cas system, such as the Cas protein operable with the sgRNA or sgRNA ^iBAR constructs. In some embodiments, the Cas protein is Cas9 nuclease.

iBAR sequences

A set of sgRNA ^iBAR construct comprises three or more sgRNA ^iBAR constructs each comprising a different iBAR sequence. In some embodiments, a set of sgRNA ^iBAR construct comprises three sgRNA ^iBAR constructs each comprising a different iBAR sequence. In some embodiments, a set of sgRNA ^iBAR construct comprises four sgRNA ^iBAR constructs each comprising a different iBAR sequence. In some embodiments, a set of sgRNA ^iBAR construct comprises five sgRNA ^iBAR constructs each comprising a different iBAR sequence. In some embodiments, a set of sgRNA ^iBAR construct comprises six or more sgRNA ^iBAR constructs each comprising a different iBAR sequence.

The iBAR sequences may have any suitable length. In some embodiments, each iBAR sequence is about 1-50 nucleotides ( “nt” ) in length, such as about any one of 1nt-40nt, 1nt-30nt, 1nt-20nt, 2nt-20nt, 3nt-18nt, 3nt-16nt, 3nt-14nt, 3nt-12nt, 3nt-10nt, 3nt-9nt, 3nt-8nt, 4nt-8nt, or 5nt-7nt. In some embodiments, each iBAR sequence is about any of 2nt, 3nt, 4nt, 5nt, 6nt, 7nt, or 8nt long. In some embodiments, the iBAR sequence in each sgRNA ^iBAR construct has the same length. In some embodiments, the iBAR sequences of different sgRNA ^iBAR constructs have different lengths. In some embodiments, the iBAR sequences within a set of sgRNA ^iBAR constructs have the same length. In some embodiments, the iBAR sequences within a set of sgRNA ^iBAR constructs have different lengths. In some embodiments, the iBAR sequences within one set of sgRNA ^iBAR constructs have different lengths from the iBAR sequences within another set of sgRNA ^iBAR constructs. In some embodiments, the iBAR sequence is about 6nt, hereinafter referred to as “iBAR ₆. ” In some embodiments, each iBAR sequence within the sgRNA ^iBAR library is about 6nt.

The iBAR sequences may have any suitable sequences. In some embodiments, the iBAR sequence is a DNA sequence made of any of A, T, C and/or G nucleotides. In some embodiments, the iBAR sequence is an RNA sequence made of any of A, U, C, and/or G nucleotides. In some embodiments, the iBAR sequence has non-conventional or modified nucleotides other than A, T/U, C, and G. In some embodiments, each iBAR sequence is 6 nucleotides long consisting of A, T, C, and G nucleotides. In some embodiments, the iBAR sequence in the encoded sgRNA ^iBAR is 6 nucleotides long consisting of A, U, C, and G nucleotides.

In some embodiments, the set of iBAR sequences associated with each set of sgRNA ^iBAR constructs in the sgRNA ^iBAR library is different from each other. In some embodiments, the iBAR sequences for at least two sgRNA ^iBAR constructs among different sets of sgRNA ^iBAR constructs are the same (e.g., the first set and the second set of sgRNA ^iBAR constructs have at least 1, 2, 3, 4, or more shared iBAR sequences among the two sets of sgRNA ^iBAR constructs, but the iBAR sequences for each sgRNA ^iBAR construct within the same set of sgRNA ^iBAR constructs are different from each other) . In some embodiments, the iBAR sequences for at least two (e.g., at least about any of 2, 3, 4, 5, 10, 50, 100, 1000, or more) sets of sgRNA ^iBAR constructs in the sgRNA ^iBAR library are the same. In some embodiments, one or more same iBAR sequences are used for one or more sgRNA ^iBAR constructs of each set of sgRNA ^iBAR constructs in the sgRNA ^iBAR library (but the iBAR sequences for each sgRNA ^iBAR construct within the same set of sgRNA ^iBAR constructs are different from each other) . In some embodiments, the same set of iBAR sequences are used for each set of sgRNA ^iBAR constructs in the sgRNA ^iBAR library. In some embodiments, it is not necessary to design different iBAR sets for different sets of sgRNA ^iBAR constructs. In some embodiments, a fixed set of iBARs is used for all sets of sgRNA ^iBAR constructs in the sgRNA ^iBAR library. In some embodiments, a plurality of iBAR sequences are randomly assigned to different sets of sgRNA ^iBAR constructs in the sgRNA ^iBAR library. The iBAR strategy with a streamlined analytic tool (MAGeCK ^iBAR; Zhu et al., Genome Biol. 2019; 20: 20) described herein can facilitate large-scale CRISPR/Cas screens for biomedical discoveries in various settings.

The iBAR sequence may be inserted (including appended) to any suitable regions in a guide RNA (e.g., sgRNA) that does not affect the efficiency of the gRNA in guiding the Cas nuclease (e.g., Cas9) to its target site. In some embodiments, the iBAR sequence is placed at the 3' end of an sgRNA. In some embodiments, the iBAR sequence is placed at the 5' end of an sgRNA. In some embodiments, the iBAR sequence is placed at an internal position in an sgRNA. For example, an sgRNA may comprise various stem loops that interact with the Cas nuclease in a CRISPR complex, and the iBAR sequence may be embedded in the loop region of any one of the stem loops. In some embodiments, each sgRNA ^iBAR sequence comprises in the 5'-to-3' direction a first stem sequence and a second stem sequence, wherein the first stem sequence hybridizes with the second stem sequence to form a double-stranded RNA (dsRNA) region that interacts with the Cas protein (e.g., Cas9) , and wherein the iBAR sequence is disposed between the 3' end of the first stem sequence and the 5' end of the second stem sequence. In some embodiments, the sgRNA ^iBAR further comprises a stem loop 1, a stem loop 2, and/or a stem loop 3, and wherein the iBAR sequence is inserted in the loop region of stem loop 1, stem loop 2, and/or stem loop 3.

For example, the guide RNA of a CRISPR/Cas9 system may comprise a guide sequence targeting a genomic locus (e.g., a target site in a hit gene) , and a guide hairpin sequence coding for a Repeat: Anti-Repeat Duplex and a tetraloop. In some embodiments, the iBAR is inserted in the tetraloop serving as internal replicates. In the context of an endogenous CRISPR/Cas9 system, the crRNA hybridizes with the trans-activating crRNA (tracrRNA) to form a crRNA: tracrRNA duplex, which is loaded onto Cas9 to direct the cleavage of cognate DNA sequences bearing appropriate protospacer-adjacent motifs (PAMs) . An endogenous crRNA sequence can be divided into guide (20 nt) and repeat (12 nt) regions, whereas an endogenous tracrRNA sequence can be divided into anti-repeat (14 nt) and three tracrRNA stem loops. In some embodiments, the sgRNA binds the target DNA to form a T-shaped architecture comprising a guide: target heteroduplex, a repeat: anti-repeat duplex, and stem loops 1-3. In some embodiments, the repeat and anti-repeat parts are connected by the tetraloop, and the repeat and anti-repeat form a repeat: anti-repeat duplex, connected with stem loop 1 by a single nucleotide (A51) , whereas stem loops 1 and 2 are connected by a 5 nt single-stranded linker (nucleotides 63-67) . In some embodiments, the guide sequence (nucleotides 1-20) and target DNA (nucleotides 10-200) form the guide: target heteroduplex via 20 Watson-Crick base pairs, and the repeat (nucleotides 21-32) and the anti-repeat (nucleotides 37-50) form the repeat: anti-repeat duplex via nine Watson-Crick base pairs (U22: A49-A26: U45 and G29: C40-A32: U37) . In some embodiments, the tracrRNA tail (nucleotides 68-81 and 82-96) forms stem loops 2 and 3 via four and six Watson-Crick base pairs (A69: U80-U72: A77 and G82: C96-G87: C91) , respectively. Nishimasu et al. describes a crystal structure of an exemplary CRISPR/Cas9 system (Nishimasu et al. “Crystal structure of cas9 in complex with guide RNA and target DNA. ” Cell. 2014; 156: 935-949) , which is incorporated herein by reference in its entirety.

In some embodiments, the iBAR sequence is inserted in the tetraloop, or the loop region of the repeat: anti-repeat stem loop of an sgRNA. In some embodiments, the iBAR sequence of each sgRNA ^iBAR within the library is inserted in the loop region of the repeat-anti-repeat stem loop. The tetraloop of the Cas9 sgRNA scaffold is outside the Cas9-sgRNA ribonucleoprotein complex, which has been subject to alterations for various purposes without affecting the activity of its upstream guide sequence (Gilbert et al. Cell 159, 647-661 (2014) ; Zhu et al. Methods Mol Biol 1656, 175-181 (2017) ) . Applicant has previously demonstrated in WO2020125762 that a 6-nt-long iBAR (iBAR ₆) may be embedded in the tetraloop of a typical Cas9 sgRNA scaffold without affecting the gene editing efficiency of the sgRNA or increasing off-target effects, and without sequence bias in the iBAR ₆. The exemplary iBAR ₆ gives rise to 4, 096 barcode combinations, which provides sufficient variations for a high throughput screen (see FIG. 1A of WO2020125762) .

Guide sequences

The guide sequence hybridizes with the target sequence (e.g., a target site in a hit gene) and directs sequence-specific binding of a CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence, when optimally aligned using a suitable alignment algorithm, is about or more than about any of 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 100%complementary) . A guide sequence that is “complementary” to a target site or a hit gene can be fully or partially complementary (e.g., at least about any of 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% complementary) to the target site or the hit gene. Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences, non-limiting example of which include the Smith-Waterman algorithm, the Needleman-Wimsch algorithm, algorithms based on the Burrows-Wheeler Transform. In certain embodiments, a guide sequence is about or more than about any of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotides in length. In some embodiments, the guide sequence comprises about 17 to about 23 nucleotides. The ability of a guide sequence to direct sequence-specific binding of a CRISPR complex to a target sequence may be assessed by any suitable assay. For example, the components of a CRISPR system sufficient to form a CRISPR complex, including the guide sequence to be tested, may be provided to a host cell having the corresponding target sequence, such as by transfection with vectors encoding the components of the CRISPR sequence, followed by an assessment of preferential cleavage within the target sequence. Similarly, cleavage of a target polynucleotide sequence may be evaluated in a test tube by providing the target sequence, components of a CRISPR complex, including the guide sequence to be tested and a control guide sequence different from the test guide sequence, and comparing binding or rate of cleavage at the target sequence between the test and control guide sequence reactions.

In some embodiments, a guide sequence can be as short as about 10 nucleotides and as long as about 30 nucleotides. In some embodiments, the guide sequence is about any one of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides long. Synthetic guide sequences can be about 20 nucleotides long, but can be longer or shorter. By way of example, a guide sequence for a CRISPR/Cas9 system may consist of 20 nucleotides complementary to a target sequence (e.g., a target site in a hit gene) , i.e., the guide sequence may be identical to the 20 nucleotides upstream of the PAM sequence except for the A/U difference between DNA and RNA. In some embodiments, the guide sequence comprises about 17 to about 23 nucleotides. In some embodiments, the guide sequence of each sgRNA or sgRNA ^iBAR within the library has the same length. In some embodiments, the guide sequences of at least two sgRNAs or sgRNAs ^iBAR within the library have different lengths. In some embodiments, the guide sequences within a set of sgRNA ^iBAR constructs have the same length. In some embodiments, the guide sequences within a set of sgRNA ^iBAR constructs have different lengths. In some embodiments, the guide sequences within one set of sgRNA ^iBAR constructs have different lengths from the guide sequences within another set of sgRNA ^iBAR constructs.

In some embodiments, the guide sequences within a set of sgRNA ^iBAR constructs are the same. In some embodiments, the guide sequences within a set of sgRNA ^iBAR constructs are the same, while the guide sequence within each set of sgRNA ^iBAR constructs is complementary to a different target site (e.g., different hit genes, or different target sites of the same hit gene) in the genome. In some embodiments, the guide sequences of at least two sets of sgRNA ^iBAR constructs are complementary to two different target sites of the same hit gene. In some embodiments, each hit gene in the genome is targeted by at least two (e.g., 2, 3, 4 or more, such as 2) guide sequences of at least two (e.g., 2, 3, 4 or more, such as 2) sets of sgRNA ^iBAR constructs in at least two (e.g., 2, 3, 4 or more, such as 2) different target sites. In some embodiments, the guide sequence within each set of sgRNA ^iBAR constructs is complementary to a different hit gene in the genome.

The guide sequence in an sgRNA construct or an sgRNA ^iBAR construct may be designed according to any known methods in the art. The guide sequence may target the coding region such as an exon or a splicing site, the 5' untranslated region (UTR) or the 3' untranslated region (UTR) of a gene of interest. For example, the reading frame of a gene could be disrupted by indels mediated by double-strand breaks (DSB) at a target site of a guide RNA. Alternatively, a guide RNA targeting the 5' end of a coding sequence may be used to produce gene knockouts with high efficiency. The guide sequence may be designed and optimized according to certain sequence features for high on-target gene-editing activity and low off-target effects. For instance, the GC content of a guide sequence may be in the range of about 20%to about 70%, and sequences containing homopolymer stretches (e.g., TTTT, GGGG) may be avoided.

The guide sequence may be designed to target any genomic locus of interest (e.g., any target site of any hit gene) . In some embodiments, the guide sequence targets a protein-coding gene. In some embodiments, the guide sequence targets a gene encoding an RNA, such as a small RNA (e.g., microRNA, piRNA, siRNA, snoRNA, tRNA, rRNA and snRNA) , a ribosomal RNA, or a long non-coding RNA (lincRNA) . In some embodiments, the guide sequence targets a non-coding region of the genome. In some embodiments, the guide sequence targets a chromosomal locus. In some embodiments, the guide sequence targets an extrachromosomal locus. In some embodiments, the guide sequence targets a mitochondrial gene. In some embodiments, the guide sequence is complementary to a target site of any annotated genes in the genome (e.g., human genome) . In some embodiments, the guide sequence targets a region without any gene annotation in the genome ( “non-gene region” ) . sgRNA or sgRNA ^iBAR constructs comprising or encoding such guide sequence complementary to a non-gene region can serve as negative control.

In some embodiments, the guide sequence is designed to repress or inactivate the expression of any hit gene or target gene of interest. The hit gene or target gene may be an endogenous gene or a transgene. In some embodiments, the hit gene or target gene may be known to be associated with a particular phenotype. In some embodiments, the hit gene or target gene is a gene that has not been implicated in a particular phenotype, such as a known gene that is not known to be associated with a particular phenotype, or an unknown gene that has not been characterized. In some embodiments, the guide sequence targeted region is located on a different chromosome as the hit gene or target gene.

Other sgRNA or sgRNA ^iBAR components

In some embodiments, the sgRNA or sgRNA ^iBAR comprises additional sequence element (s) that promotes formation of the CRISPR complex with the Cas protein. In some embodiments, the sgRNA or sgRNA ^iBAR comprises a second sequence comprising a repeat-anti-repeat stem loop. A repeat-anti-repeat stem loop comprises a tracr mate sequence fused to a tracr sequence that is complementary to the tracr mate sequence via a loop region.

Typically, in the context of an endogenous CRISPR/Cas9 system, formation of a CRISPR complex (comprising a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins) results in cleavage of one or both strands in or near (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from) the target sequence. The tracr sequence, which may comprise or consist of all or a portion of a wild-type tracr sequence (e.g., about or more than about any of 20, 26, 32, 45, 48, 54, 63, 67, 85, or more nucleotides of a wild-type tracr sequence) , may also form part of a CRISPR complex, such as by hybridization along at least a portion of the tracr sequence to all or a portion of a tracr mate sequence that is operably linked to the guide sequence. In some embodiments, the tracr sequence has sufficient complementarity to a tracr mate sequence to hybridize and participate in formation of a CRISPR complex. As with the target sequence, it is believed that complete complementarity is not needed, provided there is sufficient to be functional. In some embodiments, the tracr sequence has at least about any of 50%, 60%, 70%, 80%, 90%, 95%or 99%of sequence complementarity along the length of the tracr mate sequence when optimally aligned. Determining optimal alignment is within the purview of one of skill in the art. For example, there are publically and commercially available alignment algorithms and programs such as, but not limited to, ClustalW, Smith-Waterman in Matlab, Bowtie, Geneious, Biopython and SeqMan. In some embodiments, the tracr sequence is about or more than about any of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or more nucleotides in length. Any one of the known tracr mate sequences and tracr sequences derived from naturally occurring CRISPR system, such as the tracr mate sequence and tracr sequence from the S. pyogenes CRISPR/Cas9 system as described in US8697359 and those described herein, may be used.

In some embodiments, the tracr sequence and tracr mate sequence are contained within a single transcript, such that hybridization between the two produces a transcript having a secondary structure, such as a stem loop (also known as a hairpin) , known as the “repeat-anti-repeat stem loop. ”

In some embodiments, the loop region of the stem loop in an sgRNA construct without an iBAR sequence is four nucleotides in length, and such loop region is also referred to as the “tetraloop. ” In some embodiments, the loop region has the sequence of GAAA. However, longer or shorter loop sequences may be used, or alternative sequences may be used, such as sequences including a nucleotide triplet (for example, AAA) , and an additional nucleotide (for example C or G) . In some embodiments, the sequence of the loop region is CAAA or AAAG. In some embodiments, the iBAR is inserted in the loop region, such as the tetraloop. For example, the iBAR sequence may be inserted before the first nucleotide, between the first nucleotide or the second nucleotide, between the second nucleotide and the third nucleotide, between the third nucleotide and the fourth nucleotide, or after the fourth nucleotide in the tetraloop. In some embodiments, the iBAR sequence replaces one or more nucleotides in the loop region.

In some embodiments, the sgRNA ^iBAR comprises at least two or more stem loops. In some embodiments, the sgRNA ^iBAR has two, three, four or five stem loops. In some embodiments, the sgRNA ^iBAR has at most five hairpins. In some embodiments, the sgRNA or sgRNA ^iBAR construct further includes a transcription termination sequence, such as a polyT sequence, for example six T nucleotides.

In some embodiments, wherein the Cas protein is Cas9, each sgRNA or sgRNA ^iBAR comprises a guide sequence fused to a second sequence comprising a repeat-anti-repeat stem loop that interacts with the Cas 9. In some embodiments, the iBAR sequence is inserted in the loop region of the repeat-anti-repeat stem loop. In some embodiments, the iBAR sequence replaces one or more nucleotides in the loop region of the repeat-anti-repeat stem loop. In some embodiments, the second sequence of each sgRNA or sgRNA ^iBAR further comprises a stem loop 1, stem loop 2, and/or stem loop 3. In some embodiments, the iBAR sequence is inserted in the loop region of stem loop 1. In some embodiments, the iBAR sequence replaces one or more nucleotides in the loop region of stem loop 1. In some embodiments, the iBAR sequence is inserted in the loop region of stem loop 2. In some embodiments, the iBAR sequence replaces one or more nucleotides in the loop region of stem loop 2. In some embodiments, the iBAR sequence is inserted in the loop region of stem loop 3. In some embodiments, the iBAR sequence replaces one or more nucleotides in the loop region of stem loop 3.

In some embodiments, each sgRNA ^iBAR comprises in the 5'-to-3' direction a first stem sequence and a second stem sequence, wherein the first stem sequence hybridizes with the second stem sequence to form a double-stranded RNA (dsRNA) region that interacts with the Cas protein, and wherein the iBAR sequence is disposed between the 3' end of the first stem sequence and the 5' end of the second stem sequence.

In a CRISPR/Cas9 system, a guide RNA can be used to guide the cleavage of a genomic DNA by the Cas9 nuclease. For example, the guide RNA may be composed of a nucleotide spacer of variable sequence (guide sequence) that targets the CRISPR/Cas system nuclease to a genomic location in a sequence-specific manner, and an invariant hairpin sequence that is constant among different guide RNAs and allows the guide RNA to bind to the Cas nuclease. In some embodiments, the CRISPR/Cas guide RNA comprising a CRISPR/Cas variable guide sequence that is homologous or complementary to a target genomic sequence (e.g., target site of a hit gene) in a host cell and an invariant hairpin sequence that when transcribed is capable of binding a Cas nuclease (e.g., Cas9) , wherein the hairpin sequence codes for a Repeat: Anti-Repeat Duplex and a tetraloop, and an iBAR is embedded in the tetraloop region.

The guide sequence for a CRISPR/Cas9 guide RNA can be about any of 17-23, 18-22, or 19-21 nucleotides in length. The guide sequence can target the Cas nuclease to a genomic locus in a sequence-specific manner and can be designed following general principles known in the art. The invariant guide RNA hairpin sequences can be provided according to common knowledge in the art, for example, as disclosed by Nishimasu et al. (Nishimasu H, et al. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell. 2014; 156: 935-949) . Any invariant hairpin sequences may be used as long as they are capable of binding to a Cas nuclease once transcribed.

Previous studies showed that, although sgRNA with a 48-nt tracrRNA tail (referred to as sgRNA (+48) ) is the minimal region, for the Cas9-catalyzed DNA cleavage in vitro (Jinek et al., 2012) , sgRNAs with extended tracrRNA tails, sgRNA (+67) and sgRNA (+85) , may improve the Cas9 cleavage activity in vivo (Hsu et al., 2013) . In some embodiments, the sgRNA or sgRNA ^iBAR comprises stem loop 1, stem loop 2, and/or stem loop 3. The stem loop 1, stem loop 2 and/or stem loop 3 regions may improve editing efficiency in a CRISPR/Cas9 system. In some embodiments, the sgRNA comprises from 5' to 3': a guide sequence, a repeat-anti-repeat stem loop, a stem loop 1, a stem loop 2, and a stem loop 3. In some embodiments, the sgRNA ^iBAR comprises from 5' to 3': a guide sequence, a repeat-anti-repeat stem loop with an iBAR sequence inserted in the loop region, a stem loop 1, a stem loop 2, and a stem loop 3.

Vectors and vehicles

In some embodiments, the sgRNA construct comprises one or more regulatory elements operably linked to the guide RNA sequence. In some embodiments, the sgRNA ^iBAR construct comprises one or more regulatory elements operably linked to the guide RNA sequence and the iBAR sequence. Exemplary regulatory elements include, but are not limited to, promoters, enhancers, internal ribosomal entry sites (IRES) , and other expression control elements (e.g. transcription termination signals, such as polyadenylation signals and poly-U sequences) . Such regulatory elements are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) . Regulatory elements include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences) .

The sgRNA or sgRNA ^iBAR constructs may be present in a vector. In some embodiments, the vector is suitable for replication and integration in eukaryotic cells, such as mammalian cells (e.g., T cells) . In some embodiments, the sgRNA or sgRNA ^iBAR construct is an expression vector, such as a viral vector or a plasmid. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, lentiviral vector, retroviral vectors, herpes simplex viral vector, and derivatives thereof. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) , and in other virology and molecular biology manuals. It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression desired, etc. In some embodiments, the sgRNA or sgRNA ^iBAR construct is a lentiviral vector. In some embodiments, the sgRNA or sgRNA ^iBAR construct is a virus. In some embodiments, the sgRNA or sgRNA ^iBAR construct is an adenovirus or an adeno-associated virus. In some embodiments, the sgRNA or sgRNA ^iBAR construct is a lentivirus. In some embodiments, the vector further comprises a selection marker. In some embodiments, the vector further comprises one or more nucleotide sequences encoding one or more elements of the CRISPR/Cas system, such as a nucleotide sequence encoding a Cas nuclease (e.g., Cas9) . In some embodiments, there is provided a vector system comprising one or more vectors encoding nucleotide sequences encoding one or more elements of the CRISPR/Cas system, and a vector comprising any one of the sgRNA or sgRNA ^iBAR constructs described herein. A vector may include one or more of the following elements: an origin of replication, one or more regulatory sequences (e.g., promoters and/or enhancers) that regulate the expression of the polypeptide of interest, and/or one or more selectable marker genes (e.g., antibiotic resistance genes, or fluorescent protein-encoding genes) .

A number of viral based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The heterologous nucleic acid can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to the engineered mammalian cell in vitro or ex vivo. A number ofretroviral systems are known in the art. In some embodiments, adenovirus vectors are used. A number of adenovirus vectors are known in the art. In some embodiments, lentivirus vectors are used. In some embodiments, self-inactivating lentiviral vectors are used. Self-inactivating lentiviral vectors can be packaged into lentiviruses with protocols known in the art. The resulting lentiviruses can be used to transduce a mammalian cell (such as primary human T cells) using methods known in the art. Vectors derived from retroviruses such as lentivirus are suitable tools to achieve long-term gene transfer, because they allow long-term, stable integration of a transgene and its propagation in progeny cells. Lentiviral vectors also have low immunogenicity, and can transduce non-proliferating cells.

In some embodiments, the vector is a non-viral vector. In some embodiments, the vector is a transposon, such as a Sleeping Beauty transposon system, or a PiggyBac transposon system. In some embodiments, the vector is a polymer-based non-viral vector, including for example, poly (lactic-co-glycolic acid) (PLGA) and poly lactic acid (PLA) , poly (ethylene imine) (PEI) , and dendrimers. In some embodiments, the vector is a cationic-lipid based non-viral vector, such as cationic liposome, lipid nanoemulsion, and solid lipid nanoparticle (SLN) . In some embodiments, the vector is a peptide-based gene non-viral vector, such as poly-L-lysine. Any of the known non-viral vectors suitable for gene editing can be used for introducing the sgRNA or sgRNA ^iBAR-encoding nucleic acid to an immune effector cell (e.g., T cells) . See, for example, Yin H. et al. Nature Rev. Genetics (2014) 15: 521-555; Aronovich EL et al. “The Sleeping Beauty transposon system: a non-viral vector for gene therapy. ” Hum. Mol. Genet. (2011) R1: R14-20; and Zhao S. et al. “PiggyBac transposon vectors: the tools of the human gene editing. ” Transl. Lung Cancer Res. (2016) 5 (1) : 120-125, which are incorporated herein by reference. In some embodiments, any one or more of the nucleic acids encoding the sgRNAs or sgRNAs ^iBAR described herein is introduced to a T cell by a physical method, including, but not limited to electroporation, sonoporation, photoporation, magnetofection, hydroporation.

In some embodiments, the nucleic acid encoding the sgRNA or sgRNA ^iBAR, and the one or more nucleic acids encoding the one or more elements of the CRISPR/Cas system (e.g., Cas nuclease such as Cas9) , are on separate vectors (e.g., viral vector such as lentiviral vector) . In some embodiments, the nucleic acid encoding the sgRNA or sgRNA ^iBAR, and the one or more nucleic acids encoding the one or more elements of the CRISPR/Cas system, are on the same vector. In some embodiments, the nucleic acid encoding the sgRNA or sgRNA ^iBAR and the one or more nucleic acids encoding the one or more elements of the CRISPR/Cas system are operably controlled by separate promoters. In some embodiments, the nucleic acid encoding the sgRNA or sgRNA ^iBAR and the one or more nucleic acids encoding the one or more elements of the CRISPR/Cas system are operably controlled by the same promoter. In some embodiments, the nucleic acid encoding the sgRNA or sgRNA ^iBAR and the one or more nucleic acids encoding the one or more elements of the CRISPR/Cas system are connected by one or more linking sequences such as IRES.

The nucleic acid can be cloned into the vector using any known molecular cloning methods in the art, including, for example, using restriction endonuclease sites and one or more selectable markers. In some embodiments, the nucleic acid is operably linked to a promoter. Varieties of promoters have been explored for gene expression in mammalian cells, and any of the promoters known in the art may be used in the present invention. Promoters may be roughly categorized as constitutive promoters or regulated promoters, such as inducible promoters.

In some embodiments, the nucleic acid encoding the sgRNA or sgRNA ^iBAR and/or the one or more nucleic acids encoding the one or more elements of the CRISPR/Cas system (e.g., Cas9) is operably linked to a constitutive promoter. Constitutive promoters allow heterologous genes (also referred to as transgenes) to be expressed constitutively in the host cells. Exemplary promoters contemplated herein include, but are not limited to, cytomegalovirus immediate-early promoter (CMV IE) , human elongation factors-1alpha (hEF1α) , ubiquitin C promoter (UbiC) , phosphoglycerokinase promoter (PGK) , simian virus 40 early promoter (SV40) , chicken β-Actin promoter coupled with CMV early enhancer (CAGG) , a Rous Sarcoma Virus (RSV) promoter, a polyoma enhancer/herpes simplex thymidine kinase (MC 1) promoter, a beta actin (β-ACT) promoter, a “myeloproliferative sarcoma virus enhancer, negative control region deleted, d1587rev primer-binding site substituted (MND) ” promoter. The efficiencies of such constitutive promoters on driving transgene expression have been widely compared in a huge number of studies.

In some embodiments, the nucleic acid encoding the sgRNA or sgRNA ^iBAR and/or the one or more nucleic acids encoding the one or more elements of the CRISPR/Cas system (e.g., Cas9) is operably linked to an inducible promoter. Inducible promoters belong to the category of regulated promoters. The inducible promoter can be induced by one or more conditions, such as a physical condition, microenvironment of the engineered T cells, or the physiological state of the engineered T cells, an inducer (i.e., an inducing agent) , or a combination thereof. In some embodiments, the inducing condition does not induce the expression of endogenous genes in the engineered T cell, and/or in the subject that receives T cell therapy. In some embodiments, the inducing condition is selected from the group consisting of: inducer, irradiation (such as ionizing radiation, light) , temperature (such as heat) , redox state, tumor environment, and the activation state of the engineered T cell. In some embodiments, the inducible promoter can be an NFAT promoter, a

promoter, or an NFκB promoter.

Library

The sgRNA libraries described herein comprise one or a plurality of sgRNA constructs, wherein each sgRNA construct comprises or encodes an sgRNA, and wherein each sgRNA comprises a guide sequence that is complementary (e.g., at least about any of 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%complementary) to a target site in a corresponding hit gene in the genome. The sgRNA libraries described herein may be designed to target one or a plurality of genomic loci (e.g., a plurality of target sites in one or more hit genes in the genome) according to the needs of a genetic screen. In some embodiments, a single sgRNA construct is designed to target each hit gene. In some embodiments, a plurality of (e.g., at least about 2, 3, 4, 5, 10, 20, 100, or more) sgRNA constructs with different guide sequences targeting a single hit gene may be designed. For example, such plurality of sgRNA constructs may comprise or encode guide sequences targeting different target sites of a single hit gene, such as 2 (or about 3 to about 12) different target sites of a single hit gene.

sgRNA library comprising one or a plurality of sgRNA ^iBAR constructs are also referred to herein as sgRNA ^iBAR library, in which each sgRNA construct comprises or encodes an iBAR sequence. The sgRNA ^iBAR libraries described herein comprise one or a plurality of sgRNA ^iBAR constructs, wherein each sgRNA ^iBAR construct comprises or encodes an sgRNA ^iBAR, and wherein each sgRNA ^iBAR comprises a guide sequence that is complementary (e.g., at least about any of 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%complementary) to a target site in a corresponding hit gene in the genome. The sgRNA ^iBAR libraries described herein may be designed to target one or a plurality of genomic loci (e.g., a plurality of target sites in one or more hit genes in the genome) according to the needs of a genetic screen. In some embodiments, a single sgRNA ^iBAR construct is designed to target each hit gene. In some embodiments, a plurality of (e.g., at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, or more) sgRNA ^iBAR constructs with different guide sequences targeting a single hit gene may be designed. For example, such plurality of sgRNA ^iBAR constructs may comprise or encode guide sequences targeting different target sites of a single hit gene, such as 2 different target sites of a single hit gene.

In some embodiments, the sgRNA ^iBAR library described herein comprises one or a plurality of sets of sgRNA ^iBAR constructs, wherein each set of sgRNA ^iBAR constructs comprise three or more (e.g., 3, 4, 5, or more, such as 4) sgRNA ^iBAR constructs each comprising or encoding an sgRNA ^iBAR, wherein each sgRNA ^iBAR comprises a guide sequence and an iBAR sequence, wherein the guide sequences for the three or more sgRNA ^iBAR constructs are the same, wherein the iBAR sequence for each of the three or more sgRNA ^iBAR constructs is different from each other, and wherein the guide sequence of each set of sgRNA ^iBAR constructs is complementary (e.g., at least about any of 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%complementary) to a different target site of a corresponding hit gene (e.g., different hit genes, or different sites within the same hit gene) in the genome. In some embodiments, each set of sgRNA ^iBAR constructs comprises four sgRNA ^iBAR constructs, and wherein the iBAR sequence for each of the four sgRNA ^iBAR constructs is different from each other. In some embodiments, a single set of sgRNA ^iBAR constructs is designed to target each hit gene. In some embodiments, the sgRNA ^iBAR library comprises a plurality of (e.g., at least about 2, 3, 4, 5, 10, 20, or more) sets of sgRNA ^iBAR constructs with different guide sequences targeting a single hit gene. In some embodiments, the sgRNA ^iBAR library comprises at least 2 (such as 2) sets of sgRNA ^iBAR constructs designed to target at least 2 (such as 2) different target sites of every hit gene, wherein each set of sgRNA ^iBAR constructs comprises four sgRNA ^iBAR constructs. In some embodiments, the sgRNA ^iBAR library comprises at least about 100 sets of sgRNA ^iBAR constructs, such as at least about any of 200, 300, 400, 800, 1,000, 2,000, 3,000, 5,000, 10,000, 15,000, 19,000, 20,000, 40,000, 50,000, 100,000, 150,000, 200,000 or more sets of sgRNA ^iBAR constructs. In some embodiments, the sgRNA ^iBAR library comprises at least about 100, such as about 18,000 to about 20,000, sets of sgRNA ^iBAR constructs. In some embodiments, the sgRNA ^iBAR library comprises about 36,000 to about 40,000 sets of sgRNA ^iBAR constructs.

In some embodiments, the sgRNA library or sgRNA ^iBAR library comprises at least about any of 1, 2, 3, 4, 5, 10, 20, 50, 100, 200, 400, 500, 1,000, 2,000, 4,000, 5,000, 10,000, 15,000, 19,000, 20,000, 38,000, 39,000, 40,000, 50,000, 100,000, 150,000, 155,000, 200,000 or more sgRNA constructs or sgRNA ^iBAR constructs. In some embodiments, the sgRNA library or sgRNA ^iBAR library comprises at least about 150,000 sgRNA constructs or sgRNA ^iBAR constructs. In some embodiments, the sgRNA library comprises about 15,000 to about 200,000 sgRNA constructs, such as about 18,000 to about 20,000, about 38,000 to about 40,000, about 18,000 to about 50,000, about 50,000 to about 100,000, about 100,000 to about 200,000, about 140,000 to about 180,000, or about 150,000 to about 160,000 sgRNA constructs. In some embodiments, the sgRNA ^iBAR library comprises about 15,000 to about 200,000 sgRNA ^iBAR constructs, such as about 18,000 to about 50,000, about 18,000 to about 20,000, about 38,000 to about 40,000, about 50,000 to about 100,000, about 100,000 to about 200,000, about 140,000 to about 180,000, or about 150,000 to about 160,000 sgRNA ^iBAR constructs. In some embodiments, the sgRNA ^iBAR library comprises at least about any of 1, 2, 3, 4, 5, 10, 20, 50, 100, 200, 400, 500, 1,000, 2,000, 5,000, 10,000, 19,000, 20,000, 38,000, 50,000, 100,000, 150,000, 200,000 or more sets of sgRNA ^iBAR constructs. In some embodiments, the sgRNA library or the sgRNA ^iBAR library targets at least about any of 1, 2, 3, 4, 5, 10, 20, 50, 100, 200, 500, 1,000, 2,000, 5,000, 10,000, 15,000, 19,000, 20,000, 38,000, 50,000 or more genes in a cell or an organism. In some embodiments, the organism is human. In some embodiments, the sgRNA library or the sgRNA ^iBAR library is a whole-genome library for protein-coding genes and/or non-coding RNAs. In some embodiments, the sgRNA library or the sgRNA ^iBAR library is a whole-genome library for every annotated gene. Thus in some embodiments, the sgRNA library comprising a plurality of sgRNA constructs comprises or encodes sgRNAs with guide sequences complementary to target sites of every annotated gene in the genome, such as target sites of 19, 114 annotated genes in the human genome. In some embodiments, the sgRNA ^iBAR library comprising a plurality of sgRNA ^iBAR constructs comprises or encodes sgRNAs ^iBAR with guide sequences complementary to target sites of every annotated gene in the genome, such as target sites of 19, 114 annotated genes in the human genome. In some embodiments, the sgRNA library or the sgRNA ^iBAR library targets at least about any of 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%of the genes in a cell or an organism. In some embodiments, the sgRNA library or the sgRNA ^iBAR library is a targeted library, which targets selected genes in a signaling pathway or associated with a cellular process, such as sensitivity or resistance to immune effector cell (e.g., NK cell) -mediated killing, cell proliferation, cell cycle, transcriptional regulation, ubiquitination, apoptosis, immune response such as autoimmune, tumor metastasis, tumor malignant transformation, etc. In some embodiments, the sgRNA library or the sgRNA ^iBAR library is used for a genome-wide screen associated with a particular modulated phenotype, such as sensitivity or resistance to immune effector cell (e.g., NK cell) -mediated killing. In some embodiments, the sgRNA library or the sgRNA ^iBAR library is used for a genome-wide screen to identify at least one target gene associated with a particular modulated phenotype, such as a target gene in a T cell that modulates the activity of the T cell in response to NK cell treatment. In some embodiments, the sgRNA library or the sgRNA ^iBAR library is designed to target an eukaryotic genome, such as a mammalian genome. Exemplary genomes of interest include genomes of a rodent (mouse, rat, hamster, guinea pig) , a domesticated animal (e.g., cow, sheep, cat, dog, horse, or rabbit) , a non-human primate (e.g., monkey) , fish (e.g., zebrafish) , non-vertebrate (e.g., Drosophila melanogaster and Caenorhabditis elegans) , and human.

The guide sequences of the sgRNA libraries or the sgRNA ^iBAR libraries may be designed using any known algorithms that identify CRISPR/Cas target sites in user-defined lists with a high degree of targeting specificity in the human genome, such as Genomic Target Scan (GT-Scan) (see O'Brien et al., Bioinformatics (2014) 30: 2673-2675) ) , DeepCRISPR, CasFinder, CHOPCHOP, CRISPRscan, etc. In some embodiments, at least about any of 100, 400, 500, 1,000, 5,000, 10,000, 15,000, 19,000, 20,000, 50,000, 100,000, 150,000, 155,000, 200,000, or more sgRNA constructs or sgRNA ^iBAR constructs can be generated on a single array. In some embodiments, at least about any of 19,000, 20,000, 38,000, 50,000, 100,000, 150,000, 155,000, 200,000, or more sgRNA constructs or sgRNA ^iBAR constructs can be generated on a single array, providing sufficient coverage to comprehensively screen all genes in a human genome. This approach can also be scaled up to enable genome-wide screens by the synthesis of multiple sgRNA libraries or sgRNA ^iBAR libraries in parallel. The exact number of sgRNA constructs in an sgRNA library, or the exact number of sgRNA ^iBAR constructs (or sets of sgRNA ^iBAR constructs) in an sgRNA ^iBAR library, can depend on whether the screen 1) targets genes or regulatory elements, 2) targets the complete genome, or subgroup of the genomic genes.

In some embodiments, the sgRNA library or the sgRNA ^iBAR library is designed to target every PAM sequence overlapping a gene in a genome, wherein the PAM sequence corresponds to the Cas protein. In some embodiments, the sgRNA library or the sgRNA ^iBAR library is designed to target a subset of the PAM sequences found in the genome, wherein the PAM sequence corresponds to the Cas protein.

In some embodiments, the sgRNA library comprises one or more control sgRNA constructs that do not target any genomic loci in a genome. In some embodiments, sgRNA constructs that do not target putative genomic genes can be included in an sgRNA library as negative controls. In some embodiments, the sgRNA ^iBAR library comprises one or more control sgRNA ^iBAR constructs that do not target any genomic loci in a genome. In some embodiments, sgRNA ^iBAR constructs that do not target putative genomic genes can be included in an sgRNA ^iBAR library as negative controls.

The sgRNA constructs and libraries described herein may be prepared using any known nucleic acid synthesis and/or molecular cloning methods in the art. In some embodiments, the sgRNA library is synthesized by electrochemical means on arrays (e.g., CustomArray, Twist, Gen9) , DNA printing (e.g., Agilent) , or solid phase synthesis of individual oligos (e.g., by IDT) . The sgRNA constructs can be amplified by PCR and cloned into an expression vector (e.g., a lentiviral vector) . In some embodiments, the lentiviral vector further encodes one or more components of the CRISPR/Cas-based genetic editing system, such as the Cas protein, e.g., Cas9.

The present invention in some embodiments provides isolated nucleic acids encoding any of the sgRNA constructs, sgRNA ^iBAR constructs, sets of sgRNA ^iBAR constructs, sgRNA library, sgRNA ^iBAR library, or B2M sgRNA construct described herein. Also provided are vectors (e.g., non-viral vector, or viral vector such as lentiviral vector) and virus (e.g., lentivirus) comprising any of the nucleic acids encoding any of the sgRNA constructs, sgRNA ^iBAR constructs, sets of sgRNA ^iBAR constructs, sgRNA library, sgRNA ^iBAR library, and/or B2M sgRNA construct described herein.

Cas protein

The sgRNA constructs or sgRNA ^iBAR constructs described herein may be designed to operate with any one of the naturally-occurring or engineered CRISPR/Cas systems known in the art. In some embodiments, the sgRNA construct or the sgRNA ^iBAR construct is operable with a Type I CRISPR/Cas system. In some embodiments, the sgRNA construct or the sgRNA ^iBAR construct is operable with a Type II CRISPR/Cas system. In some embodiments, the sgRNA construct or the sgRNA ^iBAR construct is operable with a Type III CRISPR/Cas system. Exemplary CRISPR/Cas systems can be found in WO2013176772, WO2014065596, WO2014018423, WO2016011080, US8697359, US8932814, US10113167B2, the disclosures of which are incorporated herein by reference in their entireties for all purposes.

In certain embodiments, the sgRNA construct or the sgRNA ^iBAR construct is operable with a Cas protein derived from a CRISPR/Cas type I, type II, or type III system, which has an RNA-guided polynucleotide binding and/or nuclease activity. Examples of such Cas proteins are recited in, e.g., WO2014144761 WO2014144592, WO2013176772, US20140273226, and US20140273233, which are incorporated herein by reference in their entireties.

In certain embodiments, the Cas protein is derived from a type II CRISPR-Cas system. In certain embodiments, the Cas protein is or is derived from a Cas9 protein. In certain embodiments, the Cas protein is or is derived from a bacterial Cas9 protein, including those identified in WO2014144761.

In some embodiments, the sgRNA construct or the sgRNA ^iBAR construct is operable with Cas9 (also known as Csnl and Csxl2) , a homolog thereof, or a modified version thereof. In some embodiments, the sgRNA construct or the sgRNA ^iBAR construct is operable with two or more (e.g., 2, 3, 4, 5, or more) Cas proteins. In some embodiments, the sgRNA construct or the sgRNA ^iBAR construct is operable with a Cas9 protein from S. pyogenes or S. pneumoniae. Cas enzymes are known in the art; for example, the amino acid sequence of S. pyogenes Cas9 protein may be found in the SwissProt database under accession number Q99ZW2.

The Cas protein (also referred herein as “Cas nuclease” ) provides a desired activity, such as target binding, target nicking or cleaving activity. In certain embodiments, the desired activity is target binding. In certain embodiments, the desired activity is target nicking or target cleaving. In certain embodiments, the desired activity also includes a function provided by a polypeptide that is covalently fused to a Cas protein or a nuclease-deficient Cas protein. Examples of such a desired activity include a transcription regulation activity (either activation or repression) , an epigenetic modification activity, or a target visualization/identification activity.

In some embodiments, the sgRNA construct or the sgRNA ^iBAR construct is operable with a Cas nuclease that cleaves the target sequence, including double-strand cleavage and single-strand cleavage. In some embodiments, the sgRNA construct or the sgRNA ^iBAR construct is operable with a catalytically inactive Cas ( “dCas” ) . In some embodiments, the sgRNA construct or the sgRNA ^iBAR construct is operable with a dCas of a CRISPR activation ( “CRISPRa” ) system, wherein the dCas is fused to a transcriptional activator. In some embodiments, the sgRNA construct or the sgRNA ^iBAR construct is operable with a dCas of a CRISPR interference (CRISPRi) system. In some embodiments, the dCas is fused to a repressor domain, such as a KRAB domain. Such CRISPR/Cas systems can be used to modulate (e.g., induce, repress, increase, or reduce) gene expression.

In certain embodiments, the Cas protein is a mutant of a wild type Cas protein (such as Cas9) or a fragment thereof. A Cas9 protein generally has at least two nuclease (e.g., DNase) domains. For example, a Cas9 protein can have a RuvC-like nuclease domain and an HNH-like nuclease domain. The RuvC and HNH domains work together to cut both strands in a target site to make a double-stranded break in the target polynucleotide. (Jinek et al., Science 337: 816-21) . In certain embodiments, a mutant Cas9 protein is modified to contain only one functional nuclease domain (either a RuvC-like or an HNH-like nuclease domain) . For example, in certain embodiments, the mutant Cas9 protein is modified such that one of the nuclease domains is deleted or mutated such that it is no longer functional (i.e., the nuclease activity is absent) . In some embodiments where one of the nuclease domains is inactive, the mutant is able to introduce a nick into a double-stranded polynucleotide (such protein is termed a “nickase” ) but not able to cleave the double-stranded polynucleotide. In certain embodiments, the Cas protein is modified to increase nucleic acid binding affinity and/or specificity, alter an enzymatic activity, and/or change another property of the protein. In certain embodiments, the Cas protein is truncated or modified to optimize the activity of the effector domain. In certain embodiments, both the RuvC-like nuclease domain and the HNH-like nuclease domain are modified or eliminated such that the mutant Cas9 protein is unable to nick or cleave the target polynucleotide. In certain embodiments, a Cas9 protein that lacks some or all nuclease activity relative to a wild-type counterpart, nevertheless, maintains target recognition activity to a greater or lesser extent.

In certain embodiments, the Cas protein is a fusion protein comprising a naturally-occurring Cas or a variant thereof fused to another polypeptide or an effector domain. The another polypeptide or effector domain may be, for example, a cleavage domain, a transcriptional activation domain, a transcriptional repressor domain, or an epigenetic modification domain. In certain embodiments, the fusion protein comprises a modified or mutated Cas protein in which all the nuclease domains have been inactivated or deleted. In certain embodiments, the RuvC and/or HNH domains of the Cas protein are modified or mutated such that they no longer possess nuclease activity.

In certain embodiments, the effector domain of the fusion protein is a cleavage domain obtained from any endonuclease or exonuclease with desirable properties.

In certain embodiments, the effector domain of the fusion protein is a transcriptional activation domain. In general, a transcriptional activation domain interacts with transcriptional control elements and/or transcriptional regulatory proteins (i.e., transcription factors, RNA polymerases, etc. ) to increase and/or activate transcription of a gene. In certain embodiments, the transcriptional activation domain is a herpes simplex virus VP16 activation domain, VP64 (which is a tetrameric derivative of VP16) , a NFκB p65 activation domain, p53 activation domains 1 and 2, a CREB (cAMP response element binding protein) activation domain, an E2A activation domain, or an NFAT (nuclear factor of activated T-cells) activation domain. In certain embodiments, the transcriptional activation domain is Gal4, Gcn4, MLL, Rtg3, Gln3, Oaf1, Pip2, Pdr1, Pdr3, Pho4, or Leu3. The transcriptional activation domain may be wild type, or modified or truncated version of the original transcriptional activation domain.

In certain embodiments, the effector domain of the fusion protein is a transcriptional repressor domain, such as inducible cAMP early repressor (ICER) domains, Kruppel-associated box A (KRAB-A) repressor domains, YY1 glycine rich repressor domains, Spl-like repressors, E (spI) repressors, I. kappa. B repressor, or MeCP2.

In certain embodiments, the effector domain of the fusion protein is an epigenetic modification domain which alters gene expression by modifying the histone structure and/or chromosomal structure, such as a histone acetyltransferase domain, a histone deacetylase domain, a histone methyltransferase domain, a histone demethylase domain, a DNA methyltransferase domain, or a DNA demethylase domain.

In certain embodiments, the Cas protein further comprises at least one additional domain, such as a nuclear localization signal (NLS) , a cell-penetrating or translocation domain, and a marker domain (e.g., a fluorescent protein marker) .

The Cas protein can be introduced into T cells as a (i) Cas protein, or (ii) mRNA encoding the Cas protein, or (iii) a linear or circular DNA encoding the protein. The Cas protein or construct encoding the Cas protein may be purified, or non-purified in a composition. Methods of introducing a protein or nucleic acid construct into a host cell are well known in the art, and are applicable to all methods described herein which requires introduction of a Cas protein or construct thereof to a T cell. In certain embodiments, the Cas protein is delivered into a T cell as a protein. In certain embodiments, the Cas protein is constitutively expressed from an mRNA or a DNA in a host T cell. In certain embodiments, the expression of Cas protein from mRNA or DNA is inducible or induced in a host T cell. In certain embodiments, a Cas protein can be introduced into a host T cell in Cas protein: sgRNA complex using recombinant technology known in the art. Exemplary methods of introducing a Cas protein or construct thereof have been described, e.g., in WO2014144761 WO2014144592 and WO2013176772, which are incorporated herein by reference in their entireties.

In some embodiments, the method uses a CRISPR/Cas9 system. Cas9 is a nuclease from the microbial type II CRISPR (clustered regularly interspaced short palindromic repeats) system, which has been shown to cleave DNA when paired with a single-guide RNA (sgRNA) . The sgRNA directs Cas9 to complementary regions in the target genome gene, which may result in site-specific double-strand breaks (DSBs) that can be repaired in an error-prone fashion by cellular non-homologous end joining (NHEJ) machinery. Wildtype Cas9 primarily cleaves genomic sites at which the gRNA sequence is followed by a PAM sequence (-NGG) . NHEJ-mediated repair of Cas9-induced DSBs induces a wide range of mutations initiated at the cleavage site which are typically small (＜10 bp) insertion/deletions (indels) but can include larger (＞100 bp) indels.

T cell library

The T cell library described herein comprises a plurality of (e.g., at least about any of 2, 3, 4, 5, 10, 100, 1×10 ³, 1×10 ⁴, 1×10 ⁵, 1×10 ⁶, 1×10 ⁷, 2×10 ⁷, 3.5× 10 ⁷, 1×10 ⁸, or more) T cells (e.g., cytotoxic T lymphocyte or “CTL” ) , wherein each of the plurality of T cells has a mutation (e.g., inactivating mutation) at a hit gene in the genome (e.g., human genome) , and wherein the hit gene in at least two of the plurality of T cells are different from each other. In some embodiments, the T cell library further comprises a B2M mutation (e.g., inactivating B2M mutation) .

In some embodiments, the T cell library comprises a plurality of T cells that have mutations (e.g., inactivating mutations) in at least about any of 2, 3, 4, 5, 10, 20, 50, 100, 200, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, or more hit genes in a cell or organism. In some embodiments, the organism is human. In some embodiments, the T cell library comprises a plurality of T cells that have mutations (e.g., inactivating mutations) at about 15,000 to about 50,000 hit genes, such as about 18,000 to about 20,000 hit genes. In some embodiments, the T cell library comprises at least about any of 2, 3, 4, 5, 10, 20, 50, 100, 200, 500, 1,000, 2,000, 5,000, 1×10 ⁴, 2×10 ⁴, 5×10 ⁴, 1×10 ⁵, 2×10 ⁵, 1×10 ⁶, 5×10 ⁶, 1×10 ⁷, 1.5×10 ⁷, 2×10 ⁷, 3.5×10 ⁷, 1×10 ⁸, 1×10 ⁹, 1×10 ¹⁰, or more T cells. In some embodiments, at least two T cells within the T cell library have mutations (e.g., inactivating mutation) at different target sites (e.g., different hit genes, or different sites within the same hit gene) . In some embodiments, each T cell within the T cell library has a mutation (e.g., inactivating mutation) at a different hit gene. In some embodiments, each T cell within the T cell library has a mutation (e.g., inactivating mutation) at a different target site (e.g., can be within the same hit gene, or within different hit genes) . In some embodiments, the T cell library does not contain T cells that have mutation (e.g., inactivating mutation) at the same hit gene, such as inactivating mutation at the same target site of the same hit gene, or inactivating mutations at different target sites of the same hit gene. In some embodiments, the T cell library does not contain T cells that have mutation (e.g., inactivating mutation) at the same target site. In some embodiments, the plurality of (e.g., at least about 2, 3, 4, 5, 10, 100, 500, 1,000, 2000, 5,000, 10,000, 2×10 ⁷, or more) T cells within the T cell library have a mutation (e.g., inactivating mutation) at the same hit gene, such as inactivating mutation at the same target site of the same hit gene, or inactivating mutations at different target sites of the same hit gene. In some embodiments, the T cell library comprises a plurality of T cells that contain mutations (e.g., inactivating mutations) in at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 60%, 70%, 80%, 90%, 95%, or more hit genes in the genome. In some embodiments, the T cell library comprises a plurality of T cells that contain mutations (e.g., inactivating mutations) at all genes in the genome (also referred to herein as “whole-genome T cell library” ) , such as all annotated genes of the human genome. In some embodiments, for each annotated gene in the genome, or for each hit gene, there are at least two (e.g., 2, 3, 4, 5, or more, such as 2) T cells in the T cell library that each contains a mutation (e.g., inactivating mutation) in a different target site of the same hit gene, e.g., T cell A contains a mutation (e.g., inactivating mutation) in target site A' of gene X, and T cell B contains a mutation (e.g., inactivating mutation) in target site B' of gene X. In some embodiments, the T cell library is a targeted library, which contains mutations (e.g., inactivating mutations) at selected genes in a signaling pathway or associated with a cellular process, such as sensitivity or resistance to immune effector cell (e.g., NK cell) -mediated killing, cell proliferation, cell cycle, transcriptional regulation, ubiquitination, apoptosis, immune response such as autoimmune, tumor metastasis, tumor malignant transformation, etc. In some embodiments, the T cell library is used for a genome-wide screen associated with a particular modulated phenotype, such as sensitivity or resistance to immune effector cell (e.g., NK cell) -mediated killing. In some embodiments, the T cell library is used for a genome-wide screen to identify at least one target gene associated with a particular modulated phenotype, such as a target gene in a T cell that modulates the activity of the T cell in response to NK cell treatment. In some embodiments, the T cell library is an eukaryotic T cell library, such as a mammalian T cell library. Exemplary genomes of interest covered by the T cell library include genomes of a rodent (mouse, rat, hamster, guinea pig) , a domesticated animal (e.g., cow, sheep, cat, dog, horse, or rabbit) , a non-human primate (e.g., monkey) , fish (e.g., zebrafish) , non-vertebrate (e.g., Drosophila melanogaster and Caenorhabditis elegans) , and human. In some embodiments, the T cell library is a human T cell library, such as a human whole-genome T cell library.

In some embodiments, a plurality of (e.g., about 2, 3, 4, 5, 10, 100, 500, 1000, 2000, 5000, 10000, or more) T cells within a whole-genome T cell library have a mutation (e.g., inactivating mutation) at the same hit gene, such whole-genome T cell library is also referred to as “having X-fold coverage for the genome” or “having X-fold coverage for each gene, ” wherein “X” is the number of T cells with mutation (e.g., inactivating mutation) at the same hit gene. For example, a whole-genome T cell library comprising about 19, 114 T cells (each having a mutation such as inactivating mutation at a different hit gene) has averagely about 1-fold coverage for a human genome (about 19, 114 annotated genes) . A whole-genome T cell library comprising about 1.9× 10 ⁷ T cells has averagely about 1000-fold coverage for a human genome, i.e., about 1000 T cells have mutations (e.g., inactivating mutations) at the same hit gene. A whole-genome T cell library comprising about 3.56×10 ⁷ T cells, wherein about 2000 T cells have mutations (e.g., inactivating mutations) at the same hit gene (e.g., about 1000 T cells have mutations such as inactivating mutations at a first target site of the same hit gene, about 1000 T cells have mutations such as inactivating mutations at a second target site of the same hit gene; or about 2000 T cells have mutations such as inactivating mutations at the same target site of the same hit gene) , has averagely about 1000-fold coverage for a human genome. In some embodiments, the T cell library described herein has averagely at least about 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 100-fold, 200-fold, 500-fold, 1,000-fold, 2,000-fold, 5,000-fold, 10,000-fold, or more fold coverage of the genome (e.g., human genome) . In some embodiments, the T cell library described herein has averagely at least about 1,000-fold coverage of the human genome. In some embodiments, the whole-genome T cell library described herein has averagely at least about 100-fold coverage of the human genome. In some embodiments, the Cas9 ⁺ sgRNA T cell library has averagely about 100-fold to about 1000-fold coverage for each sgRNA. In some embodiments, the Cas9 ⁺ sgRNA (or mutagenic agent-induced mutation) T cell library described herein has averagely about 300-fold to about 3000-fold coverage of each hit gene. In some embodiments, the Cas9 ⁺ sgRNA ^iBAR T cell library has averagely about 25-fold to about 250-fold, such as about 100-fold, coverage for each sgRNA ^iBAR. In some embodiments, the Cas9 ⁺ sgRNA ^iBAR T cell library has averagely about 100-fold to about 1000-fold, such as about 400-fold, coverage for each set of sgRNAs ^iBAR. In some embodiments, the Cas9 ⁺ sgRNA ^iBAR T cell library described herein has averagely about 300-fold to about 3000-fold (e.g., any of about 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1500, 1800, 2000, 2400, or 3000, fold) , such as about 300-fold, coverage of each hit gene.

Mutations at hit genes

In some embodiments, all annotated genes in the genome (e.g., human genome) are selected as hit genes. In some embodiments, a hit gene is further selected based on that the encoded mRNA or protein expresses within a T cell, or that the encoded protein expresses on the T cell surface, either in heathy T cells or in T cells of disease status.

In some embodiments, the mutation at a hit gene is a pathogenic mutation or an inactivating mutation. An inactivating mutation described herein can be any mutation, such as insertion, deletion (indels) , substitution, frame shift, chromosomal rearrangement, or combinations thereof, that leads to complete abolishment or elimination of a gene's expression (transcription and/or translation) and/or function. Inactivating mutations in some embodiments can completely abolish the transcription, translation, post-translation modification, association with other molecules (e.g., other molecules in a protein complex) , and/or function (e.g., signal transduction or receptor activation) of a gene. In some embodiments, the mutation at a hit gene is a mutation that reduces (e.g., reduces at least about any of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more) or affects (e.g., disrupts) one or more of hit gene transcription, hit gene translation, hit gene mRNA processing, hit gene mRNA stability, hit gene mRNA function, hit gene protein function, association with other molecules (e.g., other molecules in a protein complex) , and hit gene post-translation modification. The mutation (e.g., inactivating mutation) at a hit gene can be within one or more of regulatory region such as enhancer, promoter, 5' untranslated region (UTR) , 3'UTR, or the coding region such as an exon or a splicing site, of a hit gene. A hit gene described herein can be any genomic sequence, such as a protein-encoding gene, a gene encoding an RNA, such as a small RNA (e.g., microRNA, piRNA, siRNA, snoRNA, tRNA, rRNA and snRNA) , a ribosomal RNA, a long non-coding RNA (lincRNA) , or a mitochondrial gene. The hit gene may be known to be associated with a particular phenotype; or has not been implicated in a particular phenotype, such as a known gene that is not known to be associated with a particular phenotype, or an unknown gene that has not been characterized. In some embodiments, the hit gene is a genomic sequence that does not encode anything, or not yet known to encode anything.

Pathogenic inactivating mutations (loss-of-function) of certain genes can be determined by review of experimental evidence within the published scientific literature and review of critical regions that may be disrupted, including but not limited to frameshift, missense mutations, truncating mutations, deletions, copy number variations, nonsense mutations, and loss or deletion of the gene. Pathogenic or inactivating mutation includes but not limited to homozygous deletions, bi-allelic (double hit) mutations, splice site mutations (e.g., a 2nd or an additional splice site mutation) , frameshift mutations, and nonsense mutations in coding region, missense mutations with confirmed impact.

In some embodiments, the T cell library is generated by subjecting (e.g., contacting) an initial population of T cells (e.g., allogeneic T cells or CAR-T cells (such as allogeneic CAR-T cells) ) to mutagenic agents. Mutagenic agents can be classified into three categories: physical (e.g., gamma rays, ultraviolet radiations) , chemical (e.g., ethyl methane sulphonate or EMS) and transposable elements (such as transposons, retrotransposons, T-DNA, retroviruses) .

In some embodiments, the T cell library is generated by subjecting an initial population of T cells (e.g., allogeneic T cells or CAR-T cells (such as allogeneic CAR-T cells) ) to gene editing (e.g., genome-wide gene editing) . Any known gene editing methods can be used for generating T cell libraries described herein, such as Zinc-finger nucleases (ZFNs) , transcription activator-like effector nucleases (TALENs) , and CRISPR/Cas-based methods for gene editing or genome engineering. See, e.g., Gaj et al. (Trends Biotechnol. 2013; 31 (7) : 397-405) . In some embodiments, the T cell library is generated by subjecting an initial population of T cells to genome-wide gene editing via CRISPR/Cas-based methods.

In some embodiments, the T cell library is generated by contacting an initial population of T cells (e.g., allogeneic T cells or CAR-T cells (such as allogeneic CAR-T cells) ) with i) an sgRNA library or an sgRNA ^iBAR library descried herein; and optionally ii) a Cas component comprising a Cas protein or a nucleic acid encoding the Cas protein (e.g., Cas9) , under a condition that allows introduction of the sgRNA constructs or sgRNA ^iBAR constructs and the optional Cas component into the initial population of T cells. Hence in some embodiments, the T cell library is generated by contacting an initial population of T cells with i) an sgRNA library comprising a plurality of sgRNA constructs, wherein each sgRNA construct comprises or encodes an sgRNA, and wherein each sgRNA comprises a guide sequence that is complementary (e.g., at least about any of 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%complementary) to a target site in a corresponding hit gene in the genome; and optionally ii) a Cas component comprising a Cas protein or a nucleic acid encoding the Cas protein, under a condition that allows introduction of the sgRNA constructs and the optional Cas component into the initial population of T cells. In some embodiments, the T cell library is generated by contacting an initial population of T cells with i) an sgRNA ^iBAR library comprising a plurality of sets of sgRNA ^iBAR constructs, wherein each set of sgRNA ^iBAR constructs comprise three or more (e.g., 3, 4, 5, or more, such as 4) sgRNA ^iBAR constructs each comprising or encoding an sgRNA ^iBAR, wherein the guide sequences for the three or more sgRNA ^iBAR constructs are the same, wherein the ^iBAR sequence for each of the three or more sgRNA ^iBAR constructs is different from each other, and wherein the guide sequence of each set of sgRNA ^iBAR constructs is complementary (e.g., at least about any of 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%complementary) to a different target site of a corresponding hit gene (e.g., different hit genes, or different sites within the same hit gene) in the genome; and optionally ii) a Cas component comprising a Cas protein or a nucleic acid encoding the Cas protein, under a condition that allows introduction of the sgRNA constructs and the optional Cas component into the initial population of T cells. In some embodiments, the condition further allows generation of mutations at the hit genes. In some embodiments, each set of sgRNA ^iBAR constructs comprises four sgRNA ^iBAR constructs, and wherein the iBAR sequence for each of the four sgRNA ^iBAR constructs is different from each other. In some embodiments, the sgRNA library or the sgRNA ^iBAR library, and the Cas component, are introduced into the initial population of T cells simultaneously. In some embodiments, the sgRNA library or the sgRNA ^iBAR library, and the Cas component, are introduced into the initial population of T cells sequentially. In some embodiments, the sgRNA library or the sgRNA ^iBAR library, and the Cas component, are introduced into the initial population of T cells via separate vectors (e.g., lentiviral vectors) or separate viruses. In some embodiments, the sgRNA library or the sgRNA ^iBAR library, and the Cas component, are introduced into the initial population of T cells via the same vector or the same virus. In some embodiments, the sgRNA library or the sgRNA ^iBAR library is introduced into the initial population of T cells via lentiviral vectors or lentiviruses, and the Cas component is introduced into the initial population of T cells as mRNA encoding the Cas component (e.g., Cas9) . In some embodiments, the initial population of T cells already each carries a Cas component (e.g., transgenic Cas9, or Cas9 introduced as mRNA; hereinafter also referred to as “Cas9 ⁺ T cells” ) , and the sgRNA library or the sgRNA ^iBAR library is then introduced into each cell, such as via a vector (e.g., lentiviral vector) or virus (e.g., lentivirus) .

In some embodiments, the T cell library only comprises the sgRNA library or the sgRNA ^iBAR library described herein and does not comprise a Cas component (e.g., Cas9) , i.e., the hit genes targeted by the sgRNA library or the sgRNA ^iBAR library have not been inactivated in the T cell library yet, until a Cas component (e.g., Cas9) is further introduced (e.g., when introducing sgRNA against B2M) . T cell libraries comprising an sgRNA library or an sgRNA ^iBAR library described herein are referred to hereinafter as “sgRNA T cell library, ” or “sgRNA ^iBAR T cell library. ” In some embodiments, the T cell library comprises both the sgRNA library or the sgRNA ^iBAR library, and the Cas component (e.g., Cas9) , i.e., the T cell library comprises inactivated hit genes. In some embodiments, the initial population of T cells express a Cas protein. In some embodiments, the T cell library is generated by contacting an initial population of T cells expressing a Cas protein with an sgRNA library or an sgRNA ^iBAR library descried herein, which will result in T cell library comprising inactivated hit genes. T cell libraries comprising an sgRNA library or an sgRNA ^iBAR library described herein, and a Cas9 component (e.g., Cas9 protein, or nucleic acid encoding thereof) are referred to hereinafter as “Cas9 ⁺ sgRNA T cell library, ” or “Cas9 ⁺ sgRNA ^iBAR T cell library. ” In some embodiments, the T cells in the initial population of T cells comprise a B2M mutation (e.g., by introducing an sgRNA against B2M, and Cas9) , such as an inactivating B2M mutation, such T cells are also referred to herein as “B2M ^-T cells, ” and the resulting T cell library is referred to as “B2M ^-T cell library. ” In some embodiments, the T cells in the initial population of T cells comprise an sgRNA construct against B2M (e.g., sgRNA against B2M, or a vector encoding thereof) . T cell libraries comprising an sgRNA library or an sgRNA ^iBAR library described herein, and an sgRNA against B2M, are referred to hereinafter as “B2M-sgRNA sgRNA T cell library, ” or “B2M-sgRNA sgRNA ^iBAR T cell library. ” T cell libraries comprising an sgRNA library or an sgRNA ^iBAR library described herein, an sgRNA against B2M, and a Cas9 component (e.g., Cas9 protein, or nucleic acid encoding thereof) are referred to hereinafter as “Cas9 ⁺ B2M ^-sgRNA T cell library, ” or “Cas9 ⁺ B2M ^-sgRNA ^iBAR T cell library, ” in which both B2M and corresponding hit genes have been inactivated.

In some embodiments, at least about 50% (such as at least about any of 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more) of the sgRNA constructs in the sgRNA library, or the sgRNA ^iBAR constructs in the sgRNA ^iBAR library, or the sets of sgRNA ^iBAR constructs in the sgRNA ^iBAR library, are introduced into the initial population of T cells, or B2M ^-T cell library, or Cas9 ⁺ B2M ^-T cell library described herein. In some embodiments, at least about 95% (e.g., at least about any of 96%, 97%, 98%, 99%, or more) of the sgRNA constructs in the sgRNA library, or the sgRNA ^iBAR constructs in the sgRNA ^iBAR library, or the sets of sgRNA ^iBAR constructs in the sgRNA ^iBAR library, are introduced into the initial population of T cells, or B2M ^-T cell library, or Cas9 ⁺ B2M ^-T cell library described herein. In some embodiments, the hit gene inactivating efficiency by the sgRNA library or the sgRNA ^iBAR library is at least about 80%, such as at least about any of 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more. In some embodiments, the hit gene inactivating efficiency by the sgRNA library or the sgRNA ^iBAR library is at least about 90%.

In some embodiments, the T cell library comprises one or a plurality of (e.g., about 2, 3, 4, 5, 8, 10, 100, 250, 400, 500, 1,000, 2,000, 5,000, 10,000, or more) T cells that comprise the same sgRNA construct or the same sgRNA ^iBAR construct, which targets the same hit gene. Such T cell library is also referred to as “having X-fold coverage for the sgRNA/sgRNA ^iBAR” or “having X-fold coverage for each sgRNA/sgRNA ^iBAR, ” wherein “X” is the number of T cells expressing the same sgRNA or sgRNA ^iBAR. In some embodiments, the T cell library has averagely about 1 to about 10,000 fold coverage of each sgRNA or sgRNA ^iBAR, or each set of sgRNA ^iBAR, such as any of about 1 to about 5,000, about 100 to about 10,000, about 1,000 to about 5,000, about 10 to about 100, about 50 to about 500, about 80 to about 200, about 100 to about 400, about 100 to about 800, about 100 to about 1,000, about 1 to about 1,000, about 10 to about 1,000, or about 300 to about 600 fold coverage of each sgRNA or sgRNA ^iBAR, or each set of sgRNAiBAB. In some embodiments, the T cell library has averagely at least about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 100-fold, 400-fold, 500-fold, 1,000-fold, 2,000-fold, 5,000-fold, 10,000-fold, or more fold coverage of each sgRNA or sgRNA ^iBAR, or each set of sgRNA ^iBAR. In some embodiments, the T cell library has at least about 100-fold (e.g., about 400-fold) coverage for each sgRNA or mutation (e.g., mutagenic agent-induced mutation) . In some embodiments, each hit gene is targeted by about 2 to about 12 different sgRNAs, or has mutations in at least 2 (e.g., about 2 to about 12) different target sites. In some embodiments, the T cell library has at least about 400-fold (e.g., about 800-fold) coverage for each set of sgRNA ^iBAR. In some embodiments, the T cell library has at least about 100-fold (e.g., about 200-fold) coverage for each sgRNA ^iBAR.

In some embodiments, the T cell library has averagely at least about 100-fold (e.g., at least about any of 200-, 400-, 500-, 1,000-, 4,000-, or more fold) coverage for each sgRNA ^iBAR. In some embodiments, the T cell library has averagely at least about 400-fold (e.g., at least about any of 800-, 1000-, 2000-, 4000-, 16,000-, or more fold) coverage for each set of sgRNA ^iBAR. In some embodiments, the T cell library has averagely at least about 100-fold (e.g., at least about any of 200-, 400-, 500-, 1,000-, 4,000-, or more fold) coverage for the sgRNAs ^iBAR library. In some embodiments, the T cell library has averagely at least about 800-fold (e.g., at least about any of 1,000-, 1,600-, 2,000-, 2,400, 3,200-, 4,000-, 10,000, 16,000-, or more fold) coverage for each hit gene. In some embodiments, the sgRNAs ^iBAR library targets every annotated gene in the genome (i.e., the sgRNAs ^iBAR library is a whole-genome sgRNAs ^iBAR library) . In some embodiments, the T cell library has at least about 100-fold (e.g., at least about any of 400-fold, 800-fold, or 1,200-fold) coverage for the whole-genome sgRNAs ^iBAR library.

B2M mutation

Beta-2 microglobulin (B2M) is a component of MHC class I molecules (α ₁, α ₂, α ₃) expressed on all nucleated cells. Host TCRαβ cells recognize MHC class I molecules and distinguish between “self” and “foreign” cells. The activity of NK cells is regulated by a complex interplay of various cell surface inhibitory and activating receptors. Inhibitory receptors include killer immunoglobulin-like receptors (KIRs) and CD94/NKG2A, recognize MHC or HLA class I molecules, allow NK cells to recognize autologous cells and prevent them from attacking the host tissue. Cells (e.g., T cells such as allogeneic T cells) with reduced or absent HLA class I expression are targeted by NK cells as “foreign, ” leading to rejection reactions (Liu et al. Curr. Res. Transl. Med. 2018; 66: 39-42) .

The T cell library described herein in some embodiments further comprises a B2M mutation ( “B2M ^-T cell library” ) . In some embodiments, the B2M mutation is an inactivating B2M mutation. An inactivating B2M mutation described herein can be any mutation, such as insertion, deletion (indels) , substitution, frame shift, chromosomal rearrangement, or combinations thereof, that leads to complete abolishment or elimination of B2M expression (transcription and/or translation) and/or function. Inactivating B2M mutations in some embodiments can completely abolish the transcription, translation, post-translation modification, association with other molecules (e.g., other molecules in MHC class I molecules) , and/or function (e.g., receptor recognition or antigen presentation) of B2M. In some embodiments, the B2M mutation is a mutation that reduces (e.g., reduces at least about any of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more) or affects (e.g., disrupts) one or more of B2M transcription, B2M translation, B2M mRNA processing, B2M mRNA stability, B2M mRNA function, B2M protein function, B2M cell surface expression, and B2M post-translation modification. In some embodiments, the B2M mutation is a mutation that reduces (e.g., reduces at least about any of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more) or affects (e.g., disrupts) one or more of MHC class I molecules cell surface expression, assembly, function, and/or ability to be recognized by NK cells. In some embodiments, the B2M mutation is a mutation within one or more of regulatory region such as enhancer, promoter, 5' untranslated region (UTR) , 3'UTR, or the coding region such as an exon or a splicing site, of B2M. In some embodiments, the B2M mutation is a mutation not within the B2M gene or corresponding regulatory components, but affects B2M expression and/or function, such as a mutation in another molecule (e.g., nucleic acid or protein) that affects B2M mRNA splicing, B2M post-translation modification, etc. Cells (e.g., T cells) that have reduced (e.g., reduces at least about any of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more) or abolished B2M expression and/or function are also referred to herein as cells that are B2M-negative or deficient. In some embodiments, the T cell library comprises one or more mutations (e.g., inactivating mutations) in the B2M gene. In some embodiments, the T cells in the initial population of T cells comprise a B2M mutation ( “B2M ^-T cells” ) , such as an inactivating B2M mutation. Such B2M ^-T cells are further used for constructing the T cell library described herein. In some embodiments, the B2M mutation (e.g., inactivating B2M mutation) is introduced after obtaining a T cell library described herein, such as Cas9 ⁺ sgRNA/sgRNA ^iBAR T cell library, or sgRNA/sgRNA ^iBAR T cell library. In some embodiments, the B2M mutation (e.g., inactivating B2M mutation) is generated by mutagenic agents, such as physical mutagenic agents (e.g., gamma rays, ultraviolet radiations) , chemical mutagenic agents (e.g., ethyl methane sulphonate or EMS) , or transposable elements (such as transposons, retrotransposons, T-DNA, retroviruses) . In some embodiments, the B2M mutation (e.g., inactivating B2M mutation) is generated by B2M gene editing. In some embodiments, the B2M mutation (e.g., inactivating B2M mutation) is generated by gene editing of a non-B2M gene that affects B2M expression and/or function. Any known gene editing methods can be used for generating B2M ^-T cell libraries described herein, such as Zinc-finger nucleases (ZFNs) , transcription activator-like effector nucleases (TALENs) , and CRISPR/Cas-based methods. In some embodiments, the B2M mutation (e.g., inactivating B2M mutation) is generated by subjecting an initial population of T cells, or a T cell library described herein, to CRISPR/Cas-based gene editing. Thus in some embodiments, the B2M mutation (e.g., inactivating B2M mutation) is generated by contacting an initial population of T cells or a T cell library described herein (e.g., Cas9 ⁺ sgRNA/sgRNA ^iBAR T cell library, or sgRNA/sgRNA ^iBAR T cell library) with i) one or more B2M sgRNA constructs, wherein each B2M sgRNA construct comprises or encodes an B2M sgRNA comprising a guide sequence that is complementary (e.g., at least about any of 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%complementary) to a target site in the B2M gene (herein also referred to as “sgRNA against B2M” , or “sgRNA targeting B2M” ) ; and optionally ii) a Cas component comprising a Cas protein or a nucleic acid encoding the Cas protein (e.g., Cas9) , under a condition that allows introduction of the one or more B2M sgRNA constructs and the optional Cas component into the initial population of T cells or T cell library. In some embodiments, the B2M mutation (e.g., inactivating B2M mutation) in the T cell library is generated with one B2M sgRNA construct. In some embodiments, the B2M mutation (s) (e.g., inactivating B2M mutation (s) ) in the T cell library is generated with two B2M sgRNA constructs, each comprises or encodes an B2M sgRNA comprising a guide sequence that is complementary to a different target site in the B2M gene. In some embodiments, the B2M sgRNA construct comprises a B2M sgRNA. In some embodiments, the B2M sgRNA construct encodes a B2M sgRNA. In some embodiments, the B2M sgRNA construct is a plasmid that encodes the B2M sgRNA. In some embodiments, the B2M sgRNA construct is a viral vector (e.g., lentiviral vector) encoding the B2M sgRNA. In some embodiments, the B2M sgRNA construct is a virus (e.g., lentivirus) encoding the B2M sgRNA.

In some embodiments, the T cell library described herein is generated by i) contacting an initial population of T cells with an sgRNA library or an sgRNA ^iBAR library described herein (e.g., via lentivirus) under a condition that allows introduction of the sgRNA constructs or the sgRNA ^iBAR constructs into the initial population of T cells ( “sgRNA T cell library” or “sgRNA ^iBAR T cell library” ) ; ii) contacting the T cells comprising the sgRNA library or the sgRNA ^iBAR library with an B2M sgRNA construct described here (e.g., B2M sgRNA) and a Cas component comprising a Cas protein or a nucleic acid encoding the Cas protein (e.g., Cas9 mRNA) , under a condition (e.g., via electrotransformation) that allows introduction of the B2M sgRNA construct and the Cas component into the T cells comprising the sgRNA library or the sgRNA ^iBAR library, thereby generating a Cas9 ⁺ B2M ^-sgRNA T cell library or a Cas9 ⁺ B2M ^- sgRNA ^iBAR T cell library, in which both B2M and corresponding hit genes have been inactivated.

The guide sequence in the B2M sgRNA construct may be designed according to any known methods in the art. The guide sequence may target the coding region such as an exon or a splicing site, the 5' UTR or the 3' UTR of B2M. For example, the reading frame of B2M could be disrupted by indels mediated by DSB at a target site of a B2M guide RNA. Alternatively, a guide RNA targeting the 5' end of the B2M coding sequence may be used to produce B2M knockout with high efficiency. The guide sequence may be designed and optimized according to certain sequence features for high on-target gene-editing activity and low off-target effects. For instance, the GC content of a guide sequence may be in the range of about 20%to about 70%, and sequences containing homopolymer stretches (e.g., TTTT, GGGG) may be avoided.

In some embodiments, at least about 50% (such as at least about any of 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more) of the B2M sgRNA construct (e.g., sgRNA against B2M) , and/or the Cas component (e.g., Cas9 mRNA) , are introduced into the initial population of T cells, or Cas9 ⁺ sgRNA/sgRNA ^iBAR T cell library described herein, or sgRNA/sgRNA ^iBAR T cell library described herein. In some embodiments, at least about 90% (such as at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) of the B2M sgRNA construct (e.g., sgRNA against B2M) , and/or the Cas component (e.g., Cas9 mRNA) , are introduced into the sgRNA/sgRNA ^iBAR T cell library described herein. In some embodiments, the B2M inactivating efficiency (e.g., by B2M gene editing, such as by CRISPR/Cas with a B2M sgRNA and a Cas component such as Cas9) is at least about 80%, such as at least about any of 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more. In some embodiments, the B2M inactivating efficiency is at least about 90%.

T cells and preparation methods

In some embodiments, there is provided a composition comprising T cells (e.g., allogeneic T cells or CAR-T cells (such as allogeneic CAR-T cells) ) comprising any one of the sgRNA or sgRNA ^iBAR constructs, molecules, sets, or libraries described herein. In some embodiments, the T cells further comprise a B2M construct described herein, or one or more B2M mutations (e.g., inactivating B2M mutations) .

In some embodiments, there is provided a method of editing a genomic locus in a T cell, comprising introducing into a host T cell (e.g., a primary T cell, or a T cell comprising a B2M mutation such as an inactivating B2M mutation) a guide RNA construct comprising a guide sequence targeting a genomic locus (e.g., a target site of a hit gene) and a guide hairpin sequence coding for a Repeat: Anti-Repeat Duplex and a tetraloop, wherein an iBAR is embedded in the tetraloop serving as internal replicates, expressing the guide RNA that targets the genomic locus in the host T cell, and thereby editing the targeted genomic locus (e.g., hit gene) in the presence of a Cas nuclease (e.g., Cas9) . In some embodiments, the method further comprises introducing a B2M sgRNA construct into the host T cell or the T cell comprising the guide RNA construct. In some embodiments, the method further comprises introducing into the T cell a Cas component comprising a Cas protein or a nucleic acid encoding the Cas protein, e.g., as Cas9 mRNA.

In some embodiments, there is provided a T cell library prepared by transfecting any one of the sgRNA libraries or the sgRNA ^iBAR libraries described herein to a plurality of host T cells (e.g., an initial population of T cells, with or without a B2M mutation such as an inactivating B2M mutation) , wherein the sgRNA constructs or the sgRNA ^iBAR constructs are present in viral vectors (e.g., lentiviral vectors) or viruses (e.g., lentiviruses) . In some embodiments, the T cell library is prepared by further transfecting an B2M sgRNA construct described herein (e.g., mRNA, viral vector, or virus) , either to the initial population of T cells, or to the T cell library comprising the sgRNA library or the sgRNA ^iBAR library. In some embodiments, the multiplicity of infection (MOI) between the viral vectors or viruses and the host T cells (e.g., initial population of T cells, or T cell library) during the transfection is at least about 1. In some embodiments, the MOI is at least about any one of 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, or higher. In some embodiments, the MOI is about 1, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, or about 10. In some embodiments, the MOI is about any one of 1-10, 1-3, 3-5, 5-10, 2-9, 3-8, 4-6, or 2-5. In some embodiments, the MOI between the viral vectors or viruses and the host T cells (e.g., initial population of T cells, or T cell library) during transfection is less than 1, such as less than about any of 0.8, 0.5, 0.3, or lower. In some embodiments, the MOI is about 0.3 to about 1. In some embodiments, the viral sgRNA library or the viral sgRNA ^iBAR library is contacted with the initial population of T cells at an MOI of at least about 2, such as at least about 3. In some embodiments, the B2M sgRNA viral construct is contacted with the initial population of T cells or the T cell library at an MOI of at least about 2, such as at least about 3.

In some embodiments, one or more vectors driving expression of one or more elements of a CRISPR/Cas system are introduced into a host T cell (e.g., the initial population of T cells, or T cell library) such that expression of the elements of the CRISPR system directs formation of a CRISPR complex with an sgRNA molecule or an sgRNA ^iBAR molecule described herein at one or more target sites of one or more hit genes. In some embodiments, the host T cell (e.g., the initial population of T cells) has been introduced a Cas nuclease (e.g., Cas9 mRNA) or is engineered to stably express CRISPR/Cas nuclease.

In some embodiments, the host T cell (e.g., the initial population of T cells) is a T cell line, such as a pre-established T cell line. The host T cells and T cell lines may be human T cells or T cell lines, or they may be non-human, mammalian T cells or T cell lines. In some embodiments, the host T cell is difficult to transfect with a viral vector, such as lentiviral vector, at a low MOI (e.g., lower than 1, 0.5, or 0.3) . In some embodiments, the host T cell is difficult to edit using a CRISPR/Cas system at low MOI (e.g., lower than 1, 0.5, or 0.3) . In some embodiments, the host T cell is available at a limited quantity. In some embodiments, the host T cell is obtained from a blood sample from an individual.

Isolation and culture of T cells

Prior to expansion and genetic modification of the T cells, a source of T cells is obtained from an individual. T cells can be obtained from a number of sources, including peripheral blood mononuclear cells (PBMCs) , bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments, any number of T cell lines available in the art, may be used. In some embodiments, T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as FICOLL ^TM separation. In some embodiments, cells from the circulating blood of an individual are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In some embodiments, the cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS) . In some embodiments, the wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations. In some embodiments, initial activation steps in the absence of calcium lead to magnified activation. As those of ordinary skill in the art would readily appreciate a washing step may be accomplished by methods known to those in the art, such as by using a semi-automated “flow-through” centrifuge (for example, the Cobe 2991 cell processor, the Baxter CytoMate, or the Haemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca ²⁺-free, Mg ²⁺-free PBS, PlasmaLyte A, or other saline solution with or without buffer. Alternatively, the undesirable components of the apheresis sample may be removed and the cells directly resuspended in culture media.

In some embodiments, the T cell is provided from an umbilical cord blood bank, a peripheral blood bank, or derived from an induced pluripotent stem cell (iPSC) , multipotent and pluripotent stem cell, or a human embryonic stem cell. In some embodiments, the T cells are derived from cell lines. The T cells in some embodiments are obtained from a xenogeneic source, for example, from mouse, rat, non-human primate, and pig. In some embodiments, the T cells are human cells. In some aspects, the T cells are primary cells, such as those isolated directly from a subject and/or isolated from a subject and frozen. In some embodiments, after blood collection, PBMCs are separated from the donor blood samples, then T cells are isolated from the PBMCs, e.g., using the immunomagnetic bead method. In some embodiments, the cells include one or more subsets of T cells, such as whole T cell populations, CD4+ cells, CD8+ cells, and subpopulations thereof, such as those defined by function, activation state, maturity, potential for differentiation, expansion, recirculation, localization, and/or persistence capacities, antigen-specificity, type of antigen receptor, presence in a particular organ or compartment, marker or cytokine secretion profile, and/or degree of differentiation. With reference to the subject to be treated, the cells may be allogeneic and/or autologous. In some cases, the T cell is allogeneic in reference to one or more intended recipients. In some cases, the T cell is suitable for transplantation, such as without inducing GvHD in the recipient. In some embodiments, the T cell is an allogeneic CAR-T cells. In some embodiments, the T cell (e.g., allogeneic T cell) is modified to express a chimeric receptor, such as CAR or engineered TCR. In some embodiments, the T cell (e.g., allogeneic T cell) is modified to knock-out endogenous TCR.

Among the sub-types and subpopulations of T cells and/or of CD4+ and/or of CD8+ T cells are naive T (T _N) cells, effector T cells (T _EFF) , memory T cells and sub-types thereof, such as stem cell memory T (TSC _M) , central memory T (TC _M) , effector memory T (T _EM) , or terminally differentiated effector memory T cells, tumor-infiltrating lymphocytes (TIL) , immature T cells, mature T cells, helper T cells, cytotoxic T cells (CTLs) , mucosa-associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha/beta T cells, and delta/gamma T cells.

In some embodiments, T cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL ^TM gradient or by counterflow centrifugal elutriation. A specific subpopulation of T cells, such as CD3+, CD28+, CD4+, CD8+, CD45RA+, and CD45RO+T cells, can be further isolated by positive or negative selection techniques. For example, in some embodiments, T cells are isolated by incubation with anti-CD3/anti-CD28 (i.e., 3×28) -conjugated beads, such as

M-450 CD3/CD28 T, for a time period sufficient for positive selection of the desired T cells. In some embodiments, the time period is about 30 minutes. In a further embodiment, the time period ranges from 30 minutes to 36 hours or longer and all values there between. In a further embodiment, the time period is at least 1, 2, 3, 4, 5, or 6 hours. In some embodiments, the time period is 10 to 24 hours. In some embodiments, the incubation time period is 24 hours. For isolation of T cells from patients with leukemia, use of longer incubation times, such as 24 hours, can increase cell yield. Longer incubation times may be used to isolate T cells in any situation where there are few T cells as compared to other cell types, such in isolating tumor infiltrating lymphocytes (TIL) from tumor tissue or from immune-compromised individuals. Further, use of longer incubation times can increase the efficiency of capture of CD8+ T cells. Thus, by simply shortening or lengthening the time T cells are allowed to bind to the CD3/CD28 beads and/or by increasing or decreasing the ratio of beads to T cells (as described further herein) , subpopulations of T cells can be preferentially selected for or against at culture initiation or at other time points during the process. Additionally, by increasing or decreasing the ratio of anti-CD3 and/or anti-CD28 antibodies on the beads or other surface, subpopulations of T cells can be preferentially selected for or against at culture initiation or at other desired time points. The skilled artisan would recognize that multiple rounds of selection can also be used. In some embodiments, it may be desirable to perform the selection procedure and use the “unselected” cells in the activation and expansion process. “Unselected” cells can also be subjected to further rounds of selection.

Enrichment of a T cell population by negative selection can be accomplished with a combination of antibodies directed to surface markers unique to the negatively selected cells. One method is cell sorting and/or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected. For example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In certain embodiments, it may be desirable to enrich for or positively select for regulatory T cells which typically express CD4+, CD25+, CD62Lhi, GITR+, and FoxP3+. Alternatively, in certain embodiments, T regulatory cells are depleted by anti-CD25 conjugated beads or other similar method of selection.

For isolation of a desired population of cells by positive or negative selection, the concentration of cells and surface (e.g., particles such as beads) can be varied. In certain embodiments, it may be desirable to significantly decrease the volume in which beads and cells are mixed together (i.e., increase the concentration of cells) , to ensure maximum contact of cells and beads. For example, in one embodiment, a concentration of 2 billion cells/mL is used. In one embodiment, a concentration of 1 billion cells/mL is used. In a further embodiment, greater than 100 million cells/mL is used. In a further embodiment, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/mL is used. In yet another embodiment, a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells/mL is used. In further embodiments, concentrations of 125 or 150 million cells/mL can be used. Using high concentrations can result in increased cell yield, cell activation, and cell expansion. Further, use of high cell concentrations allows more efficient capture of cells that may weakly express target antigens of interest, such as CD28-negative T cells, or from samples where there are many tumor cells present (i.e., leukemic blood, tumor tissue, etc. ) . Such populations of cells may have therapeutic value and would be desirable to obtain. For example, using high concentration of cells allows more efficient selection of CD8+ T cells that normally have weaker CD28 expression.

In some embodiments, it may be desirable to use lower concentrations of cells. By significantly diluting the mixture of T cells and surface (e.g., particles such as beads) , interactions between the particles and cells is minimized. This selects for cells that express high amounts of desired antigens to be bound to the particles. For example, CD4+ T cells express higher levels of CD28 and are more efficiently captured than CD8+ T cells in dilute concentrations. In some embodiments, the concentration of cells used is 5 × 10 ⁶/mL. In some embodiments, the concentration used can be from about 1 × 10 ⁵/mL to 1 × 10 ⁶/mL, and any value in between.

In some embodiments, the cells may be incubated on a rotator for varying lengths of time at varying speeds at either 2-10℃, at room temperature, or at about 37℃.

T cells for stimulation can also be frozen after a washing step. Wishing not to be bound by theory, the freeze and subsequent thaw step provides a more uniform product by removing granulocytes and to some extent monocytes in the cell population. After the washing step that removes plasma and platelets, the cells may be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and will be useful in this context, one method involves using PBS containing 20%DMSO and 8%human serum albumin, or culture media containing 10%Dextran 40 and 5%Dextrose, 20%Human Serum Albumin and 7.5% DMSO, or 31.25%Plasmalyte-A, 31.25%Dextrose 5%, 0.45%NaCl, 10%Dextran 40 and 5%Dextrose, 20%Human Serum Albumin, and 7.5%DMSO or other suitable cell freezing media containing for example, Hespan and PlasmaLyte A, the cells then are frozen to -80℃ at a rate of 1° per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing may be used as well as uncontrolled freezing immediately at -20℃ or in liquid nitrogen.

In some embodiments, cryopreserved cells are thawed and washed as described herein and allowed to rest for one hour at room temperature prior to activation.

Also contemplated in the present application is the collection of blood samples or apheresis product from a subject at a time period prior to when the expanded cells as described herein might be needed. As such, the source of the cells to be expanded can be collected at any time point necessary, and desired cells, such as T cells, isolated and frozen for later use in T cell therapy for any number of diseases or conditions that would benefit from T cell therapy, such as those described herein. In one embodiment a blood sample or an apheresis is taken from a generally healthy subject. In certain embodiments, a blood sample or an apheresis is taken from a generally healthy subject who is at risk of developing a disease, but who has not yet developed a disease, and the cells of interest are isolated and frozen for later use. In certain embodiments, the T cells may be expanded, frozen, and used at a later time. In certain embodiments, samples are collected from a patient shortly after diagnosis of a particular disease as described herein but prior to any treatments. In a further embodiment, the cells are isolated from a blood sample or an apheresis from a subject prior to any number of relevant treatment modalities, including but not limited to treatment with agents such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies, cytoxan, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, and irradiation. These drugs inhibit either the calcium dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit the p70S6 kinase that is important for growth factor induced signaling (rapamycin) (Liu et al., Cell 66: 807-815, 1991; Henderson et al., Immun 73: 316-321, 1991; Bierer et al., Curr. Opin. Immun. 5: 763-773, 1993) . In a further embodiment, the cells are isolated for a patient and frozen for later use in conjunction with (e.g., before, simultaneously or following) bone marrow or stem cell transplantation, T cell ablative therapy using either chemotherapy agents such as, fludarabine, external-beam radiation therapy (XRT) , cyclophosphamide, or antibodies such as OKT3 or CAMPATH. In another embodiment, the cells are isolated prior to and can be frozen for later use for treatment following B-cell ablative therapy such as agents that react with CD20, e.g., Rituxan.

In some embodiments, T cells are obtained from a patient directly following treatment. In this regard, it has been observed that following certain cancer treatments, in particular treatments with drugs that damage the immune system, shortly after treatment during the period when patients would normally be recovering from the treatment, the quality of T cells obtained may be optimal or improved for their ability to expand ex vivo. Likewise, following ex vivo manipulation using the methods described herein, these cells may be in a preferred state for enhanced engraftment and in vivo expansion. Thus, it is contemplated within the context of the present invention to collect blood cells, including T cells, during this recovery phase. Further, in certain embodiments, mobilization (for example, mobilization with GM-CSF) and conditioning regimens can be used to create a condition in a subject wherein repopulation, recirculation, regeneration, and/or expansion of particular cell types is favored, especially during a defined window of time following therapy.

Activation and expansion of T cells

In some embodiments, the cells are incubated and/or cultured prior to or in connection with genetic engineering. The incubation steps can include culture, cultivation, stimulation, activation, and/or propagation or expansion. In some embodiments, the compositions or cells are incubated in the presence of stimulating conditions or a stimulatory agent. Such conditions include those designed to induce proliferation, expansion, activation, and/or survival of cells in the population, to mimic antigen exposure, and/or to prime the cells for genetic engineering. The conditions can include one or more of particular media, temperature, oxygen content, carbon dioxide content, time, agents, e.g., nutrients, amino acids, antibiotics, ions, and/or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate the cells.

Whether prior to or after genetic modification of the T cells, the T cells can be activated and expanded generally using methods as described, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent Application Publication No. 20060121005.

Generally, T cells can be expanded by contact with a surface having attached thereto an agent that stimulates a CD3/TCR complex associated signal and a ligand that stimulates a co-stimulatory molecule on the surface of the T cells. In particular, T cell populations may be stimulated as described herein, such as by contact with an anti-CD3 antibody, or antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ionophore. For co-stimulation of an accessory molecule on the surface of the T cells, a ligand that binds the accessory molecule is used. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody, under conditions appropriate for stimulating proliferation of the T cells. To stimulate proliferation of either CD4+ T cells or CD8+ T cells, an anti-CD3 antibody and an anti-CD28 antibody. Examples of an anti-CD28 antibody include 9.3, B-T3, XR-CD28 (Diaclone, Besancon, France) can be used as can other methods commonly known in the art (Berg et al., Transplant Proc. 30 (8) : 3975-3977, 1998; Haanen et al., J. Exp. Med. 190 (9) : 13191328, 1999; Garland et al., J. Immunol Meth. 227 (1-2) : 53-63, 1999) .

In some embodiments, the T cells are expanded by adding to the culture-initiating composition feeder cells, such as non-dividing peripheral blood mononuclear cells (PBMC) , (e.g., such that the resulting population of cells contains at least about 5, 10, 20, or 40 or more PBMC feeder cells for each T lymphocyte in the initial population to be expanded) ; and incubating the culture (e.g. for a time sufficient to expand the numbers of T cells) . In some aspects, the non-dividing feeder cells can comprise gamma-irradiated PBMC feeder cells. In some embodiments, the PBMC are irradiated with gamma rays in the range of about 3000 to 3600 rads to prevent cell division. In some aspects, the feeder cells are added to culture medium prior to the addition of the populations of T cells.

In some embodiments, the primary stimulatory signal and the co-stimulatory signal for the T cell may be provided by different protocols. For example, the agents providing each signal may be in solution or coupled to a surface. When coupled to a surface, the agents may be coupled to the same surface (i.e., in “cis” formation) or to separate surfaces (i.e., in “trans” formation) . Alternatively, one agent may be coupled to a surface and the other agent in solution. In one embodiment, the agent providing the co-stimulatory signal is bound to a cell surface and the agent providing the primary activation signal is in solution or coupled to a surface. In certain embodiments, both agents can be in solution. In another embodiment, the agents may be in soluble form, and then cross-linked to a surface, such as a cell expressing Fc receptors or an antibody or other binding agent which will bind to the agents. In this regard, see for example, U.S. Patent Application Publication Nos. 20040101519 and 20060034810 for artificial antigen presenting cells (aAPCs) that are contemplated for use in activating and expanding T cells in the present invention.

In some embodiments, the T cells, are combined with agent-coated beads, the beads and the cells are subsequently separated, and then the cells are cultured. In an alternative embodiment, prior to culture, the agent-coated beads and cells are not separated but are cultured together. In a further embodiment, the beads and cells are first concentrated by application of a force, such as a magnetic force, resulting in increased ligation of cell surface markers, thereby inducing cell stimulation.

By way of example, cell surface proteins may be ligated by allowing paramagnetic beads to which anti-CD3 and anti-CD28 are attached (3×28 beads) to contact the T cells. In one embodiment the cells (for example, 10 ⁴ to 10 ⁹ T cells) and beads (for example,

M-450 CD3/CD28 T paramagnetic beads) are combined in a buffer, preferably PBS (without divalent cations such as, calcium and magnesium) . Again, those of ordinary skill in the art can readily appreciate any cell concentration may be used. For example, the target cell may be very rare in the sample and comprise only 0.01%of the sample or the entire sample (i.e., 100%) may comprise the target cell of interest. Accordingly, any cell number is within the context of the present invention. In certain embodiments, it may be desirable to significantly decrease the volume in which particles and cells are mixed together (i.e., increase the concentration of cells) , to ensure maximum contact of cells and particles. For example, in one embodiment, a concentration of about 2 billion cells/mL is used. In another embodiment, greater than 100 million cells/mL is used. In a further embodiment, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/mL is used. In yet another embodiment, a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells/mL is used. In further embodiments, concentrations of 125 or 150 million cells/mL can be used. Using high concentrations can result in increased cell yield, cell activation, and cell expansion. Further, use of high cell concentrations allows more efficient capture of cells that may weakly express target antigens of interest, such as CD28-negative T cells. Such populations of cells may have therapeutic value and would be desirable to obtain in certain embodiments. For example, using high concentration of cells allows more efficient selection of CD8+ T cells that normally have weaker CD28 expression. In some embodiments, about 30 million cultured T cells are used for activation and expansion.

In some embodiments, the mixture may be cultured for several hours (about 3 hours) to about 14 days or any hourly integer value in between. In another embodiment, the mixture may be cultured for 21 days. In one embodiment of the invention the beads and the T cells are cultured together for about eight days. In another embodiment, the beads and T cells are cultured together for 2-3 days. Several cycles of stimulation may also be desired such that culture time of T cells can be 60 days or more. Conditions appropriate for T cell culture include an appropriate media (e.g., Minimal Essential Media or RPMI Media 1640 or, X-vivo 15 (Lonza) ) that may contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine or human serum) , interleukin-2 (IL-2) , insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFβ, and TNF-α or any other additives for the growth of cells known to the skilled artisan. Other additives for the growth of cells include, but are not limited to, surfactant, plasmanate, and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanol. Media can include RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, with added amino acids, sodium pyruvate, and vitamins, either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and/or an amount of cytokine (s) sufficient for the growth and expansion of T cells. Antibiotics, e.g., penicillin and streptomycin, are included only in experimental cultures, not in cultures of cells that are to be infused into a subject. The target cells are maintained under conditions necessary to support growth, for example, an appropriate temperature (e.g., 37 ℃) and atmosphere (e.g., air plus 5%CO ₂) . T cells that have been exposed to varied stimulation times may exhibit different characteristics. For example, typical blood or apheresis peripheral blood mononuclear cell products have a helper T cell population (TH, CD4+) that is greater than the cytotoxic or suppressor T cell population (TC, CD8) . Ex vivo expansion of T cells by stimulating CD3 and CD28 receptors produces a population of T cells that prior to about days 8-9 consists predominately of TH cells, while after about days 8-9, the population of T cells comprises an increasingly greater population of TC cells. Accordingly, depending on the purpose of treatment, infusing a subject with a T cell population comprising predominately of TH cells may be advantageous. Similarly, if an antigen-specific subset of TC cells has been isolated it may be beneficial to expand this subset to a greater degree.

Further, in addition to CD4 and CD8 markers, other phenotypic markers vary significantly, but in large part, reproducibly during the course of the cell expansion process. Thus, such reproducibility enables the ability to tailor an activated T cell product for specific purposes.

In some embodiments, the methods include assessing expression of one or more markers on the surface of the modified cells or cells to be engineered. In one embodiment, the methods include assessing surface expression of TCR or CD3ε, for example, by affinity-based detection methods such as by flow cytometry. In some aspects, where the method reveals surface expression of the antigen or other marker, the gene encoding the antigen or other marker is disrupted or expression otherwise repressed for example, using the methods described herein.

Isolation and enrichment of modified T cells

In some embodiments, the methods described herein further comprise isolating or enriching T cells comprising the mutation (e.g., inactivating mutation) in a hit gene, and/or the B2M mutation such as inactivating B2M mutation. In some embodiments, the methods described herein further comprise isolating or enriching T cells comprising the Cas component, the sgRNA construct, the sgRNA ^iBAR construct, and/or the B2M sgRNA construct described herein. In some embodiments, the method described herein further comprises isolating or enriching CD8+ T cells from the modified T cells.

In some embodiments, the isolation methods include the separation of different cell types based on the absence or presence in the cell of one or more specific molecules, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acid (e.g., sgRNA, sgRNA ^iBAR, B2M sgRNA, and/or nucleic acid encoding Cas) . In some embodiments, any known method for separation based on such markers may be used. In some embodiments, the separation is affinity-or immunoaffinity-based separation. For example, the isolation in some aspects includes separation of cells and cell populations based on the cells’ expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such markers, followed generally by washing steps and separation of cells having bound the antibody or binding partner, from those cells having not bound to the antibody or binding partner. In some embodiments, the isolation comprises separation of cells and cell populations based on the cells’ expression of selectable marker genes (e.g., antibiotic resistance genes such as puromycin, or fluorescent protein-encoding genes) . Such separation steps can be based on positive selection, in which the cells having bound the reagents, resistant to antibiotics, or expressing fluorescent proteins are retained for further use, and/or negative selection, in which the cells having not bound to the antibody or binding partner or not expressing fluorescent proteins are retained. In some examples, both fractions are retained for further use. In some aspects, negative selection can be particularly useful where no antibody is available that specifically identifies a cell type in a heterogeneous population, such that separation is best carried out based on markers expressed by cells other than the desired population.

The separation need not result in 100%enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection of or enrichment for cells of a particular type, such as those expressing a marker, refers to increasing the number or percentage of such cells, but need not result in a complete absence of cells not expressing the marker. Likewise, negative selection, removal, or depletion of cells of a particular type, such as those expressing a marker, refers to decreasing the number or percentage of such cells, but need not result in a complete removal of all such cells.

In some examples, multiple rounds of separation steps are carried out, where the positively or negatively selected fraction from one step is subjected to another separation step, such as a subsequent positive or negative selection. In some examples, a single separation step can deplete cells expressing multiple markers simultaneously, such as by incubating cells with a plurality of antibodies or binding partners, each specific for a marker targeted for negative selection. Likewise, multiple cell types can simultaneously be positively selected by incubating cells with a plurality of antibodies or binding partners expressed on the various cell types.

For example, in some aspects, specific subpopulations of T cells, such as cells positive or expressing high levels of one or more surface markers, e.g., CD28 ⁺, CD62L ⁺, CCR7 ⁺, CD27 ⁺, CD127 ⁺, CD4 ⁺, CD8 ⁺, CD45RA ⁺, and/or CD45RO ⁺ T cells, are isolated by positive or negative selection techniques. In some embodiments, T cells not expressing certain markers, e.g., markers encoded by one or more hit genes, and/or B2M, are isolated.

For example, CD3 ⁺, CD28 ⁺ T cells can be positively selected using CD3/CD28 conjugated magnetic beads (e.g.,

M-450 CD3/CD28 T Cell Expander) .

In some embodiments, isolation is carried out by enrichment for a particular cell population by positive selection, or depletion of a particular cell population, by negative selection. In some embodiments, positive or negative selection is accomplished by incubating cells with one or more antibodies or other binding agent that specifically bind to one or more surface markers expressed or expressed (marker ⁺) at a relatively higher level (marker ^high) on the positively or negatively selected cells, respectively.

In some aspects, the sample or composition of cells to be separated is incubated with small, magnetizable or magnetically responsive material, such as magnetically responsive particles or microparticles, such as paramagnetic beads (e.g., such as Dynabeads or MACS beads) . The magnetically responsive material, e.g., particle, generally is directly or indirectly attached to a binding partner, e.g., an antibody, that specifically binds to a molecule, e.g., surface marker, present on the cell, cells, or population of cells that it is desired to separate, e.g., that it is desired to negatively or positively select.

In some embodiments, the magnetic particle or bead comprises a magnetically responsive material bound to a specific binding member, such as an antibody or other binding partner. There are many well-known magnetically responsive materials used in magnetic separation methods. Suitable magnetic particles include those described in Molday, U.S. Pat. No. 4,452,773, and in European Patent Specification EP 452342 B, which are hereby incorporated by reference. Colloidal sized particles, such as those described in Owen U.S. Pat. No. 4,795,698, and Liberti et al., U.S. Pat. No. 5,200,084 are other examples.

The incubation generally is carried out under conditions whereby the antibodies or binding partners, or molecules, such as secondary antibodies or other reagents, which specifically bind to such antibodies or binding partners, which are attached to the magnetic particle or bead, specifically bind to cell surface molecules ifpresent on cells within the sample.

In some embodiments, the sample is placed in a magnetic field, and those cells having magnetically responsive or magnetizable particles attached thereto will be attracted to the magnet and separated from the unlabeled cells. For positive selection, cells that are attracted to the magnet are retained; for negative selection, cells that are not attracted (unlabeled cells) are retained. In some aspects, a combination of positive and negative selection is performed during the same selection step, where the positive and negative fractions are retained and further processed or subject to further separation steps.

In certain embodiments, the magnetically responsive particles are coated in primary antibodies or other binding partners, secondary antibodies, lectins, enzymes, or streptavidin. In certain embodiments, the magnetic particles are attached to cells via a coating of primary antibodies specific for one or more markers. In certain embodiments, the cells, rather than the beads, are labeled with a primary antibody or binding partner, and then cell-type specific secondary antibody-or other binding partner (e.g., streptavidin) -coated magnetic particles, are added. In certain embodiments, streptavidin-coated magnetic particles are used in conjunction with biotinylated primary or secondary antibodies.

In some embodiments, the magnetically responsive particles are left attached to the cells that are to be subsequently incubated, cultured and/or engineered; in some aspects, the particles are left attached to the cells for administration to a patient. In some embodiments, the magnetizable or magnetically responsive particles are removed from the cells. Methods for removing magnetizable particles from cells are known and include, e.g., the use of competing non-labeled antibodies, magnetizable particles or antibodies conjugated to cleavable linkers, etc. In some embodiments, the magnetizable particles are biodegradable.

In some embodiments, the affinity-based selection is via magnetic-activated cell sorting (MACS) (Miltenyi Biotec, Auburn, Calif. ) . Magnetic Activated Cell Sorting (MACS) systems are capable of high-purity selection of cells having magnetized particles attached thereto. In certain embodiments, MACS operates in a mode wherein the non-target and target species are sequentially eluted after the application of the external magnetic field. That is, the cells attached to magnetized particles are held in place while the unattached species are eluted. Then, after this first elution step is completed, the species that were trapped in the magnetic field and were prevented from being eluted are freed in some manner such that they can be eluted and recovered. In certain embodiments, the non-target cells are labelled and depleted from the heterogeneous population of cells.

In certain embodiments, the isolation or separation is carried out using a system, device, or apparatus that carries out one or more of the isolation, cell preparation, separation, processing, incubation, culture, and/or formulation steps of the methods. In some aspects, the system is used to carry out each of these steps in a closed or sterile environment, for example, to minimize error, user handling and/or contamination. In one example, the system is a system as described in International Patent Application, Publication Number WO2009/072003, or US20110003380.

In some embodiments, the system or apparatus carries out one or more, e.g., all, of the isolation, processing, engineering, and formulation steps in an integrated or self-contained system, and/or in an automated or programmable fashion. In some aspects, the system or apparatus includes a computer and/or computer program in communication with the system or apparatus, which allows a user to program, control, assess the outcome of, and/or adjust various aspects of the processing, isolation, engineering, and formulation steps.

In some aspects, the separation and/or other steps is carried out using CliniMACS system (Miltenyi Biotec) , for example, for automated separation of cells on a clinical-scale level in a closed and sterile system. Components can include an integrated microcomputer, magnetic separation unit, peristaltic pump, and various pinch valves. The integrated computer in some aspects controls all components of the instrument and directs the system to perform repeated procedures in a standardized sequence. The magnetic separation unit in some aspects includes a movable permanent magnet and a holder for the selection column. The peristaltic pump controls the flow rate throughout the tubing set and, together with the pinch valves, ensures the controlled flow of buffer through the system and continual suspension of cells.

The CliniMACS system in some aspects uses antibody-coupled magnetizable particles that are supplied in a sterile, non-pyrogenic solution. In some embodiments, after labelling of cells with magnetic particles the cells are washed to remove excess particles. A cell preparation bag is then connected to the tubing set, which in turn is connected to a bag containing buffer and a cell collection bag. The tubing set consists of pre-assembled sterile tubing, including a pre-column and a separation column, and are for single use only. After initiation of the separation program, the system automatically applies the cell sample onto the separation column. Labelled cells are retained within the column, while unlabeled cells are removed by a series of washing steps. In some embodiments, the cell populations for use with the methods described herein are unlabeled and are not retained in the column. In some embodiments, the cell populations for use with the methods described herein are labeled and are retained in the column. In some embodiments, the cell populations for use with the methods described herein are eluted from the column after removal of the magnetic field, and are collected within the cell collection bag.

In certain embodiments, separation and/or other steps are carried out using the CliniMACS Prodigy system (Miltenyi Biotec) . The CliniMACS Prodigy system in some aspects is equipped with a cell processing unity that permits automated washing and fractionation of cells by centrifugation. The CliniMACS Prodigy system can also include an onboard camera and image recognition software that determines the optimal cell fractionation endpoint by discerning the macroscopic layers of the source cell product. For example, peripheral blood is automatically separated into erythrocytes, white blood cells and plasma layers. The CliniMACS Prodigy system can also include an integrated cell cultivation chamber which accomplishes cell culture protocols such as, e.g., cell differentiation and expansion, antigen loading, and long-term cell culture. Input ports can allow for the sterile removal and replenishment of media and cells can be monitored using an integrated microscope.

In some embodiments, a cell population described herein is collected and enriched (or depleted) via flow cytometry, in which cells stained for multiple cell surface markers are carried in a fluidic stream. In some embodiments, a cell population described herein is collected and enriched (or depleted) via preparative scale (FACS) -sorting. In certain embodiments, a cell population described herein is collected and enriched (or depleted) by use of microelectromechanical systems (MEMS) chips in combination with a FACS-based detection system (see, e.g., WO 2010/033140, Cho et al. (2010) Lab Chip 10, 1567-1573; and Godin et al. (2008) J Biophoton. 1 (5) : 355-376. In both cases, cells can be labeled with multiple markers, allowing for the isolation of well-defined T cell subsets at high purity.

In some embodiments, the antibodies or binding partners are labeled with one or more detectable marker, to facilitate separation for positive and/or negative selection. For example, separation may be based on binding to fluorescently labeled antibodies. In some examples, separation of cells based on binding of antibodies or other binding partners specific for one or more cell surface markers are carried in a fluidic stream, such as by fluorescence-activated cell sorting (FACS) , including preparative scale (FACS) and/or microelectromechanical systems (MEMS) chips, e.g., in combination with a flow-cytometric detection system. Such methods allow for positive and negative selection based on multiple markers simultaneously.

NK cell treatments and obtaining T cells that are sensitive or resistant to the killing of the NK cells

The methods described herein comprise subjecting the T cell library described herein (e.g., Cas9 ⁺ B2M ^- sgRNA T cell library, Cas9 ⁺ B2M ^- sgRNA ^iBAR T cell library, Cas9 ⁺ sgRNA T cell library, or Cas9 ⁺ sgRNA ^iBAR T cell library) to treatment with Natural Killer (NK) cells, and obtaining a T cell from the T cell library that is sensitive or resistant to the killing of the NK cells. In some embodiments, subjecting the T cell library to treatment with NK cells comprises growing the T cell library in the presence of the NK cells.

In some embodiments, treatment with NK cells (hereinafter also referred to as “the NK cell treatment step, ” “the NK cell treatment step b) , ” or “step b) ” ) comprises: i) an initial treatment step comprising contacting the T cell library with the NK cells ( “the initial treatment step” ) ; ii) an optional first enrichment step comprising sorting the mixture of treated cells to obtain a first T cell subpopulation that is sensitive or resistant to the killing of the NK cells ( “the first enrichment step” ) ; iii) an optional first recovery step comprising culturing the first T cell subpopulation ( “the first recovery step” ) ; and iv) an optional second treatment step comprising contacting the first T cell subpopulation with the NK cells ( “the second treatment step” ) . In some embodiments, treatment with NK cells step b) comprises a single (e.g., initial) treatment step comprising contacting the T cell library with the NK cells. In some embodiments, treatment with NK cells step b) comprises: i) a single (e.g., initial) treatment step comprising contacting the T cell library with the NK cells; and ii) a first recovery step comprising culturing the mixture of treated cells. In some embodiments, treatment with NK cells step b) comprises: i) an initial treatment step comprising contacting the T cell library with the NK cells; ii) a first recovery step comprising culturing the mixture of treated cells; and iii) a second treatment step comprising contacting the recovered mixture of treated cells with the NK cells. In some embodiments, treatment with NK cells step b) comprises: i) a single (e.g., initial) treatment step comprising contacting the T cell library with the NK cells; ii) a first enrichment step comprising sorting the mixture of treated cells to obtain a first T cell subpopulation that is resistant to the killing of the NK cells; and iii) a first recovery step comprising culturing the first T cell subpopulation. In some embodiments, treatment with NK cells step b) comprises: i) an initial treatment step comprising contacting the T cell library with the NK cells; ii) a first enrichment step comprising sorting the mixture of treated cells to obtain a first T cell subpopulation that is resistant to the killing of the NK cells; iii) a first recovery step comprising culturing the first T cell subpopulation; and iv) a second treatment step comprising contacting the first T cell subpopulation with the NK cells.

In some embodiments, obtaining the T cell from the T cell library that is sensitive or resistant to the killing of the NK cells (hereinafter also referred to as “the T cell obtaining step, ” “the T cell obtaining step c) , ” or “step c) ” ) comprises: i) a sorting step comprising sorting the cells obtained from “the NK cell treatment step b) ” to obtain a second T cell subpopulation that is sensitive or resistant to the killing of the NK cells ( “the harvest sorting step” ) ; and ii) an optional second recovery step comprising culturing the second T cell subpopulation before harvesting the cells ( “the second recovery step” ) . In some embodiments, the T cell obtaining step c) comprises a sorting step comprising sorting the cells obtained from “the NK cell treatment step b) ” to obtain a second T cell subpopulation that is sensitive or resistant to the killing of the NK cells. In some embodiments, the T cell obtaining step c) comprises: i) a sorting step comprising sorting the cells obtained from “the NK cell treatment step b) ” to obtain a second T cell subpopulation that is resistant to the killing of the NK cells; and ii) a second recovery step comprising culturing the second T cell subpopulation before harvesting the cells.

In some embodiments, the NK cell treatment step b) and the T cell obtaining step c) comprises: i) an initial treatment step comprising contacting the T cell library with the NK cells for about 72 hours at a ratio of NK cells to T cells of about 0.5∶ 1; ii) an enrichment step comprising sorting the mixture of treated cells that are B2M-negative (or deficient) and alive, thus obtaining a first T cell subpopulation that is resistant to the killing of the NK cells; iii) a recovery step comprising culturing the first T cell subpopulation for about 48 hours; iv) a second treatment step comprising contacting the first T cell subpopulation post-recovery with the NK cells for about 96 hours at a ratio of NK cells to T cells of about 0.3∶ 1; and v) a sorting step comprising sorting the final mixture of treated cells that are B2M-negative (or deficient) and alive, thus obtaining a second T cell subpopulation that is resistant to the killing of the NK cells.

In some embodiments, the NK cell treatment step b) and the T cell obtaining step c) comprises: i) a treatment step comprising contacting the T cell library with the NK cells for about 10 days at a ratio of NK cells to T cells of about 0.5∶ 1; and ii) a sorting step comprising sorting the mixture of treated cells that are B2M-negative (or deficient) and alive, thus obtaining a T cell subpopulation that is resistant to the killing of the NK cells.

In some embodiments, the NK cell treatment step b) and the T cell obtaining step c) comprises: i) a treatment step comprising contacting the T cell library with the NK cells for about 48 hours at a ratio of NK cells to T cells of about 1∶ 1; ii) a sorting step comprising sorting the mixture of treated cells that are B2M-negative (or deficient) and alive, thus obtaining a T cell subpopulation that is resistant to the killing of the NK cells; and iii) a recovery step comprising culturing the T cell subpopulation for about 48 hours before harvesting the cells.

In some embodiments, the NK cell treatment step b) and the T cell obtaining step c) comprises: i) a treatment step comprising contacting the T cell library with the NK cells for about 48 hours at a ratio of NK cells to T cells of about 1∶ 1; ii) an enrichment step comprising sorting the mixture of treated cells that are B2M-negative (or deficient) and alive, thus obtaining a first T cell subpopulation that is resistant to the killing of the NK cells; iii) a recovery step comprising culturing the first T cell subpopulation for about 48 hours; and iv) a sorting step comprising sorting the first T cell subpopulation post-recovery that are B2M-negative (or deficient) and alive, thus obtaining a second T cell subpopulation that is resistant to the killing of the NK cells.

Initial treatment step

In some embodiments, the initial treatment step comprises contacting the T cell library with the NK cells (e.g., growing the T cell library in the presence of the NK cells) for at least about 48 hours, such as at least about any of 50 hours, 52 hours, 54 hours, 56 hours, 58 hours, 60 hours, 62 hours, 64 hours, 66 hours, 68 hours, 70 hours, 72 hours, 74 hours, 76 hours, 78 hours, 80 hours, 84 hours, 96 hours, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 12 days, 14 days, 16 days, 18 days, 20 days, or longer. In some embodiments, the initial treatment step comprises contacting the T cell library with the NK cells for at least about 48 hours. In some embodiments, the initial treatment step comprises contacting the T cell library with the NK cells for at least about 72 hours. In some embodiments, the initial treatment step comprises contacting the T cell library with the NK cells for at least about 5 days. In some embodiments, the initial treatment step comprises contacting the T cell library with the NK cells for at least about 10 days.

In some embodiments, the ratio of the NK cells and the T cells in the T cell library in the initial treatment step is about 0.1∶ 1 to about 100∶ 1, such as any of about 0.1∶ 1 to about 1∶ 1, about 0.3∶ 1 to about 1∶ 1, about 0.1∶ 1 to about 0.5∶ 1, about 0.5∶ 1 to about 1∶ 1, about 1∶ 1 to about 5∶ 1, about 1∶ 1 to about 10∶ 1, about 5∶ 1 to about 10∶ 1, about 1∶ 1 to about 50∶ 1, about 1∶ 1 to about 20∶ 1, about 10∶ 1 to about 100∶ 1, about 0.1∶ 1 to about 20∶ 1, about 0.5∶ 1 to about 20∶ 1, about 0.1∶ 1 to about 10∶ 1, or about 0.2∶ 1 to about 2∶ 1. In some embodiments, the ratio of the NK cells and the T cells in the T cell library in the initial treatment step is about 0.5∶ 1. In some embodiments, the ratio of the NK cells and the T cells in the T cell library in the initial treatment step is about 1∶ 1.

The longer the NK cell contacting time, and/or the higher ratio of NK cells to T cells, the harsher the treatment condition.

First enrichment step

In some embodiments, the method comprises a first enrichment step after the initial treatment step, comprising sorting the mixture of treated cells (comprising NK cells and treated T cell library) to obtain a first T cell subpopulation that is sensitive or resistant to the killing of the NK cells. In some embodiments, the first enrichment step comprises sorting the mixture of treated cells that are T cells (or not NK cells) and alive, thus obtaining the first T cell subpopulation that is resistant to the killing of the NK cells (herein also referred to as “first alive enrichment” ) . In some embodiments, the first enrichment step comprises sorting the mixture of treated cells that are T cells (or not NK cells) and dead, thus obtaining the first T cell subpopulation that is sensitive to the killing of the NK cells (herein also referred to as “first dead enrichment” ) .

In some embodiments, the first enrichment step further comprises staining the mixture of treated cells with an antibody specifically recognizing a T cell-specific marker or an NK cell specific marker before sorting, to tell apart T cells from NK cells. For example, in some embodiments, the first enrichment step comprises staining the mixture of treated cells with an anti-CD3 antibody and/or an anti-CD56 antibody, and sorting the mixture of treated cells that are CD3+ and/or CD56- (i.e., T cells) .

In some embodiments, the first enrichment step further comprises staining the mixture of treated cells with a cell viability marker (e.g., dye) before sorting. Methods and reagents for assessing cell viability are well known in the art, e.g., fluorescent based or colorimetric (enzymatic) based. For example, membrane permeability-based assays such as staining with DAPI, propidium iodide (PI) , 7-AAD, or amine-reactive dyes indicates dead cells; while acridine orange stains viable cells more efficiently. Carboxyfluorescein diacetate (CFDA) is a nonfluorescent, cell permeable dye that is hydrolyzed to form the fluorescent molecule carboxyfluorescein by nonspecific intracellular esterases present only in viable cells. CFDA-SE is a derivative of CFDA that is better retained upon hydrolysis, in viable cells. Tetramethylrhodamine ethyl esters (TMRE) and Tetramethylrhodamine methyl esters (TMRM) localize to mitochondria in healthy cells and to the cytoplasm in dying cells. JC-1 is a commonly used potentiometric dye. In healthy cells JC-1 localizes to the mitochondria, where it forms red fluorescent aggregates. Upon breakdown of the mitochondrial membrane potential, JC-1 diffuses throughout the cell and exists as a green fluorescent monomer. BrdU incorporation into newly synthesized DNA indicates live cells.

In some embodiments, the first enrichment step further comprises staining the mixture of treated cells with propidium iodide (PI) before sorting, wherein PI staining indicates cell death. Thus in some embodiments, the first enrichment step comprises sorting the mixture of treated cells that are T cells (e.g., CD3+ and/or CD56-) and PI-negative (no PI staining) , thus obtaining the first T cell subpopulation that is resistant to the killing of the NK cells. In some embodiments, the first enrichment step comprises sorting the mixture of treated cells that are T cells (e.g., CD3+ and/or CD56-) and PI-positive (PI staining indicates cell death) , thus obtaining the first T cell subpopulation that is sensitive to the killing of the NK cells.

In some embodiments, the T cell library described herein comprises a B2M mutation (e.g., comprises a B2M sgRNA construct) , such as an inactivating B2M mutation. Thus in some embodiments, the first enrichment step comprises sorting the mixture of treated cells that are B2M-negative or deficient (i.e., T cells) and alive (e.g., PI-) , thus obtaining the first T cell subpopulation that is resistant to the killing of the NK cells ( “first alive enrichment” ) . In some embodiments, the first enrichment step comprises sorting the mixture of treated cells that are B2M-negative or deficient (i.e., T cells) and dead (e.g., PI+) , thus obtaining the first T cell subpopulation that is sensitive to the killing of the NK cells ( “first dead enrichment” ) . The presence or absence of B2M mutation (e.g., inactivating B2M mutation) can be assessed by anti-B2M antibody staining, assessing the presence of the B2M sgRNA construct (e.g., the sgRNA vector backbone, or B2M sgRNA) , assessing the presence of the sgRNA construct targeting another gene that affects B2M expression and/or function, or detecting B2M mutation such as by PCR or sequencing (e.g., PCR or sequencing of the B2M locus, or PCR or sequencing of another gene that affects B2M expression and/or function) . In some embodiments, the first enrichment step further comprises staining the mixture of treated cells with an anti-B2M antibody before sorting. Thus in some embodiments, the first enrichment step comprises staining the mixture of treated cells with an anti-B2M antibody and PI, and sorting the mixture of stained treated cells that are: i) B2M- (or less B2M expression) and PI-, thus obtaining the first T cell subpopulation that is resistant to the killing of the NK cells; or ii) B2M- (or less B2M expression) and PI+, thus obtaining the first T cell subpopulation that is sensitive to the killing of the NK cells.

Any cell sorting methods can be used herein, such as FACS, MACS, microfluidic cell-sorting, buoyancy-activated cell sorting (BACS) , etc. Sorting the mixture of treated cells by cell type and viability can be done in one sorting step, or separate sorting steps. For example, T cells (alive and dead) can be sorted from the mixture of treated cells, then alive T cells (or dead T cells) are sorted from the mixture of T cells; or, alive (or dead) cells (mixture of T cells and NK cells) can be sorted from the mixture of treated cells first, then alive (or dead) T cells are sorted from the mixture of T cells and NK cells.

First recovery step

In some embodiments, the method comprises a first recovery step comprising culturing the mixture of treated cells (NK cells and treated T cell library) , after the initial treatment step comprising contacting the T cell library with the NK cells. In some embodiments, the method comprises a first recovery step comprising culturing the first T cell subpopulation, after the first enrichment step comprising sorting the mixture of treated cells to obtain a first T cell subpopulation that is resistant to the killing of the NK cells (i.e., alive T cells) . In some embodiments, the first recovery step comprises culturing the mixture of treated cells (NK cells and treated T cell library) or the first T cell subpopulation for at least about 24 hours, such as at least about any of 26 hours, 28 hours, 30 hours, 32 hours, 34 hours, 36 hours, 38 hours, 40 hours, 48 hours, 52 hours, 56 hours, 60 hours, 64 hours, 68 hours, 72 hours, 78 hours, 84 hours, 96 hours, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 12 days, 14 days, 16 days, 18 days, 20 days, or longer. In some embodiments, the first recovery step comprises culturing the mixture of treated cells (NK cells and treated T cell library) or the first T cell subpopulation for about 48 hours.

The culturing condition is suitable for T cell growth and/or proliferation. In some embodiments, the culturing condition does not induce T cells to a specific phenotype during expansion. Such culture conditions are well known in the art. For example, in 37℃, 5%CO ₂ incubator. Also see Master et al. ( “T Cell Media: A Comprehensive Guide to Key Components, ” 2018) . In some embodiments, the culture medium is a T cell complete medium. In some embodiments, the culture condition is the same as that for the T cell library before NK cell treatment. In some embodiments, the culture condition is suitable for adoptive T cell therapy, such as CAR-T cells (e.g., allogeneic CAR-T cells) . The type of culture media for successful cultivation can vary depending on the subset of T cells. For T cells, interleukin-2 (IL-2) is a potent cytokine which modulates proliferation and differentiation into effector and memory T cells. Culture conditions may be further refined to polarize T cells to a specific phenotype during expansion. For example, IL-4, IL-7 and IL-15 have been reported to be essential for induction, survival or turnover of memory T cells, respectively. The most widely used medium for culturing T cells in research laboratories is RPMI 1640 supplemented with FBS, whereas for the biomanufacturing of T cells for adoptive cell therapy, “complete” formulations such as X-VIVO 15 (Lonza, Inc) and CTS OpTimizer (Thermofisher, Inc) supplemented with human serum are more common. In some embodiments, the culture medium is further supplemented with an agent for selectable markers, e.g., to select T cells that do not lose transgenes or mutations during proliferation.

Second treatment step

In some embodiments, the method comprises a second treatment step comprising contacting the mixture of treated cells (NK cells and treated T cell library) with NK cells, after the initial treatment step comprising contacting the T cell library with the NK cells (with or without further cultured during a recovery step) . In some embodiments, the method comprises a second treatment step comprising contacting the first T cell subpopulation after the first enrichment step (with or without further cultured during a recovery step) with NK cells, wherein the first T cell subpopulation is resistant to the killing of the NK cells during the initial treatment step. In some embodiments, contacting the T cells with NK cells comprises growing the T cells in the presence of the NK cells.

In some embodiments, the second treatment step comprises contacting the mixture of treated cells (NK cells and treated T cell library) , or the first T cell subpopulation that is resistant to the killing of the NK cells during the initial treatment step, with or without further cultured during a recovery step, with NK cells for at least about 48 hours, such as at least about any of 50 hours, 52 hours, 54 hours, 56 hours, 58 hours, 60 hours, 62 hours, 64 hours, 66 hours, 68 hours, 70 hours, 72 hours, 74 hours, 76 hours, 78 hours, 80 hours, 84 hours, 96 hours, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 12 days, 14 days, 16 days, 18 days, 20 days, or longer. In some embodiments, the second treatment step comprises contacting with NK cells for the same or similar (e.g., at most about 30 minutes more or less) amount of time as compared to the initial treatment step. In some embodiments, the second treatment step comprises contacting with NK cells for less amount of time as compared to the initial treatment step, such as about any of 35 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, 96 hours, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days less as compared to the initial treatment step. In some embodiments, the second treatment step comprises contacting with NK cells for more amount of time as compared to the initial treatment step, such as about any of 35 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, 96 hours, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days longer as compared to the initial treatment step. In some embodiments, the second treatment step comprises contacting the mixture of treated cells (NK cells and treated T cell library) , or the first T cell subpopulation that is resistant to the killing of the NK cells during the initial treatment step, with or without further cultured during a recovery step, with NK cells for about 96 hours.

In some embodiments, in the second NK cell treatment step, the ratio of the NK cells and the T cells in the first T cell subpopulation that is resistant to the killing of the NK cells during the initial treatment step, or the ratio of the NK cells and the T cells from the first T cell subpopulation after the recovery step, is about 0.1∶ 1 to about 100∶ 1, such as any of about 0.1∶ 1 to about 1∶ 1, about 0.3∶ 1 to about 1∶ 1, about 0.1∶ 1 to about 0.5∶ 1, about 0.5∶ 1 to about 1∶ 1, about 1∶ 1 to about 5∶ 1, about 1∶ 1 to about 10∶ 1, about 1∶ 1 to about 50∶ 1, about 1∶ 1 to about 20∶ 1, about 10∶ 1 to about 100∶ 1, about 0.1∶ 1 to about 20∶ 1, about 0.5∶ 1 to about 20∶ 1, about 0.1∶ 1 to about 10∶ 1, about 5∶ 1 to about 10∶ 1, or about 0.2∶ 1 to about 2∶ 1. In some embodiments, the second treatment step comprises contacting the mixture of treated cells (NK cells and treated T cell library) , the first T cell subpopulation that is resistant to the killing of the NK cells during the initial treatment step, or the T cells from the first T cell subpopulation after the recovery step, with the same ratio of NK cells to T cells. In some embodiments, the second treatment step comprises contacting the mixture of treated cells (NK cells and treated T cell library) , the first T cell subpopulation that is resistant to the killing of the NK cells during the initial treatment step, or the T cells from the first T cell subpopulation after the recovery step, with higher ratio of NK cells to T cells, such as at least about any of 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, or 20-fold higher ratio of NK cells to T cells, compared to that in the initial treatment step. In some embodiments, the second treatment step comprises contacting the mixture of treated cells (NK cells and treated T cell library) , the first T cell subpopulation that is resistant to the killing of the NK cells during the initial treatment step, or the T cells from the first T cell subpopulation after the recovery step, with lower ratio of NK cells to T cells, such as at least about any of 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, or 20-fold lower ratio of NK cells to T cells, compared to that in the initial treatment step. In some embodiments, in the second NK cell treatment step, the ratio of the NK cells and the T cells in the first T cell subpopulation that is resistant to the killing of the NK cells during the initial treatment step, or the ratio of the NK cells and the T cells from the first T cell subpopulation after the recovery step, is about 0.3∶ 1.

Optional additional recovery step

In some embodiments, the method further comprises an additional recovery step comprising culturing the mixture of treated cells (NK cells and treated first T cell subpopulation) after the second treatment step. In some embodiments, the additional recovery step has the same culturing condition as in the first recovery step. In some embodiments, the additional recovery step has a different culturing condition as in the first recovery step. In some embodiments, the additional recovery step is longer than the first recovery step, such as at least about any of 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, 96 hours, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days longer than the first recovery step. In some embodiments, the additional recovery step is shorter than the first recovery step, such as at least about any of 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, 96 hours, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days shorter than the first recovery step.

Harvest sorting step

In some embodiments, obtaining the T cell from the T cell library that is sensitive or resistant to the killing of the NK cells ( “the T cell obtaining step c) ” ) comprises a sorting step comprising sorting the cells obtained from “the NK cell treatment step b) ” to obtain a second T cell subpopulation that is sensitive or resistant to the killing of the NK cells ( “the harvest sorting step” ) .

In some embodiments, the cells obtained from “the NK cell treatment step b) ” are a mixture of treated cells (NK cells and treated T cell library) after the initial treatment step. In some embodiments, the cells obtained from “the NK cell treatment step b) ” are a mixture of treated cells (NK cells and treated T cell library) after the initial treatment step and after a first recovery step comprising culturing the mixture of treated cells. In some embodiments, the cells obtained from “the NK cell treatment step b) ” are a mixture of treated cells (NK cells and treated first T cell subpopulation) after the second treatment step. In some embodiments, the cells obtained from “the NK cell treatment step b) ” are a mixture of treated cells (NK cells and treated first T cell subpopulation) after the second treatment step and after the additional recovery step comprising culturing the mixture of treated cells. For such embodiments, the harvest sorting step is the same as or similar to the first enrichment step described above.

For example, in some embodiments, the harvest sorting step comprises sorting the mixture of treated cells (with or without further cultured in a recovery step) obtained from “the NK cell treatment step b) ” that are T cells (or not NK cells) and alive, thus obtaining the second T cell subpopulation that is resistant to the killing of the NK cells (herein also referred to as “harvest alive sorting” ) . In some embodiments, the harvest sorting step comprises sorting the mixture of treated cells (with or without further cultured in a recovery step) obtained from “the NK cell treatment step b) ” that are T cells (or not NK cells) and dead, thus obtaining the second T cell subpopulation that is sensitive to the killing of the NK cells (herein also referred to as “harvest dead sorting” ) .

In some embodiments, the harvest sorting step further comprises staining the mixture of treated cells (with or without further cultured in a recovery step) obtained from “the NK cell treatment step b) ” with an antibody specifically recognizing a T cell-specific marker or an NK cell specific marker before sorting, to tell apart T cells from NK cells. For example, in some embodiments, the harvest sorting step comprises staining the mixture of treated cells obtained from “the NK cell treatment step b) ” with an anti-CD3 antibody and/or an anti-CD56 antibody, and sorting the mixture of treated cells that are CD3+ and/or CD56- (i.e., T cells) .

In some embodiments, the harvest sorting step further comprises staining the mixture of treated cells (with or without further cultured in a recovery step) obtained from “the NK cell treatment step b) ” with a cell viability marker (e.g., dye) before sorting. Any agents and/or methods described in the “first enrichment step” subsection above can be used herein.

In some embodiments, the harvest sorting step further comprises staining the mixture of treated cells (with or without further cultured in a recovery step) obtained from “the NK cell treatment step b) ” with PI before sorting, wherein PI staining indicates cell death. Thus in some embodiments, the harvest sorting step comprises sorting the mixture of treated cells (with or without further cultured in a recovery step) obtained from “the NK cell treatment step b) ” that are T cells (e.g., CD3+ and/or CD56-) and PI-negative (no PI staining) , thus obtaining the second T cell subpopulation that is resistant to the killing of the NK cells. In some embodiments, the harvest sorting step comprises sorting the mixture of treated cells (with or without further cultured in a recovery step) obtained from “the NK cell treatment step b) ” that are T cells (e.g., CD3+ and/or CD56-) and PI-positive (PI staining indicates cell death) , thus obtaining the second T cell subpopulation that is sensitive to the killing of the NK cells.

In some embodiments, the T cell library described herein comprises a B2M mutation (e.g., comprises a B2M sgRNA construct) , such as an inactivating B2M mutation. Thus in some embodiments, the harvest sorting step comprises sorting the mixture of treated cells (with or without further cultured in a recovery step) obtained from “the NK cell treatment step b) ” that are B2M-negative or deficient (i.e., T cells) and alive (e.g., PI-) , thus obtaining the second T cell subpopulation that is resistant to the killing of the NK cells ( “harvest alive sorting” ) . In some embodiments, the harvest sorting step comprises sorting the mixture of treated cells (with or without further cultured in a recovery step) obtained from “the NK cell treatment step b) ” that are B2M-negative or deficient (i.e., T cells) and dead (e.g., PI+) , thus obtaining the second T cell subpopulation that is sensitive to the killing of the NK cells ( “harvest dead sorting” ) . The presence or absence of B2M mutation (e.g., inactivating B2M mutation) can be assessed by anti-B2M antibody staining, assessing the presence of the B2M sgRNA construct (e.g., the sgRNA vector backbone, or B2M sgRNA) , assessing the presence of the sgRNA construct targeting another gene that affects B2M expression and/or function, or detecting B2M mutation such as by PCR or sequencing (e.g., PCR or sequencing of the B2M locus, or PCR or sequencing of another gene that affects B2M expression and/or function) . In some embodiments, the harvest sorting step further comprises staining the mixture of treated cells (with or without further cultured in a recovery step) obtained from “the NK cell treatment step b) ” with an anti-B2M antibody before sorting. Thus in some embodiments, the harvest sorting step comprises staining the mixture of treated cells (with or without further cultured in a recovery step) obtained from “the NK cell treatment step b) ” with an anti-B2M antibody and PI, and sorting the mixture of stained treated cells that are: i) B2M- (or less B2M expression) and PI-, thus obtaining the second T cell subpopulation that is resistant to the killing of the NK cells; or ii) B2M- (or less B2M expression) and PI+, thus obtaining the second T cell subpopulation that is sensitive to the killing of the NK cells.

Sorting the mixture of treated cells (with or without further cultured in a recovery step) obtained from “the NK cell treatment step b) ” by cell type and viability can be done in one sorting step, or separate sorting steps. For example, T cells (alive and dead) can be sorted from the mixture of treated cells (with or without further cultured in a recovery step) obtained from “the NK cell treatment step b) ” , then alive T cells (or dead T cells) are sorted from the mixture of T cells; or, alive (or dead) cells (mixture of T cells and NK cells) can be sorted from the mixture of treated cells (with or without further cultured in a recovery step) obtained from “the NK cell treatment step b) ” first, then alive (or dead) T cells are sorted from the mixture of T cells and NK cells.

Second recovery step

In some embodiments, the method comprises a second recovery step after the harvest sorting step. Thus in some embodiments, the “T cell obtaining step c) ” comprises: i) a sorting step comprising sorting the cells obtained from “the NK cell treatment step b) ” to obtain a second T cell subpopulation that is resistant to the killing of the NK cells; and ii) a second recovery step comprising culturing the second T cell subpopulation before harvesting the cells.

In some embodiments, the second recovery step is the only recovery step in the methods described herein. In some embodiments, the second recovery step comprises culturing the second T cell subpopulation that is resistant to the killing of the NK cells for at least about 24 hours, such as at least about any of 26 hours, 28 hours, 30 hours, 32 hours, 34 hours, 36 hours, 38 hours, 40 hours, 48 hours, 52 hours, 56 hours, 60 hours, 64 hours, 68 hours, 72 hours, 78 hours, 84 hours, 96 hours, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 12 days, 14 days, 16 days, 18 days, 20 days, or longer. In some embodiments, the second recovery step comprises culturing the second T cell subpopulation that is resistant to the killing of the NK cells for about 48 hours.

The culturing condition is suitable for T cell growth and/or proliferation. Any culturing condition and/or method described above in the “first recovery step” subsection can be used herein.

In some embodiments, the second recovery step has the same culturing condition as in the first recovery step (and/or the optional additional recovery step) . In some embodiments, the second recovery step has a different culturing condition as in the first recovery step (and/or the optional additional recovery step) . In some embodiments, the second recovery step is longer than the first recovery step (and/or the optional additional recovery step) , such as at least about any of 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, 96 hours, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days longer than the first recovery step (and/or the optional additional recovery step) . In some embodiments, the second recovery step is shorter than the first recovery step (and/or the optional additional recovery step) , such as at least about any of 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, 96 hours, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days shorter than the first recovery step (and/or the optional additional recovery step) .

T cell harvest step

In some embodiments, after obtaining the second T cell subpopulation that is sensitive or resistant to the killing of the NK cells from the harvest sorting step, or after obtaining the second T cell subpopulation and cultured in the second recovery step, the obtained T cells (alive or dead) are harvested. The T cell harvest step in some embodiments comprises collecting the T cells into a container (e.g., Falcon tubes, EP tubes, or centrifugation tubes) for storage or for later experiments. In some embodiments, the T cell harvest step comprises washing the obtained T cells, so that the T cells are in suitable condition for storage (e.g., 4℃, -20℃, or -80℃ storage) or later experiments (e.g., cell lysis, and PCR or sequencing) .

NK cells and preparation methods

NK cells are lymphoid cells that participate in immune reactions. They have the functions of killing of tumor cells, cells undergoing oncogenic transformation and other abnormal cells in a living body, and are important components of innate immunological surveillance mechanisms. NK cells possess mechanisms distinguishing between “foreign” or potential target cells and healthy “self” cells via a multitude of inhibitory and activating receptors that engage MHC class I molecules, MHC class I-like molecules, and molecules unrelated to MHC (Caliguiri, Blood 2008, 112: 461-69) . Cells (e.g., T cells such as allogeneic T cells) with reduced or absent HLA class I expression are targeted by NK cells as “foreign, ” leading to rejection reactions (Liu et al. Curr. Res. Transl. Med. 2018; 66: 39-42) .

NK cells express characteristic NK cell surface receptors, and lack both TCR rearrangement and T cell, B cell, monocyte and/or macrophage cell surface markers. NK cells exhibit cytotoxicity by releasing small cytoplasmic granules of proteins (perforin and granzyme) that cause the target cell to die by apoptosis. Killing is triggered in a contact-dependent, non-phagocytotic process which does not require prior sensitization to an antigen. Human NK cells are characterized by the presence of the cell-surface markers CD16 and CD56, and the absence of the T cell receptor (CD3) . Human bone marrow-derived NK cells are further characterized by the CD2+CD16+CD56+CD3-phenotype, further containing the T-cell receptor zeta-chain [zeta (Q-TCR] , and often characterized by NKp46, NKp30 or NKp44. Inhibitory NK cell receptors include HLA-E (CD94/NKG2A) ; HLA-C (group 1 or 2) , KIR2DL; KIR3DL (HLA-B Bw4) and HLA-A3 or A4 + peptide. Activating NK cell receptors include HLA-E (CD94/NKG2C) ; KIR2DS (HLA-C) and KIR3DS (HLA-Bw4) . Other receptors include the NK cell receptor protein-1 (termed NK1.1 in mice) and the low affinity receptor for the Fc portion of IgG (FcyRIII; CD 16) .

Methods of isolation, culture, induction, expansion, and enrichment of NK cells are well known in the art, e.g., US9,938,498, or Magee et al. ( “Chapter Nine -Isolation, culture and propagation of natural killer cells, ” Natural Killer Cells, Basic Science and Clinical Application, 2010, Pages 125-135) . Also see “T cells and preparation methods” section above, the methods of which can be adapted for the preparation of NK cells. For example, FACS with antibodies against NK cell specific markers can be used for NK cell isolation and/or enrichment.

NK cells of the present invention may be derived from any source which comprises such cells. NK cells are found in many tissues, and can be obtained, for example, from lymph nodes, spleen, liver, lungs, intestines, deciduas and can also be obtained from iPS cells or embryonic stem cells (ESC) . Typically, cord blood, peripheral blood, mobilized peripheral blood and bone marrow, which contain heterogeneous lymphocyte cell populations, are used to provide large numbers of NK cells for research and clinical use. In some embodiments, the method comprises culturing a population of NK cells derived from one of cord blood, peripheral blood or bone marrow. In some embodiments, NK cells are cultured from a heterogeneous cell population comprising NK cells, CD3-cells and CD3+ cells. In one embodiment the CD3+ fraction is greater than the CD3-NK cell fraction, as is typical of bone marrow, cord blood or peripheral blood. In some embodiments, the NK cell population is selected or enriched for NK cells. In some embodiments NK cells can be propagated from fresh cell populations, while other embodiments propagate NK cells from stored cell populations (such as cyropreserved and thawed cells) or previously cultured cell populations. In some embodiments, NK cells are from a cell line, such as

In some embodiments, the NK cells are a homogenous NK cell population (i.e., express the same cell surface markers) . In some embodiments, the NK cells are a heterogeneous NK cell population. In some embodiments, a population of cells comprising NK cells is used for treating the T cell library described herein. In some embodiments, the NK cells are a selected NK cell population, e.g., CD56+CD3-NK cells, CD56+CD16+CD3-NK cells, or CD56+CD 16-CD3-NK cells. Methods for selection of NK cells according to phenotype are well known in the art, e.g., immunodetection or FACS analysis.

Methods for enriching and isolating lymphocytes are well known in the art, and appropriate methods can be selected based on the desired population. For example, in one approach, the source material is enriched for lymphocytes by removing red blood cells. In its simplest form, removal of red blood cells can involve centrifugation of unclotted whole blood or bone marrow. Based on density red blood cells are separated from lymphocytes and other cells. The lymphocyte rich fractions can then be selectively recovered. Lymphocytes and their progenitors can also be enriched by centrifugation using separation mediums such as standard Lymphocyte Separation Medium (LSM) available from a variety of commercial sources. Alternatively, lymphocytes/progenitors can be enriched using various affinity based procedures. Numerous antibody mediated affinity preparation methods are known in the art such as antibody conjugated magnetic beads. Lymphocyte enrichment can also be performed using commercially available preparations for negatively selecting unwanted cells, such as FICOLL-HYPAQUE ^TM and other density gradient mediums formulated for the enrichment of whole lymphocytes, T cells or NK cells.

Hit gene identification

The method described herein comprises identifying the hit gene in the T cell obtained from the T cell library (e.g., Cas9 ⁺ B2M ^-sgRNA T cell library, Cas9 ⁺ B2M ^-sgRNA ^iBAR T cell library, Cas9 ⁺ sgRNA T cell library, or Cas9 ⁺ sgRNA ^iBAR T cell library) that is sensitive or resistant to the killing of the NK cells ( “hit gene identification step” ) . In some embodiments, the hit gene identified from the T cell obtained from the T cell library (or post-treatment T cell population) that is sensitive or resistant to the killing of the NK cells is considered as the target gene whose mutation makes the T cell sensitive or resistant to the killing of the NK cells, respectively.

In some embodiments, the hit gene identification step comprises: i) identifying a sequence comprising the hit gene mutation (e.g., inactivating mutation) in the T cell obtained from “the T cell obtaining step c) ” (or post-treatment T cell population) ; and ii) identifying the hit gene corresponding to the sequence comprising the hit gene mutation (e.g., inactivating mutation) . In some embodiments, the sequence comprising the hit gene mutation (e.g., inactivating mutation) is identified by sequencing, e.g., PCR-sequencing (e.g., Sanger sequencing) , or genome-sequencing (or DNA-seq, such as next-generation sequencing or “NGS” ) . For example, in some embodiments, the sequences (nucleic acid fragments, PCR fragments, or whole-genome) of the T cells obtained from the T cell library (or post-treatment T cell population) that are sensitive or resistant to the killing of the NK cells are identified by sequencing, by comparing to the wild-type genomic sequence, or by comparing to the genomic sequence of the initial population of T cells, and the sequence (s) comprising the hit gene mutation (s) (e.g., inactivating mutation (s) ) can be identified and mapped to the hit gene (s) . In some embodiments, the hit gene identification step further comprises isolating genomic DNA or RNA from the T cells obtained from “the T cell obtaining step c) ” (or post-treatment T cell population) . In some embodiments, the hit gene identification step further comprises PCR amplification of nucleic acid sequence comprising the hit gene mutation (e.g., inactivating mutation) .

In some embodiments, the T cell library described herein comprises the sgRNA constructs or the sgRNA ^iBAR constructs against hit genes described herein. Thus in some embodiments, the hit gene identification step comprises: i) identifying the sgRNA sequence or the sgRNA ^iBAR sequence in the T cell obtained from “the T cell obtaining step c) ” (or post-treatment T cell population) ; and ii) identifying the hit gene corresponding to (targeted by) the guide sequence of the sgRNA or the sgRNA ^iBAR. In some embodiments, the sgRNA sequence or the sgRNA ^iBAR sequence is identified by RNA sequencing (RNA-seq) , e.g., RNA NGS. In some embodiments, the hit gene identification step comprises: i) identifying the nucleic acid sequence encoding the sgRNA or the sgRNA ^iBAR in the T cell obtained from “the T cell obtaining step c) ” (or post-treatment T cell population) ; and ii) identifying the hit gene corresponding to the guide sequence encoded by the nucleic acid sequence. In some embodiments, the nucleic acid sequence encoding the sgRNA or the sgRNA ^iBAR is identified by sequencing, e.g., PCR-sequencing (e.g., Sanger sequencing) , or genome-sequencing (DNA-seq) , e.g., NGS. In some embodiments, the ^iBAR sequences can be used for identifying the sgRNA ^iBAR sequences or the nucleic acid sequences encoding the sgRNA ^iBAR. In some embodiments, the hit gene identification step further comprises isolating genomic DNA or RNA from the T cells obtained from “the T cell obtaining step c) ” (or post-treatment T cell population) . In some embodiments, the hit gene identification step further comprises PCR amplification of nucleic acid sequence encoding the sgRNA or the sgRNA ^iBAR.

Methods for DNA-seq, RNA-seq, PCR-sequencing (e.g., Sanger sequencing) , DNA/RNA extraction, cDNA preparation, and data analysis are well known in the art, and can be used herein as appropriate to identify the hit gene (s) in the T cell (s) from the T cell library (or post-treatment T cell population) that is/are sensitive or resistant to the killing of the NK cells. The sequencing data can be analyzed and aligned to the genome using any known methods in the art.

Target gene identification

In some embodiments, the hit gene identified in the T cell from the T cell library (or post-treatment T cell population) that is sensitive or resistant to the killing of the NK cells is considered as the target gene in the T cell that modulates the activity of the T cell. For example, in some embodiments, the hit genes identified in the T cells from the T cell library that are sensitive to the killing of the NK cells (i.e., dead T cell subpopulation) are target genes whose mutation (e.g., inactivation) makes the T cells sensitive to NK cell killing. In some embodiments, the hit genes identified in the T cells from the T cell library that are resistant to the killing of the NK cells (i.e., alive T cell subpopulation) are target genes whose mutation (e.g., inactivation) makes the T cells resistant to NK cell killing.

In some embodiments, the hit gene identified in the T cell from the T cell library (or post-treatment T cell population) that is sensitive or resistant to the killing of the NK cells is further compared to a control, and/or is further ranked and/or filtered with a predetermined threshold level. In some embodiments, identifying the target gene comprises: i) obtaining sequences comprising the hit gene mutations (e.g., inactivating mutations) in the final T cell subpopulation obtained from “the T cell obtaining step c) ” ; ii) ranking the sequences comprising the hit gene mutations (e.g., inactivating mutations) based on sequence counts; and iii) identifying the hit gene corresponding to a sequence comprising the hit gene mutation (e.g., inactivating mutation) ranked above a predetermined threshold level. In some embodiments, the ranking step comprises adjusting the rank of each sequence comprising the hit gene mutation (e.g., inactivating mutation) based on data consistency among all sequences comprising the hit gene mutation (e.g., inactivating mutation) corresponding to the same hit gene (or same target site of the same hit gene) . For example, data inconsistency (such as different directions of fold changes relative to control) will increase variance of the sequences comprising the hit gene mutation (e.g., inactivating mutation) corresponding to the same hit gene and lower the rank of such hit gene. In some embodiments, the hit gene is identified to correspond to sequence (s) comprising the hit gene mutations (e.g., inactivating mutation (s) ) that rank consistently better than expected for permuted sequences under null hypothesis based on an RRA or α-RRA algorithm. In some embodiments, the predetermined threshold level is an FDR of value “X” (e.g., 0.15 or 0.05) , and the hit gene corresponding to a sequence comprising the hit gene mutation (e.g., inactivating mutation) with FDR ≤ “X” is identified as the target gene. In some embodiments, the predetermined threshold level is an enrichment or depletion of value “X” -fold (e.g., about 2-fold) , and the hit gene corresponding to a sequence comprising the hit gene mutation (e.g., inactivating mutation) with enrichment or depletion ≥ “X” -fold is identified as the target gene. In some embodiments, the sequence comprising the hit gene mutation (e.g., inactivating mutation) is identified by sequencing, e.g., Sanger-sequencing or genome-sequencing (or DNA-seq, such as NGS) .

In some embodiments, the T cell library described herein comprises the sgRNA constructs or the sgRNA ^iBAR constructs against hit genes described herein. Thus in some embodiments, identifying the target gene comprises: i) obtaining sgRNA sequences or sgRNA ^iBAR sequences in the final T cell subpopulation obtained from “the T cell obtaining step c) ”; ii) ranking the corresponding guide sequences of the sgRNA sequences or the sgRNA ^iBAR sequences based on sequence counts; and iii) identifying the hit gene corresponding to a guide sequence ranked above a predetermined threshold level. In some embodiments, the ranking comprises adjusting the rank of each guide sequence of the sgRNA sequence or the sgRNA ^iBAR sequence based on data consistency among all guide sequences corresponding to the same hit gene (or same target site of the same hit gene) . For example, data inconsistency (such as different direction of fold change relative to control) will increase variance of the guide sequences corresponding to the same hit gene and lower the rank of such hit gene. In some embodiments, the hit gene is identified to correspond to guide sequence (s) that rank consistently better than expected for permuted guide sequences under null hypothesis based on an RRA or α-RRA algorithm. In some embodiments, the predetermined threshold level is an FDR of value “X” (e.g., 0.15 or 0.05) , and the hit gene corresponding to a guide sequence with FDR ≤ “X” is identified as the target gene. In some embodiments, the predetermined threshold level is an enrichment or depletion of value “X” -fold (e.g., about 2-fold) , and the hit gene corresponding to a guide sequence with enrichment or depletion ≥ “X” -fold is identified as the target gene. In some embodiments, the sgRNA sequence or the sgRNA ^iBAR sequence is identified by RNA-seq, e.g., RNA NGS. In some embodiments, the nucleic acid sequences encoding the sgRNAs or the sgRNAs ^iBAR are identified by genome-sequencing (DNA-seq) , e.g., NGS.

In some embodiments, the T cell library described herein comprises the sgRNA ^iBAR constructs against hit genes described herein. In some embodiments, identifying the target gene comprises: i) obtaining sgRNA ^iBAR sequences in the final T cell subpopulation obtained from “the T cell obtaining step c) ” ; ii) ranking the corresponding guide sequences of the sgRNA ^iBAR sequences based on sequence counts, wherein the ranking comprises adjusting the rank of each guide sequence based on data consistency among the iBAR sequences in the sgRNA ^iBAR sequences corresponding to the guide sequence; and iii) identifying the hit gene corresponding to a guide sequence ranked above a predetermined threshold level. In some embodiments, the hit gene is identified to correspond to guide sequence (s) that rank (s) consistently better than expected for permuted guide sequences under null hypothesis based on an RRA or α-RRA algorithm. In some embodiments, the predetermined threshold level is an FDR of value “X” (e.g., 0.15 or 0.05) , and the hit gene corresponding to a guide sequence with FDR ≤ “X” is identified as the target gene. In some embodiments, the predetermined threshold level is at least about 2-fold enrichment or depletion.

In some embodiments, the sequence counts of sequences comprising the hit gene mutations (e.g., inactivating mutations) or guide RNAs are determined from statistical analysis. In some embodiments, the sequence counts of guide RNAs and the corresponding iBAR sequences are determined from statistical analysis. See FIG. 5 for exemplary target gene identification workflow. Statistical methods may be used to determine the identity of the sequences comprising the hit gene mutations (e.g., inactivating mutations) , the sgRNA molecules, or the sgRNA ^iBAR molecules that are enriched or depleted in the final T cell subpopulation. In some embodiments, more than one (e.g., 2, 3, or more) biological or technical replicate is conducted for an NK cell treated T cell library. In some embodiments, more than one (e.g., 2, 3, or more) biological or technical replicate is conducted for a control T cell library or a subpopulation of control T cells. In some embodiments, sequences comprising the hit gene mutations (e.g., inactivating mutations) or guide RNAs from the two or more (e.g., 2, 3, 4, or more) replicates of the NK cell treated group (or control group) are combined to calculate mean and variance among replicates of the NK cell treated group (or control group) . Exemplary statistical methods include, but are not limited to, linear regression, generalized linear regression and hierarchical regression. In some embodiments, the sequence counts are subject to normalization methods, such as total count normalization, or median ratio normalization. In some embodiments, e.g., for positive screens, median ratio normalization is preferred. In some embodiments, for example, for sequence counts that follow a normal distribution, the sequence counts are subject to median ratio normalization followed by mean-variance modeling. In some embodiments, MAGeCK (Li, W. et al. MAGeCK enables robust identification of essential genes from genome-scale CRISPR/Cas9 knockout screens. Genome Biol 15, 554 (2014) ) is used to rank sequences comprising the hit gene mutations (e.g., inactivating mutations) or guide RNA sequences, and/or to identify target genes. In some embodiments, MAGeCK ^iBAR (Zhu et al., Genome Biol. 2019; 20: 20) is used to rank sequences comprising the hit gene mutations (e.g., inactivating mutations) or guide RNA sequences, and/or to identify target genes.

In some embodiments, identifying the target gene whose mutation makes the T cell sensitive or resistant to NK cell killing is based on the difference between the profiles of sgRNAs (or sgRNAs ^iBAR) or hit gene mutations in the T cell obtained from the T cell library that is sensitive or resistant to the killing of the NK cells in step c) (or post-treatment T cell population) and a control T cell (or a control T cell population) . In some embodiments, the identification of the target gene is based on the difference between the profiles of hit gene mutations in the T cell obtained from the T cell library that is sensitive or resistant to the killing of the NK cells in step c) (or post-treatment T cell population) and a control T cell (or a control T cell population) . In some embodiments, the identification of the target gene is based on the difference between the profiles of sgRNAs (or sgRNAs ^iBAR) in the T cell obtained from the T cell library that is sensitive or resistant to the killing of the NK cells in step c) (or post-treatment T cell population) and a control T cell (or a control T cell population) . In some embodiments, the control T cell population is obtained from the same T cell library cultured in the same condition without subjected to treatment with NK cells, and optionally subjected to the same obtaining method in step c) . In some embodiments, the profiles of sgRNAs (or sgRNAs ^iBAR) or hit gene mutations in the T cell obtained from the T cell library that is sensitive or resistant to the killing of the NK cells in step c) (or post-treatment T cell population) and the control T cell (or control T cell population) are identified by next generation sequencing (NGS) , such as DNA-seq or RNA-seq. In some embodiments, the profiles of sgRNAs (or sgRNAs ^iBAR) comprise sequence counts of the sgRNAs (or sgRNAs ^iBAR) , or sequence counts of the corresponding guide sequences of the sgRNAs (or sgRNAs ^iBAR) . In some embodiments, the profiles of sgRNAs (or sgRNAs ^iBAR) comprise sequence counts of the nucleic acids encoding the sgRNAs (or sgRNAs ^iBAR) , or sequence counts of the nucleic acids encoding the guide sequences of the corresponding sgRNAs (or sgRNAs ^iBAR) . In some embodiments, the profiles of the hit gene mutations comprise sequence counts of the sequences comprising the hit gene mutations. In some embodiments, the methods described herein further comprise culturing a same T cell library under the same condition without subjected to treatment with NK cells, and optionally subjected to the same obtaining method in step c) .

In some embodiments, the sequence counts (e.g., the sequence counts of sgRNAs or sgRNAs ^iBAR or guide sequences thereof, the sequence counts of nucleic acid sequences encoding the sgRNAs or sgRNAs ^iBAR or guide sequences thereof, or sequence counts of sequences comprising the hit gene mutations) obtained from the final T cell subpopulation obtained from “the T cell obtaining step c) ” (or post-treatment T cell population) are compared to corresponding sequence counts obtained from a subpopulation of control T cells or a control T cell library, e.g., to provide fold changes (e.g., actual fold changes, or derivatives of fold changes such as log2 or log 10 fold changes) , for significance tests (e.g., FDR, p-value) , for distribution statistics, and/or to provide gene or sequence rankings via scoring and/or deriving. In some embodiments, the subpopulation of control T cells (or control T cell population) are obtained from a same T cell library cultured in the same condition without subjected to treatment with NK cells, e.g., continuously cultured under the same culture condition for the same amount of time as the test group (treated with NK cells) from test beginning till final sample harvest (see FIG. 2) . In some embodiments, the subpopulation of control T cells is the entire T cell library cultured in the same condition without subjected to treatment with NK cells, and without subjecting to any selecting or obtaining method in “the T cell obtaining step c) ” , hereinafter also referred to as “control T cell library. ” In some embodiments, the subpopulation of control T cells are obtained from a same T cell library cultured in the same condition without subjected to treatment with NK cells, and subjected to the same obtaining method in “the T cell obtaining step c) ” .

In some embodiments, the methods described herein further comprise culturing a same T cell library under the same condition without subjected to treatment with NK cells, and optionally subjected to the same obtaining method in step c) to obtain a control T cell population. In some embodiments, the method further comprises identifying a sequence comprising the hit gene mutation (e.g., inactivating mutation) or the guide sequence of the sgRNA or sgRNA ^iBAR from the control T cell population or control T cell library. In some embodiments, the absence of identifying the hit gene corresponding to the sequence comprising the hit gene mutation (e.g., inactivating mutation) or the guide sequence of the sgRNA or sgRNA ^iBAR from the control T cell population or control T cell library, but presence of identifying from the T cell sensitive or resistant to the killing of the NK cells (or post-treatment T cell population) obtained from step c) , identifies the hit gene as the target gene.. In some embodiments, the presence of identifying the hit gene corresponding to the sequence comprising the hit gene mutation (e.g., inactivating mutation) or the guide sequence of the sgRNA or sgRNA ^iBAR from the control T cell population or control T cell library, but absence of identifying from the T cell sensitive or resistant to the killing of the NK cells (or post-treatment T cell population) obtained from step c) , identifies the hit gene as the target gene. For example, for a T cell library comprising mutations A, B, and C in separate T cells, if only mutation A is identified from the post-treatment T cell population (e.g., alive) , the absence of identifying mutations B and C from this post-treatment T cell population (e.g., alive) indicates hit genes B and C are the target genes, e.g., conferring sensitivity to NK cell killing when mutated. For another example, if only mutation A is identified from the post-treatment T cell population (e.g., dead) , the absence of identifying mutations B and C from this post-treatment T cell population (e.g., dead) indicates hit genes B and C are the target genes, e.g., conferring resistance to NK cell killing when mutated.

In some embodiments, the post-treatment T cell population obtained is alive T cells, which are resistant to NK cell killing. In some embodiments, identifying the target gene comprises comparing the sgRNA (or sgRNA ^iBAR or guide sequence thereof, or nucleic acid encoding sgRNA or sgRNA ^iBAR or guide sequence thereof) sequence counts obtained from the post-treatment T cell population with sgRNA (or sgRNA ^iBAR or guide sequence thereof, or nucleic acid encoding sgRNA or sgRNA ^iBAR or guide sequence thereof) sequence counts obtained from the control T cell population, wherein: i) the hit genes whose corresponding sgRNA (or sgRNA ^iBAR) guide sequences are identified as enriched in the post-treatment T cell population (e.g., alive, resistant to NK cell killing) compared to the control T cell population in at least one treatment with NK cells with an FDR ≤ 0.05 (e.g., FDR ≤ any of 0.04, 0.03, 0.02, 0.01, 0.001, or less) , or in at least two separate different treatments with NK cells with FDR ≤ 0.15 (e.g., FDR ≤ any of 0.1, 0.05, 0.01, 0.001, or less) , (and/or with at least about 2-fold enrichment, such as at least about any of 3-, 4-, 5-, 10-, 20-, 50-, 100-fold, or more enrichment) are identified as target genes whose mutations make the T cells resistant to NK cell killing; and/or ii) the hit genes whose corresponding sgRNA (or sgRNA ^iBAR) guide sequences are identified as depleted in the post-treatment T cell population (e.g., alive, resistant to NK cell killing) compared to the control T cell population in at least one treatment with NK cells with an FDR ≤ 0.01 (e.g., FDR ≤ any of 0.009, 0.007, 0.005, 0.001, 0.0005, or less) , or in at least two separate different treatments with NK cells with FDR ≤ 0.05 (e.g., FDR ≤ any of 0.04, 0.03, 0.02, 0.01, 0.001, or less) , (and/or with at least about 2-fold depletion, such as at least about any of 3-, 4-, 5-, 10-, 20-, 50-, 100-fold, or more depletion) are identified as target genes whose mutations make the T cells sensitive to NK cell killing. In some embodiments, the sgRNA (or sgRNA ^iBAR or guide sequence thereof, or nucleic acid encoding sgRNA or sgRNA ^iBAR or guide sequence thereof) sequence counts are subject to median ratio normalization followed by mean-variance modeling. In some embodiments, identifying the target gene comprises comparing the hit gene mutation sequence counts obtained from the post-treatment T cell population with hit gene mutation sequence counts obtained from the control T cell population, wherein: i) the hit genes whose corresponding hit gene mutation sequences are identified as enriched in the post-treatment T cell population (e.g., alive, resistant to NK cell killing) compared to the control T cell population in at least one treatment with NK cells with an FDR ≤ 0.05 (e.g., FDR ≤ any of 0.04, 0.03, 0.02, 0.01, 0.001, or less) , or in at least two separate different treatments with NK cells with FDR ≤ 0.15 (e.g., FDR ≤ any of 0.1, 0.05, 0.01, 0.001, or less) , (and/or with at least about 2-fold enrichment, such as at least about any of 3-, 4-, 5-, 10-, 20-, 50-, 100-fold, or more enrichment) are identified as target genes whose mutations make the T cells resistant to NK cell killing; and/or ii) the hit genes whose corresponding hit gene mutation sequences are identified as depleted in the post-treatment T cell population (e.g., alive, resistant to NK cell killing) compared to the control T cell population in at least one treatment with NK cells with an FDR ≤ 0.01 (e.g., FDR ≤ any of 0.009, 0.007, 0.005, 0.001, 0.0005, or less) , or in at least two separate different treatments with NK cells with FDR ≤ 0.05 (e.g., FDR ≤ any of 0.04, 0.03, 0.02, 0.01, 0.001, or less) , (and/or with at least about 2-fold depletion, such as at least about any of 3-, 4-, 5-, 10-, 20-, 50-, 100-fold, or more depletion) are identified as target genes whose mutations make the T cells sensitive to NK cell killing. In some embodiments, the hit gene mutation sequence counts are subject to median ratio normalization followed by mean-variance modeling.

In some embodiments, the post-treatment T cell population obtained is dead T cells, which are sensitive to NK cell killing. In some embodiments, identifying the target gene comprises comparing the sgRNA (or sgRNA ^iBAR or guide sequence thereof, or nucleic acid encoding sgRNA or sgRNA ^iBAR or guide sequence thereof) sequence counts obtained from the post-treatment T cell population with sgRNA (or sgRNA ^iBAR or guide sequence thereof, or nucleic acid encoding sgRNA or sgRNA ^iBAR or guide sequence thereof) sequence counts obtained from the control T cell population, wherein: i) the hit genes whose corresponding sgRNA (or sgRNA ^iBAR) guide sequences are identified as enriched in the post-treatment T cell population (e.g., dead, sensitive to NK cell killing) compared to the control T cell population in at least one treatment with NK cells with an FDR ≤ 0.01 (e.g., FDR ≤ any of 0.009, 0.007, 0.005, 0.001, 0.0005, or less) , or in at least two separate different treatments with NK cells with FDR ≤ 0.05 (e.g., FDR ≤ any of 0.04, 0.03, 0.02, 0.01, 0.001, or less) , (and/or with at least about 2-fold enrichment, such as at least about any of 3-, 4-, 5-, 10-, 20-, 50-, 100-fold, or more enrichment) are identified as target genes whose mutations make the T cells sensitive to NK cell killing; and/or ii) the hit genes whose corresponding sgRNA (or sgRNA ^iBAR) guide sequences are identified as depleted in the post-treatment T cell population (e.g., dead, sensitive to NK cell killing) compared to the control T cell population in at least one treatment with NK cells with an FDR ≤ 0.05 (e.g., FDR ≤ any of 0.04, 0.03, 0.02, 0.01, 0.001, or less) , or in at least two separate different treatments with NK cells with FDR ≤ 0.15 (e.g., FDR ≤ any of 0.1, 0.05, 0.01, 0.001, or less) , (and/or with at least about 2-fold depletion, such as at least about any of 3-, 4-, 5-, 10-, 20-, 50-, 100-fold, or more depletion) are identified as target genes whose mutations make the T cells resistant to NK cell killing. In some embodiments, the sgRNA (or sgRNA ^iBAR or guide sequence thereof, or nucleic acid encoding sgRNA or sgRNA ^iBAR or guide sequence thereof) sequence counts are subject to median ratio normalization followed by mean-variance modeling. In some embodiments, identifying the target gene comprises comparing the hit gene mutation sequence counts obtained from the post-treatment T cell population with hit gene mutation sequence counts obtained from the control T cell population, wherein: i) the hit genes whose corresponding hit gene mutation sequences are identified as enriched in the post-treatment T cell population (e.g., dead, sensitive to NK cell killing) compared to the control T cell population in at least one treatment with NK cells with an FDR ≤ 0.01 (e.g., FDR ≤ any of 0.009, 0.007, 0.005, 0.001, 0.0005, or less) , or in at least two separate different treatments with NK cells with FDR ≤0.05 (e.g., FDR ≤ any of 0.04, 0.03, 0.02, 0.01, 0.001, or less) , (and/or with at least about 2-fold enrichment, such as at least about any of 3-, 4-, 5-, 10-, 20-, 50-, 100-fold, or more enrichment) are identified as target genes whose mutations make the T cells sensitive to NK cell killing; and/or ii) the hit genes whose corresponding hit gene mutation sequences are identified as depleted in the post-treatment T cell population (e.g., dead, sensitive to NK cell killing) compared to the control T cell population in at least one treatment with NK cells with an FDR ≤ 0.05 (e.g., FDR ≤ any of 0.04, 0.03, 0.02, 0.01, 0.001, or less) , or in at least two separate different treatments with NK cells with FDR ≤ 0.15 (e.g., FDR ≤ any of 0.1, 0.05, 0.01, 0.001, or less) , (and/or with at least about 2-fold depletion, such as at least about any of 3-, 4-, 5-, 10-, 20-, 50-, 100-fold, or more depletion) are identified as target genes whose mutations make the T cells resistant to NK cell killing. In some embodiments, the hit gene mutation sequence counts are subject to median ratio normalization followed by mean-variance modeling.

In some embodiments, the sgRNA library is an sgRNA ^iBAR library. In some embodiments, the variance of each guide sequence is adjusted based on data consistency among the iBAR sequences in the sgRNA ^iBAR sequences corresponding to the guide sequence. In some embodiments, the variance of each guide sequence or sequence comprising the hit gene mutation (e.g., inactivating mutation) is adjusted based on data consistency among the same gene. “Data consistency” as used herein refers to consistency of sequencing results of the same guide sequences (e.g., sequence counts, normalized sequence counts, rankings, or fold changes) corresponding to different iBAR sequences in a screening experiment; or consistency of sequencing results of different hit gene mutations such as inactivating mutations (e.g., at different target sites of the same hit gene) or different sgRNA sequences corresponding to the same gene. A true hit from a screen theoretically should have biologically relevant performance similarities, such as similar normalized sequence counts, rankings, and/or fold changes corresponding to sgRNA ^iBAR constructs having the same guide sequence, but different iBARs; and/or similar normalized sequence counts, rankings, and/or fold changes corresponding to the same gene but different hit gene mutation sequences such as inactivating mutation sequences (e.g., at different target sites of the hit gene) or different sgRNA sequences. Also see WO2020125762 for how mean-variance modeling can be conducted, and how the variance of each guide sequence is adjusted based on data consistency among the iBAR sequences in the sgRNA ^iBAR sequences corresponding to the guide sequence.

In some embodiments, the data consistency among the iBAR sequences in the sgRNA ^iBAR sequences corresponding to each guide sequence is determined based on the direction of the fold change of each iBAR sequence, wherein the variance of the guide sequence is increased if the fold changes of the iBAR sequences are in different directions (e.g., increased vs. reduced, increased vs. unchanged, or reduced vs. unchanged are all considered as different directions) with respect to each other. In some embodiments, the data consistency among the different hit gene mutation (e.g., inactivating mutation) sequences or different sgRNA sequences corresponding to the same gene is determined based on the direction of the fold change of each hit gene mutation (e.g., inactivating mutation) sequence or each sgRNA sequence, wherein the variance of the hit gene mutation (e.g., inactivating mutation) sequence or the guide sequence is increased if the fold changes of the different hit gene mutation (e.g., inactivating mutation) sequences or the different sgRNA sequences are in different directions with respect to each other. Such data inconsistency-resulted variance increase can help rule out rare but dramatically changed hit gene mutation (e.g., inactivating mutation) /sgRNA/sgRNA ^iBAR sequences in positive screens under high MOI. For example, for the iBAR system, due to the high MOI during library construction, there can be “free riders” of false-positive sgRNAs associated with sgRNAs against true-positive hit genes. The “free rider” described herein refers to sgRNAs targeting irrelevant sequences (e.g., irrelevant hit genes) that are mis-associated with sgRNAs targeting true-positive hit genes to enter the same T cells. In some embodiments, the variance of sgRNAs ^iBAR is modified based on the enrichment directions of different iBARs for each guide sequence within a set of sgRNA ^iBAR constructs. If all iBARs of one set of sgRNA ^iBAR constructs (i.e., all iBARs corresponding to the same guide sequence) present the same direction of fold change, i.e., all greater or less than that of the control group, then the variance of the set of sgRNA ^iBAR constructs (or the variance of the guide sequence) would be unchanged. If iBARs of one set of sgRNA ^iBAR constructs (or iBARs corresponding to the same guide sequence) reveal inconsistent directions of fold change relative to control, then the corresponding guide sequence is penalized by increasing its variance. In some embodiments, the final adjusted variance for inconsistent sgRNAs ^iBAR is the model-estimated variance (e.g., by mean-variance modeling) plus the experimental variance calculated from the NK cell treated sample (s) and the control group (s) . In some embodiments, a hit gene comprises two or more (e.g., 2, 3, 4, 5, or more, such as 2) hit gene mutations (e.g., inactivating mutations) , or a hit gene is targeted by two or more (e.g., 2, 3, 4, 5, or more, such as 2) different guide sequences at different target sites (e.g., two or more different sgRNAs, or two or more sets of sgRNA ^iBAR constructs each comprising a guide sequence targeting different target sites) . In some embodiments, the data consistency among the iBAR sequences in the sgRNA ^iBAR sequences corresponding to each guide sequence and to the same hit gene is both determined based on the direction of the fold change of each iBAR sequence, wherein the variance of the guide sequence is increased if the fold changes of the corresponding iBAR sequences are in different directions with respect to each other, and the variance of the guide sequence (or the variance of the hit gene) is further increased if the two or more (e.g., 2, 3, 4, 5, or more, such as 2) different guide sequences targeting the same hit gene have fold changes in different directions with respect to each other. For example, for sgRNA A and sgRNA B targeting different target sites of the same hit gene X, if the guide sequences of both sgRNA A and sgRNA B are enriched or depleted compared to control, the variance of each guide sequence or the hit gene do not change; if the guide sequence of sgRNA A is enriched while the guide sequence of sgRNA B is depleted compared to control, the variance of each guide sequence or the hit gene is increased. In some embodiments, the data consistency among the iBAR sequences in the sgRNA ^iBAR sequences corresponding to the same hit gene is determined based on the direction of the fold change of each iBAR sequence, wherein the variance of each guide sequence targeting the same hit gene is increased if the fold changes of the iBAR sequences corresponding to the same hit gene are in different directions with respect to each other, and the variance of each guide sequence targeting the same hit gene (or the variance of the hit gene) is increased. For example, if 2 sets of sgRNAs ^iBAR (4 sgRNAs ^iBAR in each set) target 2 different target sites of the same hit gene, if all 8 iBAR sequences are identified as enriched compared to control, the variances of both 2 guide sequences remain unchanged; if some iBAR sequences are identified as enriched while others are identified as unchanged or depleted compared to control, the variances of both 2 guide sequences are increased.

In some embodiments, the sequences comprising hit gene mutations (e.g., inactivating mutations) at different target sites of the same hit gene whose fold changes among corresponding target sites are shown in different directions, the sgRNAs or sgRNAs ^iBAR targeting different target sites of the same hit gene whose fold changes among corresponding target sites are shown in different directions, or the sgRNAs whose fold changes among corresponding iBARs are shown in different directions, can be penalized through the increased variance leading to lower scores and rankings for certain hit genes. For example, if 2 sets of sgRNAs ^iBAR (4 sgRNAs ^iBAR in each set) target 2 different target sites of the same hit gene, if all 8 iBAR sequences are identified as enriched compared to control, the hit gene has low variance and hence high ranking and/or score (e.g., high ranking sensitive gene to NK cell killing, with high sensitivity score) ; if some iBAR sequences are identified as enriched while others are identified as unchanged or depleted compared to control, the hit gene has high variance and hence low ranking and/or score (e.g., low ranking resistant gene to NK cell killing, with low resistance score) .

In a set of sgRNA ^iBAR constructs, the ranking for the guide sequence may be adjusted based on the consistency of enrichment directions of a pre-determined threshold number m of different iBAR sequences in the set, wherein m is an integer between 1 and n. For example, if at least m iBAR sequences of the sgRNA ^iBAR set present the same direction of fold change, i.e., all greater or less than that of the subpopulation of control T cells, then the ranking (or variance) of the guide sequence is unchanged. However, if more than n-m different iBAR sequences revealed inconsistent directions of fold change, then the sgRNA ^iBAR set would be penalized by lowering its ranking, e.g., by increasing its variance. In some embodiments, the ranking for the sequences containing the hit gene mutations (e.g., inactivating mutations) or the guide sequences may be adjusted (or further adjusted) based on the consistency of enrichment directions of a pre-determined threshold number m of different hit gene mutations (e.g., inactivating mutations) or different guide sequences corresponding to the same hit gene, wherein m is an integer between 1 and n. For example, ifat least m hit gene mutations (e.g., inactivating mutations) or m guide sequences corresponding to the same hit gene present the same direction of fold change, i.e., all greater or less than that of the subpopulation of control T cells, then the ranking (or variance) is unchanged. However, if more than n-m different hit gene mutations (e.g., inactivating mutations) or more than n-m different guide sequences revealed inconsistent directions of fold change, then the sequences comprising the hit gene mutations (e.g., inactivating mutations) or the guide sequences would be penalized by lowering their ranking, e.g., by increasing their variance.

In some embodiments, the P-value of each sequence comprising a hit gene mutation (e.g., inactivating mutation) , or the P-value of each guide sequence of sgRNA or sgRNA ^iBAR, is calculated using the mean and variance (e.g., experimental variance, model-estimated variance, or modified variance based on data inconsistency) of the treatment group compared to those of the control group.

Robust Rank Aggregation (RRA; Kolde R et al. Bioinformatics. 2012; 28: 573-580) or modified RRA (e.g., α-RRA in MAGeCK; Li W et al. Genome Biol. 2014; 15: 554) is one of available tools for statistics and ranking in the art, which can detect genes that are ranked consistently better than expected under null hypothesis ofuncorrelated inputs and assign a significance score for each gene, and combine ranking lists into a single ranking. It assumes that all informative normalized ranks come from a distribution strongly skewed toward zero, and gets the binomial probability calculated from the supposed uniform distribution of ranks to detect these distributions. And a P-value assigned to each element in the aggregated list is used to rank genes and describe how much better it was ranked than expected, making the randomly ranked genes less significant. The underlying probabilistic model makes the RRA algorithm parameter free and robust to outliers, noise and errors. Significance scores also provide a rigorous way to keep only the statistically relevant genes in the final list. These properties make this approach robust and compelling for many settings. Briefly, in RRA and α-RRA, for each sequence comprising a hit gene mutation (e.g., inactivating mutation) , each sgRNA guide sequence, or each sgRNA ^iBAR guide sequence (hereinafter also referred to as “hit gene mutation (e.g., inactivating mutation) /sgRNA guide/sgRNA ^iBAR guide sequence” ) corresponding to a hit gene (e.g., when there are two sgRNAs targeting the same hit gene) , the algorithm looks at how such sequence is positioned in a normalized ranked list of all hit gene mutation (e.g., inactivating mutation) /sgRNA guide/sgRNA ^iBAR guide sequences obtained from a T cell library (NK cell treated T cell library, or control T cells/control T cell library) and compares this to the baseline case where all hit gene mutation (e.g., inactivating mutation) /sgRNA guide/sgRNA ^iBAR guide sequences are randomly shuffled ( “permuted sequences” ) . As a result, a P-value is assigned for all hit gene mutation (e.g., inactivating mutation) /sgRNA guide/sgRNA ^iBAR guide sequences corresponding to their hit genes, showing how much better it is positioned in the ranked lists than expected by chance. This P-value is used both for re-ranking the hit gene mutation (e.g., inactivating mutation) /sgRNA guide/sgRNA ^iBAR guide sequences corresponding to hit genes and deciding their significance. A skilled person in the art can understand that other tools can also be used for this statistics and ranking. In some embodiments, RRA or α-RRA is employed to calculate the final score of each hit gene in order to obtain the ranking of hit genes based on mean and variance (e.g., modified variance) of every hit gene.

In some embodiments, sequences comprising the hit gene mutations (e.g., inactivating mutations) , sgRNA guide sequences, or sgRNA ^iBAR guide sequences (hit gene mutation (e.g., inactivating mutation) /sgRNA guide/sgRNA ^iBAR guide sequences) were ranked based on P-values calculated using the mean and variance (e.g., modified variance adjusted for data inconsistency) from the negative binomial (NB) distribution model, which is used to estimate probability of every hit gene mutation (e.g., inactivating mutation) /sgRNA guide/sgRNA ^iBAR guide sequence across biological/experimental replicates and treatment vs. control groups, then RRA or α-RRA algorithm is applied to identify positively or negatively selected hit genes corresponding to the top ranking (e.g., top α%such as top 5%) hit gene mutation (e.g., inactivating mutation) /sgRNA guide/sgRNA ^iBAR guide sequence. A lower RRA score corresponded to a stronger enrichment of the hit genes. In some embodiments, the P-values of such top-ranking hit gene mutation (e.g., inactivating mutation) /sgRNA guide/sgRNA ^iBAR guide sequence lower than a threshold (e.g., P-value≤0.25) are selected, and the corresponding hit genes are identified as the target gene. In some embodiments, the FDRs of such top ranking hit gene mutation (e.g., inactivating mutation) /sgRNA guide/sgRNA ^iBAR guide sequence lower than a threshold (e.g., FDR≤0.05) are selected, and the corresponding hit genes are identified as the target gene. In some embodiments, when multiple hit gene mutation (e.g., inactivating mutation) /sgRNA guide/sgRNA ^iBAR guide sequences are designed for the same hit gene, only the top hit gene mutation (e.g., inactivating mutation) /sgRNA guide/sgRNA ^iBAR guide sequences of one gene is considered in the RRA or α-RRA calculation. RRA or α-RRA assumes if a hit gene has no effect on T cell sensitivity/resistance to NK cell treatment, then hit gene mutation (e.g., inactivating mutation) /sgRNA guide/sgRNA ^iBAR guide sequences corresponding to such hit gene should be uniformly distributed across the ranked list of all hit gene mutation (e.g., inactivating mutation) /sgRNA guide/sgRNA ^iBAR guide sequences obtained from the T cell library. In some embodiments, all hit gene mutation (e.g., inactivating mutation) /sgRNA guide/sgRNA ^iBAR guide sequences are ranked and compared by RRA or α-RRA among treatment and control groups according to their relative ranking in each group and the different distributions of the groups. All T cell library covered hit genes are ranked by comparing the skew in beta distribution of the hit gene mutation (e.g., inactivating mutation) /sgRNA guide/sgRNA ^iBAR guide sequences to the uniform null hypothesis model, and hit genes whose corresponding hit gene mutation (e.g., inactivating mutation) /sgRNA guide/sgRNA ^iBAR guide sequence rankings are consistently higher than expected with statistical significance (P-value) by permutation test and/or acceptable FDR by the Benjamini-Hochberg Procedure, are prioritized in RRA or α-RRA (lower RRA score) . Such RRA or α-RRA analysis can significantly reduce or eliminate false positives due to perturbations in experiments or sampling. In some embodiments, hit genes are ranked based on ranking scores of corresponding hit gene mutation (e.g., inactivating mutation) /sgRNA guide/sgRNA ^iBAR guide sequence obtained by median ratio normalization followed by mean-variance modeling. In some embodiments, hit genes are further ranked by RRA or α-RRA taking into consideration of multiple hit gene mutation (e.g., inactivating mutation) /sgRNA guide/sgRNA ^iBAR guide sequences for the same hit gene.

In some embodiments, the predetermined threshold level is an FDR value from a permutation test of all hit gene mutation (e.g., inactivating mutation) /sgRNA guide/sgRNA ^iBAR guide sequences obtained from an experiment (treatment or control) . In some embodiments, the FDR value is determined by considering the maximum potential true target genes in a specific screen (e.g., a specific pathway involved in response to NK cell treatment) . In some embodiments, the threshold is top β %of sequence counts (normalized or not) obtained from a T cell library, and the corresponding hit gene is identified as target gene.

Any target identification methods known in the art can be used herein. For example, the Empirical Bayesian method (identifies target by likelihood) or algorithm based therefrom, such as casTLE (cas9 High Throughput maximum Likelihood Estimator) which uses an Empirical Bayesian framework to account for multiple sources of variability, including reagent efficacy and off-target effects for the analysis of large scale genomic perturbation screens, and provides casTLE scores for ranking and threshold cutoff (Morgens, D. W. et al. (2016) Nat Biotechnol 34, 634-636) . In some embodiments, log2 ratio difference and p-value from t-test can be used to identify target genes. For example, RIGER (Luo, J. et al. (2009) . Cell 137, 835-848) , which ranks shRNAs according to their differential effects between two classes of samples, then identifies the genes targeted by the shRNAs at the top of the list, thereby identifying genes essential to the difference between the classes. LFC and P-value can be used for ranking and threshold cutoff. In some embodiments, probability mass function of binomial distribution (or algorithm based therefrom) can be used for target gene identification. For example, STARS (Doench, J. G., et al. (2016) Nat Biotechnol 34, 184-191) , in which STAR Scores can be used for ranking and threshold cutoff. In some embodiments, Negative Binomial model-based and α-RRA algorithm can be used for target gene identification, such as MAGeCK (Li, W. et al. (2014) Genome Biol 15, 554) , and RRA Scores can be used for ranking and threshold cutoff. In some embodiments, fi-binomial modeling based algorithm can be used for target gene identification, such as CRISPRBetaBinomial (CB2) (Jeong, H.H. et al. (2019) . Genome Res 29, 999-1008) , P-value or FDR can be used for ranking and threshold cutoff. In some embodiments, such as during stringent positive screens, sgRNA or sgRNA ^iBAR raw read count ranking, normalized read count ranking, and/or log2 fold change between treatment group and control group can be used for target gene identification, e.g., hit genes corresponding to top X%of read counts are identified as target genes.

In some embodiments, the target gene identification is a positive screening, i.e., by identifying hit gene mutation (e.g., inactivating mutation) sequences or guide sequences that are enriched in the final T cell subpopulation. In some embodiments, the target gene identification is a negative screening, i.e., by identifying hit gene mutation (e.g., inactivating mutation) sequences or guide sequences that are depleted in the final T cell subpopulation. Hit gene mutation (e.g., inactivating mutation) sequences or guide sequences that are enriched in the final T cell subpopulation rank high based on sequence counts or fold changes, while hit gene mutation (e.g., inactivating mutation) sequences or guide sequences that are depleted in the final T cell subpopulation rank low based on sequence counts or fold changes. In some embodiments, the enrichment or depletion is relative to the total sequence counts obtained from the final T cell subpopulation. In some embodiments, the enrichment or depletion is relative to the corresponding sequence counts in a subpopulation of control T cells or control T cell library, such as a subpopulation of T cells obtained from a same T cell library not treated with NK cells. In some embodiments, the enrichment or depletion is calculated based on RRA or α-RRA algorithm.

In some embodiments, the method comprises subjecting the T cell library to at least two (e.g., at least 3, 4, 5, 6, 7, 7, 8, 10, or more) separate different treatments with NK cells in step b) , and in step c) obtaining the T cells that are sensitive or resistant to the killing of the NK cells from each treatment, for target gene identification. In some embodiments, the method comprises identifying one or more hit genes in the post-treatment T cell population from step c) obtained from each treatment, and i) obtaining one or more hit genes identified from all treatments whose mutation makes the T cell sensitive to NK cell killing, thereby identifying the target gene in the T cell whose mutation makes the T cell sensitive to NK cell killing; or ii) obtaining one or more hit genes identified from all treatments whose mutation makes the T cell resistant to NK cell killing, thereby identifying the target gene in the T cell whose mutation makes the T cell resistant to NK cell killing. In some embodiments, the method comprises identifying one or more hit genes in the post-treatment T cell population from step c) obtained from each treatment, and i) combining the one or more hit genes identified from all treatments whose mutation makes the T cell sensitive to NK cell killing, thereby identifying the target gene in the T cell whose mutation makes the T cell sensitive to NK cell killing; or ii) combining the one or more hit genes identified from all treatments whose mutation makes the T cell resistant to NK cell killing, thereby identifying the target gene in the T cell whose mutation makes the T cell resistant to NK cell killing. In some embodiments, identifying the target gene comprises identifying the hit genes in the T cells obtained from the at least two (e.g., at least 3, 4, 5, 6, 7, 7, 8, 10, or more) separate different treatments with NK cells, wherein: i) the hit genes that are identified as depleted from the final T cell subpopulation resistant to the killing of the NK cells in at least one treatment with NK cells with FDR ≤ 0.01 (e.g., FDR ≤ any of 0.009, 0.007, 0.005, 0.001, 0.0005, or less) , or in at least two separate different treatments with NK cells with FDR ≤ 0.05 (e.g., FDR ≤ any of 0.04, 0.03, 0.02, 0.01, 0.005, 0.001, or less) (and/or with at least about 2-fold depletion, such as at least about any of 3-, 4-, 5-, 10-, 20-, 50-, 100-fold, or more depletion) are identified as target genes whose mutation (e.g., inactivation) make the T cells sensitive to NK cell killing; ii) the hit genes that are identified as enriched from the final T cell subpopulation resistant to the killing of the NK cells in at least one treatment with NK cells with FDR ≤ 0.05 (e.g., FDR ≤ any of 0.04, 0.03, 0.02, 0.01, 0.005, 0.001, or less) , or in at least two separate different treatments with NK cells with FDR≤ 0.15 (e.g., FDR≤ any of 0.14, 0.13, 0.12, 0.11, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, 0.01, or less) , (and/or with at least about 2-fold enrichment, such as at least about any of 3-, 4-, 5-, 10-, 20-, 50-, 100-fold, or more enrichment) are identified as target genes whose mutation (e.g., inactivation) make the T cells resistant to NK cell killing; iii) the hit genes that are identified as depleted from the final T cell subpopulation sensitive to the killing of the NK cells in at least one treatment with NK cells with FDR ≤ 0.05 (e.g., FDR ≤ any of 0.04, 0.03, 0.02, 0.01, 0.005, 0.001, or less) , or in at least two separate different treatments with NK cells with FDR ≤ 0.15 (e.g., FDR ≤ any of 0.14, 0.13, 0.12, 0.11, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, 0.01, or less) , (and/or with at least about 2-fold depletion, such as at least about any of 3-, 4-, 5-, 10-, 20-, 50-, 100-fold, or more depletion) are identified as target genes whose mutation (e.g., inactivation) make the T cells resistant to NK cell killing; and/or; iv) the hit genes that are identified as enriched from the final T cell subpopulation sensitive to the killing of the NK cells in at least one treatment with NK cells with FDR ≤ 0.01 (e.g., FDR ≤ any of 0.009, 0.007, 0.005, 0.001, 0.0005, or less) , or in at least two separate different treatments with NK cells with FDR ≤ 0.05 (e.g., FDR ≤ any of 0.04, 0.03, 0.02, 0.01, 0.005, 0.001, or less) (and/or with at least about 2-fold enrichment, such as at least about any of 3-, 4-, 5-, 10-, 20-, 50-, 100-fold, or more enrichment) are identified as target genes whose mutation (e.g., inactivation) make the T cells sensitive to NK cell killing.

In some embodiments, the methods described herein comprise subjecting the T cell library to at least two of the four separate Trials for target gene identification: (I) Trial I: i) an initial treatment step comprising contacting the T cell library with the NK cells for about 72 hours at a ratio of NK cells to T cells of about 0.5∶1; ii) an enrichment step comprising sorting the mixture of treated cells that are T cells (e.g., B2M-negative or deficient, or CD3+) and alive, thus obtaining a first T cell subpopulation that is resistant to the killing of the NK cells; iii) a recovery step comprising culturing the first T cell subpopulation for about 48 hours; iv) a second treatment step comprising contacting the first T cell subpopulation post-recovery with the NK cells for about 96 hours at a ratio of NK cells to T cells of about 0.3∶1; and v) a sorting step comprising sorting the final mixture of treated cells that are T cells (e.g., B2M-negative or deficient, or CD3+) and alive, thus obtaining a second T cell subpopulation that is resistant to the killing of the NK cells; (II) Trial II: i) a treatment step comprising contacting the T cell library with the NK cells for about 10 days at a ratio of NK cells to T cells of about 0.5∶1; and ii) a sorting step comprising sorting the mixture of treated cells that are T cells (e.g., B2M-negative or deficient, or CD3+) and alive, thus obtaining a T cell subpopulation that is resistant to the killing of the NK cells; (III) Trial III: i) a treatment step comprising contacting the T cell library with the NK cells for about 48 hours at a ratio of NK cells to T cells of about 1∶1; ii) a sorting step comprising sorting the mixture of treated cells that are T cells (e.g., B2M-negative or deficient, or CD3+) and alive, thus obtaining a T cell subpopulation that is resistant to the killing of the NK cells; and iii) a recovery step comprising culturing the T cell subpopulation for about 48 hours before harvesting the cells; and (IV) Trial IV: i) a treatment step comprising contacting the T cell library with the NK cells for about 48 hours at a ratio of NK cells to T cells of about 1∶1; ii) an enrichment step comprising sorting the mixture of treated cells that are T cells (e.g., B2M-negative or deficient, or CD3+) and alive, thus obtaining a first T cell subpopulation that is resistant to the killing of the NK cells; iii) a recovery step comprising culturing the first T cell subpopulation for about 48 hours; and iv) a sorting step comprising sorting the first T cell subpopulation post-recovery that are T cells (e.g., B2M-negative or deficient, or CD3+) and alive, thus obtaining a second T cell subpopulation that is resistant to the killing of the NK cells. In some embodiments, identifying the target gene comprises identifying the hit genes from the at least two of the four separate Trials, wherein: i) the hit genes that are identified as depleted from the final T cell subpopulation in at least one Trial with FDR ≤ 0.01, or in at least two Trials with FDR ≤ 0.05 (e.g., FDR ≤ any of 0.04, 0.03, 0.02, 0.01, 0.005, 0.001, or less) (and/or with at least about 2-fold depletion, such as at least about any of 3-, 4-, 5-, 10-, 20-, 50-, 100-fold, or more depletion) are identified as target genes whose mutation (e.g., inactivation) makes the T cells sensitive to NK cell killing; and/or; ii) the hit genes that are identified as enriched from the final T cell subpopulation in at least one Trial with FDR ≤ 0.05, or in at least two Trials with FDR ≤0.15 (e.g., FDR ≤ any of 0.14, 0.13, 0.12, 0.11, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, 0.01, or less) , (and/or with at least about 2-fold enrichment, such as at least about any of 3-, 4-, 5-, 10-, 20-, 50-, 100-fold, or more enrichment) are identified as target genes whose mutation (e.g., inactivation) makes the T cells resistant to NK cell killing.

In some embodiments, the method further comprises ranking the identified target genes, wherein the target gene ranking is based on the degree of enrichment or depletion (e.g., fold of enrichment, fold of depletion, enrichment FDR, or depletion FDR) of the sgRNA or sgRNA ^iBAR guide sequences or hit gene mutations in the post-treatment T cell population (T cells obtained from step c) ) compared to the control T cell population. In some embodiments, the target gene ranking is further adjusted based on data consistency among all sequences comprising the hit gene mutation (e.g., inactivating mutation) corresponding to the same target gene. In some embodiments, the sgRNA library is an sgRNA ^iBAR library, and the target gene ranking is further adjusting based on data consistency among the iBAR sequences in the sgRNA ^iBAR sequences corresponding to the guide sequence of the target gene, and/or based on data consistency among all guide sequences corresponding to (e.g., same or different target sites) of the same target gene. In some embodiments, RRA or α-RRA algorithm is used for ranking the identified target genes. In some embodiments, the ranking of the identified target genes is i) based on data consistency among all sequences comprising the hit gene mutation (e.g., inactivating mutation) corresponding to the same target gene; or ii) based on data consistency among the iBAR sequences in the sgRNA ^iBAR sequences corresponding to the guide sequence of the target gene; and/or iii) based on data consistency among all guide sequences of sgRNAs or sgRNAs ^iBAR corresponding to (e.g., same or different target sites) of the same target gene; wherein the identified target genes are ranked from high to low based on the degree of data consistency from high to low. In some embodiments, the post-treatment T cell population (T cells obtained from step c) ) is an alive population, i.e., resistant to NK cell killing. In some embodiments, the method further comprises assigning a sensitivity score or a resistance score to the identified target gene, wherein target genes whose mutations make the T cells resistant to NK cell killing are ranked from high to low based on the fold of enrichment (or based on enrichment FDR -the smaller the FDR, the higher the ranking; or based on the degree of data consistency -the higher the degree of data consistency, the higher the ranking) of the sgRNA or sgRNA ^iBAR guide sequences or hit gene mutations in the post-treatment T cell population (e.g., alive, resistant to NK cell killing) compared to the control T cell population, and each target gene is assigned a resistance score from high to low accordingly; and/or wherein target genes whose mutations make the T cells sensitive to NK cell killing are ranked from high to low based on the fold of depletion (or based on depletion FDR -the smaller the FDR, the higher the ranking; or based on the degree of data consistency -the higher the degree of data consistency, the higher the ranking) of the sgRNA or sgRNA ^iBAR guide sequences or hit gene mutations in the post-treatment T cell population (e.g., alive, resistant to NK cell killing) compared to the control T cell population, and each target gene is assigned a sensitivity score from high to low accordingly. In some embodiments, the post-treatment T cell population (T cells obtained from step c) ) is a dead population, i.e., sensitive to NK cell killing. In some embodiments, the method further comprises assigning a sensitivity score or a resistance score to the identified target gene, wherein target genes whose mutations make the T cells sensitive to NK cell killing are ranked from high to low based on the fold of enrichment (or based on enrichment FDR -the smaller the FDR, the higher the ranking; or based on the degree of data consistency -the higher the degree of data consistency, the higher the ranking) of the sgRNA or sgRNA ^iBAR guide sequences or hit gene mutations in the post-treatment T cell population (e.g., dead, sensitive to NK cell killing) compared to the control T cell population, and each target gene is assigned a sensitivity score from high to low accordingly; and/or wherein target genes whose mutations make the T cells resistant to NK cell killing are ranked from high to low based on the fold of depletion (or based on depletion FDR -the smaller the FDR, the higher the ranking; or based on the degree of data consistency -the higher the degree of data consistency, the higher the ranking) of the sgRNA or sgRNA ^iBAR guide sequences or hit gene mutations in the post-treatment T cell population (e.g., dead, sensitive to NK cell killing) compared to the control T cell population, and each target gene is assigned a resistance score from high to low accordingly.

In some embodiments, the method further comprising validating the identified target gene by: a) modifying a T cell by creating a mutation (e.g., inactivating mutation) in the target gene in the T cell; b) determining the sensitivity or resistance of the modified T cell to the killing of NK cells. In some embodiments, the method comprises subjecting the modified T cell to any of the NK cell treatment steps b) and optionally any of the T cell obtaining step c) described herein. Any cell viability assays known in the art and described herein can be used to determine the sensitivity or resistance of the modified T cell to the killing of NK cells. When the modified T cells are a homogenous population (i.e., comprising the same mutation (s) such as inactivating mutation (s) ) , more cell viability assays can be used, such as metabolic activity-based assays, e.g., resazurin (oxidation-reduction (redox) indicator) , tetrazolium salts MTT and XTT, Dihydrorhodamines, -calceins, or -fluoresceins, luminescent ATP assays. In some embodiments, the validating method further comprises creating a mutation (e.g., inactivating mutation) in B2M in the T cell, or in the target gene-modified T cell. Creating the mutation (e.g., inactivating mutation) in the target gene and B2M can be carried out simultaneously or sequentially, with the same or different mutation generating method (e.g., both using CRISPR/Cas-mediated gene editing) . The mutation (e.g., inactivating mutation) in the target gene and/or B2M can be generated by any methods known in the art and described herein, such as by mutagenic agent, or TALEN-, ZFN-, or CRISPR/Cas-mediated gene editing (e.g., using Cas, sgRNA against the target gene, and/or B2M sgRNA) . In some embodiments, the T cell before creating a mutation (e.g., inactivating mutation) in the target gene contains a mutation (e.g., inactivating mutation) in B2M. In some embodiments, the method comprises: a) modifying a T cell by creating a mutation (e.g., inactivating mutation) in the target gene in the T cell; b) an optional enrichment step of target gene-modified T cell; and c) modifying the target gene-modified T cell by creating a mutation (e.g., inactivating mutation) in B2M. In some embodiments, the method comprises: a) modifying a T cell by creating a mutation (e.g., inactivating mutation) in B2M; b) an optional enrichment step of B2M-modified T cell; and c) modifying the B2M-modified T cell by creating a mutation (e.g., inactivating mutation) in the target gene.

III. Methods of generating modified T cells

One aspect of the present invention provides methods of generating modified T cells, such as modified T cells with higher resistance to NK cell killing. In some embodiments, the method of generating a modified T cell comprises inactivating one or more target genes identified by any of the screening methods described herein ina host T cell (e.g., allogeneic T cell, precursor T cell, PBMC-derived T cell, or CAR-T cell (such as allogeneic CAR-T cell) ) . In some embodiments, the host T cell further comprises a mutation (e.g., inactivating mutation) in B2M. In some embodiments, the method further comprises generating one or more mutations (e.g., inactivating mutations) in B2M in the host T cell or the modified T cell. In some embodiments, the host T cell expresses a CAR. In some embodiments, the method further comprises introducing into the host T cell or the modified T cell a nucleic acid or vector encoding a CAR. Further provided are modified T cells generated by any of the methods described herein.

In some embodiments, the method of generating a modified T cell comprises creating one or more mutations (e.g., inactivating mutations) at one or more target genes identified by any of the screening methods described herein. In some embodiments, the method comprises contacting a host T cell (e.g., allogeneic T cells, precursor T cells, PBMC-derived T cells, or CAR-T cells (such as allogeneic CAR-T cells) ) with a mutagenic agent, and selecting modified T cells comprising one or more mutations (e.g., inactivating mutations) at one or more target genes identified herein. Methods of detecting such mutations are well known in the art, such as by PCR. In some embodiments, the method comprises creating one or more mutations (e.g., inactivating mutations) at one or more target genes identified herein in a host T cell (e.g., allogeneic T cells, precursor T cells, PBMC-derived T cells, or CAR-T cells (such as allogeneic CAR-T cells) ) by gene editing, such as any gene editing methods known in the art or described herein. For example, non-homologous end joining (NHEJ) -or homologous recombination-mediated gene disruption, or ZFN-, TALEN-, or CRISPR/Cas-mediated gene disruption. In some embodiments, the method of generating a modified T cell comprises introducing an sgRNA construct into a host T cell (e.g., allogeneic T cells, precursor T cells, PBMC-derived T cells, or CAR-T cells (such as allogeneic CAR-T cells) ) , wherein the sgRNA construct comprises or encodes an sgRNA (e.g., an sgRNA, or a vector (e.g., viral vector such as lentiviral vector) carrying a nucleic acid encoding the sgRNA) , wherein the sgRNA comprises a guide sequence that is complementary (e.g., at least about any of 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%complementary) to a target site in a target gene identified herein. In some embodiments, there is provided a method of generating a modified T cell, comprising introducing an sgRNA library into a host T cell (e.g., allogeneic T cells, precursor T cells, PBMC-derived T cells, or CAR-T cells (such as allogeneic CAR-T cells) ) , wherein the sgRNA library comprises one or more sgRNA constructs, wherein each sgRNA construct comprises or encodes an sgRNA, and wherein each sgRNA comprises a guide sequence that is complementary (e.g., at least about any of 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%complementary) to a target site in a target gene selected from the group consisting of TACC2, HES1, STON1, GJD2, LILRB4, PLS1, KLHL24, FANCB, ARNTL, AMY2A, SIX1, USP17L13, PIK3R6, ATP6V1H, TPM3, OR5H14, TFG, SMAD6, PDE4C, SRRM3, LRRC69, KCNJ13, C8orf34, and PACS2. In some embodiments, the method further comprises introducing a vector (e.g., viral vector such as lentiviral vector) carrying a nucleic acid encoding a Cas protein (e.g., Cas9) , or a Cas (e.g., Cas9) mRNA, into the host T cell or the host T cell comprising said sgRNA construct. In some embodiments, the method further comprises introducing a B2M sgRNA construct into the host T cell or the host T cell comprising the sgRNA construct against the target gene, wherein the B2M sgRNA construct comprises or encodes a B2M sgRNA (e.g., a B2M sgRNA, or a vector (e.g., viral vector such as lentiviral vector) carrying a nucleic acid encoding the B2M sgRNA) , wherein the B2M sgRNA comprises a guide sequence that is complementary (e.g., at least about any of 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%complementary) to a target site in B2M. In some embodiments, the host T cell comprises a B2M mutation (e.g., inactivating B2M mutation) . In some embodiments, the sgRNA construct against the target gene, the B2M sgRNA construct, and/or the Cas component comprising a Cas protein or a nucleic acid encoding the Cas protein (e.g., vector, or mRNA) , (and/or nucleic acid (s) encoding a chimeric receptor such as CAR or engineered TCR, e.g., for generating CAR-T or TCR-T cells) are introduced into the host T cell simultaneously. In some embodiments, the nucleic acid encoding the target gene sgRNA, the nucleic acid encoding the B2M sgRNA, and/or the nucleic acid encoding the Cas protein, (and/or nucleic acid (s) encoding a chimeric receptor such as CAR or engineered TCR, e.g., for generating CAR-T or TCR-T cells) are on the same vector, either under the same promoter control, or under separate promoter controls. In some embodiments, the nucleic acid encoding the target gene sgRNA, the nucleic acid encoding the B2M sgRNA, and/or the nucleic acid encoding the Cas protein (and/or nucleic acid (s) encoding a chimeric receptor such as CAR or engineered TCR, e.g., for generating CAR-T or TCR-T cells) are connected by one or more IRES linking sequences and under the same promoter control. In some embodiments, the nucleic acid encoding the target gene sgRNA, the nucleic acid encoding the B2M sgRNA, and/or the nucleic acid encoding the Cas protein, (and/or nucleic acid (s) encoding a chimeric receptor such as CAR or engineered TCR, e.g., for generating CAR-T or TCR-T cells) are on different vectors. In some embodiments, the host T cell comprises a B2M mutation (e.g., inactivating B2M mutation) . In some embodiments, the sgRNA construct against the target gene, the B2M sgRNA construct, and/or the Cas component comprising a Cas protein or a nucleic acid encoding the Cas protein (e.g., vector, or mRNA) , (and/or nucleic acid (s) encoding a chimeric receptor such as CAR or engineered TCR, e.g., for generating CAR-T or TCR-T cells) are introduced into the host T cell sequentially.

In some embodiments, when a population of host T cells (or initial population of T cells) are used for the production of modified T cells described herein, the methods also include one or more isolation and/or enrichment steps, for example, isolating and/or enriching T cells that comprise one or more mutations (e.g., inactivating mutations) in the target gene and/or B2M, the target gene sgRNA construct, the B2M sgRNA construct, or the Cas component, from the population of T cells contacted with any of the modifying agents described herein. In some embodiments, the method further comprises isolating and/or enriching T cells that express a chimeric receptor, such as CAR or engineered TCR. Such isolation and/or enrichment steps can be performed using any known techniques in the art and described herein, such as FACS or magnetic-activated cell sorting (MACS) . Also see methods described in “Isolation and enrichment of modified T cells, ” “first enrichment step, ” and “harvest sorting step” subsections above.

In some embodiments, the host T cells are derived from the blood, bone marrow, lymph, or lymphoid organs. In some aspects, the host T cells are human T cells. In some embodiments, the host T cells are derived from T cell lines. The host T cells in some embodiments are obtained from a xenogeneic source, for example, from mouse, rat, non-human primate, or pig. In some embodiments, the host T cell is an engineered T cell, such as an engineered T cell comprising a mutation (e.g., B2M mutation) , a CAR-T cell (such as allogeneic CAR-T cells) , a T cell with endogenous TCR knock-out, or a T cell expressing an exogenous Nef protein.

In some embodiments, the target gene sgRNA construct, the B2M sgRNA construct, and/or the Cas component, are introduced into the host T cells by transducing/transfecting the nucleic acid (DNA or RNA) or vector encoding thereof (e.g., non-viral vector, or viral vector such as lentiviral vector) , or a virus (e.g., lentivirus) comprising a nucleic acid encoding thereof. In some embodiments, the Cas component (e.g., Cas9 protein) is introduced into the host T cells by inserting proteins into the cell membrane while passing cells through a microfluidic system, such as CELL

(see, for example, U.S. Patent Application Publication No. 20140287509) .

Methods of introducing vectors (e.g., viral vectors) or isolated nucleic acids into a mammalian cell are known in the art. The nucleic acids or vectors described herein can be transferred into a T cell by physical, chemical, or biological methods.

Physical methods for introducing a vector (e.g., viral vector) into a T cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well-known in the art. See, for example, Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York. In some embodiments, the vector (e.g., viral vector) is introduced into the T cell by electroporation.

Biological methods for introducing a vector into a T cell include the use of DNA and RNA vectors. Viral vectors have become the most widely used method for inserting genes into mammalian, e.g., human cells.

Chemical means for introducing a vector (e.g., viral vector) into a T cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro is a liposome (e.g., an artificial membrane vesicle) .

In some embodiments, RNA molecules (e.g., sgRNA, or mRNA encoding Cas9) may be prepared by a conventional method (e.g., in vitro transcription) and then introduced into the T cell via known methods such as mRNA electroporation. See, e.g., Rabinovich et al., Human Gene Therapy 17: 1027-1035.

In some embodiments, the viral vectors (lentiviral vector) or viruses (e.g., lentiviruses) comprising the nucleic acid encoding any of the target gene sgRNAs, garget gene sgRNAs ^iBAR, B2M sgRNA, and/or Cas protein described herein are contacted with the host T cell (or initial T cell population) , e.g., at an MOI of at least about 1, such as at least about any of 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 8, 9, or 10. In some embodiments, the viral vectors (lentiviral vector) or viruses (e.g., lentiviruses) comprising the nucleic acid encoding any of the target gene sgRNAs, target gene sgRNAs ^iBAR, B2M sgRNA, and/or Cas protein described herein are contacted with the host T cell (or initial T cell population) at an MOI of about 3.

In some embodiments, the transduced/transfected T cell is propagated ex vivo after introduction of the vector or isolated nucleic acid. In some embodiments, the transduced/transfected T cell is cultured to propagate for at least about any of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, or 14 days. In some embodiments, the transduced/transfected T cell is further evaluated or screened to select desired modified T cells described herein.

Reporter genes may be used for identifying potentially transfected/transduced cells and for evaluating the functionality of regulatory sequences. In general, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA/RNA has been introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein (GFP) gene (e.g., Ui-Tei et al. FEBS Letters 479: 79-82 (2000) ) . Suitable expression systems are well known and may be prepared using known techniques or obtained commercially. Antibiotic selection markers can also be used to identifying potentially transfected/transduced cells.

Other methods to confirm the presence of any of the nucleic acids described herein (e.g., sgRNA construct) or the presence of a mutation (e.g., inactivating mutation) in a target gene in a modified T cell, include, for example, molecular biological assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR, PCR, DNA-seq, or RNA-seq; biochemical assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological methods (such as ELISAs and Western blots) , Fluorescence-activated cell sorting (FACS) , or Magnetic-activated cell sorting (MACS) .

In some embodiments, the method further comprises formulating the modified T cells (e.g., modified T cells that are more resistant to NK cell killing) with at least one pharmaceutically acceptable carrier. In some embodiments, the method further comprises administering to an individual (e.g., human) an effective amount of the modified T cells (e.g., modified CAR-T cells (such as allogeneic CAR-T cells) that are more resistant to NK cell killing) , or an effective amount of the pharmaceutical formulation thereof. In some embodiments, the individual has cancer. In some embodiments, the individual is histoincompatible with the donor of the host T cell from which the modified T cell is derived.

IV. Pharmaceutical compositions comprising modified T cells

Further provided by the present application are pharmaceutical compositions comprising any one of the modified T cells comprising one or more mutations (e.g., inactivating mutations) in one or more target genes identified herein (e.g., modified T cells that are more resistant to NK cell killing) , and optionally a pharmaceutically acceptable carrier. Also provided are methods of using the modified T cells (e.g., modified CAR-T cells, or modified allogeneic CAR-T cells) or pharmaceutical compositions thereof described herein (e.g., which are resistant to NK cell killing) in the treatment of a disease (e.g., cancer, immune disease such as infection, etc. ) in an individual (e.g., human) , comprising administering an effective amount of the modified T cells or pharmaceutical compositions thereof to the individual. In some embodiments, the modified T cell (e.g., resistant to NK cell killing) is a CAR-T cell. In some embodiments, the CAR specifically recognizes an antigen, such as cancer/tumor antigen, an antigen of an infectious agent (e.g., virus, bacteria, fungus, parasitic worm, etc. ) .

In some embodiments, there is provided a modified T cell (e.g., allogeneic T cell or CAR-T (such as allogeneic CAR-T cells) ) comprising one or more mutations (e.g., inactivating mutations such as knock-out) in one or more target genes, wherein the target gene is selected from the group consisting of TACC2, HES1, STON1, GJD2, LILRB4, PLS1, KLHL24, FANCB, ARNTL, AMY2A, SIX1, USP17L13, PIK3R6, ATP6V1H, TPM3, OR5H14, TFG, SMAD6, PDE4C, SRRM3, LRRC69, KCNJ13, C8orf34, and PACS2. In some embodiments, there is provided a modified T cell (e.g., allogeneic T cell or CAR-T (such as allogeneic CAR-T cells) ) comprising one or more mutations (e.g., inactivating mutations such as knock-out) in PSCS2. In some embodiments, there is provided a pharmaceutical composition comprising: i) one or more modified T cells (e.g., allogeneic T cell or CAR-T (such as allogeneic CAR-T cells) ) comprising one or more mutations (e.g., inactivating mutations such as knock-out) in one or more target genes, wherein the target gene is selected from the group consisting of TACC2, HES1, STON1, GJD2, LILRB4, PLS1, KLHL24, FANCB, ARNTL, AMY2A, SIX1, USP17L13, PIK3R6, ATP6V1H, TPM3, OR5H14, TFG, SMAD6, PDE4C, SRRM3, LRRC69, KCNJ13, C8orf34, and PACS2; and ii) an optional pharmaceutically acceptable carrier. In some embodiments, there is provided a pharmaceutical composition comprising: i) one or more modified T cells (e.g., allogeneic T cell or CAR-T (such as allogeneic CAR-T cells) ) comprising one or more mutations (e.g., inactivating mutations such as knock-out) in PSCS2; and ii) an optional pharmaceutically acceptable carrier. In some embodiments, the modified T cell comprises mutations (e.g., inactivating mutations such as knock-out) in all target genes. In some embodiments, the modified T cell further comprises a mutation (e.g., inactivating mutation) in B2M. In some embodiments, the modified T cell has higher resistance to NK cell killing in a histoincompatible individual as compared to a primary T cell isolated from the donor of the host T cell from which the modified T cell is derived. In some embodiments, the modified T cell is at least about 1.2-fold more resistant (e.g., at least about any of 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 500-fold, 1000-fold, or more resistant) to NK cell killing in a histoincompatible individual as compared to a primary T cell isolated from the donor of the host T cell from which the modified T cell is derived. In some embodiments, the amount of modified T cells killed by NK cells in a histoincompatible individual is at least about 10%less (such as at least about any of 15%, 20%, 30%, 40%, 50%, 60%, 80%, 80%, 90%, or 95%less) than that of primary T cells isolated from the donor of the host T cells from which the modified T cell is derived. In some embodiments, at most about 70% (such as at most about any of 60%, 50%, 40%, 30%, 20%, 10%, or 5%) of the modified T cells are killed by NK cells in a histoincompatible individual.

Pharmaceutical compositions can be prepared by mixing a population of modified T cells described herein with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington′s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980) ) , in the form of aqueous solutions. In some embodiments, the population of modified T cells are homogenous (i.e., comprising the same mutations such as inactivating mutation (s) ) . In some embodiments, the population of modified T cells are heterogeneous (i.e., comprising at least one different mutation such as inactivating mutation) . In some embodiments, at least about 70% (such as at least about any of 75%, 80%, 85%, 90%, or 95%) of the population of modified T cells comprise the same mutation (s) , such as inactivating mutation (s) .

Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers, antioxidants including ascorbic acid, methionine, Vitamin E, sodium metabisulfite; preservatives, isotonicifiers, stabilizers, metal complexes (e.g. Zn-protein complexes) ; chelating agents such as EDTA and/or non-ionic surfactants.

Buffers are used to control the pH in a range which optimizes the therapeutic effectiveness, especially if stability is pH dependent. Suitable buffering agents for use with the present invention include both organic and inorganic acids and salts thereof. For example, citrate, phosphate, succinate, tartrate, fumarate, gluconate, oxalate, lactate, acetate. Additionally, buffers may comprise histidine and trimethylamine salts such as Tris.

Preservatives are added to retard microbial growth, and are typically present in a range from 0.2%-1.0% (w/v) . Suitable preservatives for use with the present invention include octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium halides (e.g., chloride, bromide, iodide) , benzethonium chloride; thimerosal, phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol, 3-pentanol, and m-cresol.

Additional excipients can include agents that prevent adherence to the container wall.

Non-ionic surfactants or detergents (also known as “wetting agents” ) can be present too. Suitable non-ionic surfactants include polysorbates (20, 40, 60, 65, 80, etc. ) , polyoxamers (184, 188, etc. ) ,

polyols,

polyoxyethylene sorbitan monoethers (

etc. ) , lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, sucrose fatty acid ester, methyl cellulose and carboxymethyl cellulose. Anionic detergents that can be used include sodium lauryl sulfate, dioctyle sodium sulfosuccinate and dioctyl sodium sulfonate. Cationic detergents include benzalkonium chloride or benzethonium chloride.

In order for the pharmaceutical compositions to be used for in vivo administration, they must be sterile. The pharmaceutical composition may be rendered sterile by filtration through sterile filtration membranes. The pharmaceutical compositions herein generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

The route of administration is in accordance with known and accepted methods, such as by single or multiple bolus or infusion over a long period of time in a suitable manner, e.g., injection or infusion by subcutaneous, intravenous, intratumoral, intraperitoneal, intramuscular, intraarterial, intralesional or intraarticular routes, or by sustained release or extended-release means. Suitable examples of sustained-release preparations include semi-permeable matrices of solid hydrophobic polymers containing the antagonist, which matrices are in the form of shaped articles, e.g. films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly (2-hydroxyethyl-methacrylate) , or poly (vinylalcohol) ) , polylactides (U.S. Pat. No. 3,773,919) , copolymers of L-glutamic acid and. ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT ^TM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate) , and poly-D- (-) -3-hydroxybutyric acid.

The active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly- (methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington′s Pharmaceutical Sciences 18th edition.

The pharmaceutical compositions described herein may also contain more than one active compound or agent as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Alternatively, or in addition, the composition may comprise a cytotoxic agent, chemotherapeutic agent, cytokine, immunosuppressive agent, or growth inhibitory agent. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.

V. Kits and articles of manufacture

The present application further provides kits and articles of manufacture for use in any embodiment of the methods of identifying a target gene in a T cell described herein, such as using the sgRNA libraries or sgRNA ^iBAR libraries described herein. Also provided are kits and articles of manufacture for generating modified T cells with higher resistance to NK cell killing.

In some embodiments, there is provided a kit for identifying a target gene in a T cell that modulates the activity of the T cell (e.g., sensitivity or resistance to NK cell treatment) , comprising any of the sgRNA libraries or sgRNA ^iBAR libraries described herein. In some embodiments, the kit further comprises a Cas protein or a nucleic acid encoding the Cas protein. In some embodiments, the kit further comprises an sgRNA construct that comprises or encodes an sgRNA whose guide sequence is complementary (e.g., at least about any of 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%complementary) to a target site in B2M (e.g., a viral vector that encodes B2M sgRNA) . In some embodiments, the kit further comprises one or more positive and/or negative control sets of sgRNA ^iBAR constructs, or one or more positive and/or negative control of sgRNA constructs. In some embodiments, the kit further comprises NK cells, and/or initial population of T cells, such as allogeneic T cells, PBMC-derived T cells, precursor T cells, CAR-T cells (such as allogeneic CAR-T cells) , or T cells comprising a B2M mutation (e.g., inactivating B2M mutation) . In some embodiments, the kit further comprises data analysis software. In some embodiments, the kit comprises instructions for carrying out any one of the methods described herein.

In some embodiments, there is provided a kit for identifying a target gene in a T cell that modulates the activity of the T cell (e.g., sensitivity or resistance to NK cell treatment) , comprising any of the T cell libraries described herein, such as T cell libraries comprising mutations (e.g., inactivating mutations) in some or all hit genes in the genome, or T cell libraries comprising any of the sgRNA libraries or sgRNA ^iBAR libraries described herein. In some embodiments, the kit further comprises a Cas protein or a nucleic acid encoding the Cas protein. In some embodiments, the T cell library further comprises a mutation (e.g., inactivating mutation) in B2M. In some embodiments, the T cell library further comprises an sgRNA construct that comprises or encodes an sgRNA whose guide sequence is complementary (e.g., at least about any of 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%complementary) to a target site in B2M. In some embodiments, the kit further comprises NK cells. In some embodiments, the kit further comprises control T cell library, such as having one or more mutations (e.g., inactivating mutations) at non-gene region in the genome, or comprising one or more positive and/or negative control of sgRNA constructs or one or more positive and/or negative control sets of sgRNA ^iBAR constructs. In some embodiments, the kit further comprises data analysis software. In some embodiments, the kit comprises instructions for carrying out any one of the methods described herein.

In some embodiments, there is provided a kit for generating modified T cells with higher resistance to NK cell killing, comprising an sgRNA library (or an sgRNA ^iBAR library) comprising one or more sgRNA constructs, wherein each sgRNA construct comprises or encodes an sgRNA, and wherein each sgRNA comprises a guide sequence that is complementary (e.g., at least about any of 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%complementary) to a target site in a target gene selected from the group consisting of TACC2, HES1, STON1, GJD2, LILRB4, PLS1, KLHL24, FANCB, ARNTL, AMY2A, SIX1, USP17L13, PIK3R6, ATP6V1H, TPM3, OR5H14, TFG, SMAD6, PDE4C, SRRM3, LRRC69, KCNJ13, C8orf34, and PACS2. In some embodiments, the kit further comprises an sgRNA construct comprising or encoding an sgRNA whose guide sequence is complementary (e.g., at least about any of 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%complementary) to a target site in B2M. In some embodiments, the kit further comprises a Cas protein or a nucleic acid encoding the Cas protein. In some embodiments, the kit further comprises an initial population of T cells, such as allogeneic T cells, PBMC-derived T cells, precursor T cells, CAR-T cells (such as allogeneic CAR-T cells) , or T cells comprising a B2M mutation (e.g., inactivating B2M mutation) . In some embodiments, the kit comprises instructions for carrying out the modified T cell generating methods.

In some embodiments, there is provided a kit comprising a modified T cell or pharmaceutical composition thereof, wherein the modified T cell comprises one or more mutations (e.g., inactivating mutations such as knock-out) in one or more target genes selected from the group consisting of TACC2, HES1, STON1, GJD2, LILRB4, PLS1, KLHL24, FANCB, ARNTL, AMY2A, SIX1, USP17L13, PIK3R6, ATP6V1H, TPM3, OR5H14, TFG, SMAD6, PDE4C, SRRM3, LRRC69, KCNJ13, C8orf34, and PACS2. In some embodiments, there is provided a kit comprising a modified T cell or pharmaceutical composition thereof, wherein the modified T cell comprises one or more mutations (e.g., inactivating mutations such as knock-out) in PSCS2. In some embodiments, the modified T cell further comprises a mutation (e.g., inactivating mutation) in B2M. In some embodiments, the modified T cells have higher resistance to NK cell killing. In some embodiments, the kit further comprises instructions for use. In some embodiments, the kit comprises a homogeneous population of modified T cells. In some embodiments, the kit comprises a heterogeneous population of modified T cells. In some embodiments, the modified T cell further comprises a CAR.

The kit may contain additional components, such as containers, reagents, culturing media, primers, buffers, enzymes, and the like to facilitate execution of any one of the screening methods described herein. In some embodiments, the kit comprises reagents, buffers and vectors for introducing the sgRNA library or sgRNA ^iBAR library and the Cas protein or nucleic acid encoding the Cas protein to the T cell. In some embodiments, the kit comprises primers, reagents and enzymes (e.g., polymerase) for preparing a sequencing library of sequences comprising hit gene mutations (e.g., inactivating mutations) , sgRNA sequences, or sgRNA ^iBAR sequences extracted from selected subpopulation of T cells.

The kits of the present application are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags) , and the like. Kits may optionally provide additional components such as buffers and interpretative information. The present application thus also provides articles of manufacture, which include vials (such as sealed vials) , bottles, jars, flexible packaging, and the like.

The article of manufacture can comprise a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The containers may be formed from a variety of materials such as glass or plastic. Generally, the container holds a composition (e.g., modified T cells with higher resistance to NK cell killing) , and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle) . In some embodiments, the label or package insert indicates that the composition is used for treating a particular condition or enhancing an immune response in an individual. The label or package insert will further comprise instructions for administering the composition to the individual. Package insert refers to instructions customarily included in commercial packages of therapeutic products that contain information about the indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic products. Additionally, the article of manufacture may further comprise a second container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI) , phosphate-buffered saline, Ringer′s solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

The kits or article of manufacture may include multiple unit doses of the pharmaceutical composition and instructions for use, packaged in quantities sufficient for storage and use in pharmacies, for example, hospital pharmacies and compounding pharmacies.

EXAMPLES

The examples and exemplary embodiments below are intended to be purely exemplary of the invention and should therefore not be considered to limit the invention in any way. The following examples and detailed description are offered by way of illustration and not by way of limitation.

Example 1. Identification of T-cell modulating genes

This example provides exemplary methods for identifying T-cell modulating genes. Briefly, a T cell library carrying sgRNA ^iBAR targeting each human gene, and an sgRNA targeting B2M, was constructed for Cas9-mediated gene knock-out (KO) . B2M is a component of MHC class I molecules. T cells with B2M KO will be killed by NK cells. By examining NK cell killing efficacy of the B2M ^-sgRNA ^iBAR T cell library constructed, genes conferring resistant phenotype or sensitive phenotype to NK cell killing after KO can be identified. FIG. 1 shows the workflow.

1. T cell isolation and culture

After blood collection, PBMCs were separated from the donor blood samples. T cells were isolated from the PBMCs using the immunomagnetic bead method, then cultured in 37℃, 5%CO ₂ incubator in X-VIVO ^TM 15 media containing 10%FBS, 1%GlutaMAX, and 0.1%recombinant human IL-2 (hereinafter referred to as “T cell complete medium” ) .

2. T cell activation and expansion

800 μL

Human T-Activator CD3/CD28 were separated into four 1.5 mL Eppendorf tubes, 200 μL/tube. 1 mL PBS was added into each tube,

were resuspended well by pipetting, the 1.5 mL tubes were placed onto a magnetic rack to let stand for 1 minute, then supernatant was removed. This washing step was repeated twice. Then 1 mL T cell complete medium was added to each tube to resuspend washed

by gently pipetting.

3.2×10 ⁷ cultured T cells were transferred into a T150 cell culture flask, then 4 mL resuspended

were added into the T cells and gently mixed. The mixture was cultured in 37℃, 5%CO ₂ incubator for T cell activation and expansion. T cell library was constructed after activated T cells were expanded to sufficient amount.

3. Construction of human genome-scale CRISPR sgRNA ^iBAR library and T cell library

Human genome-scale CRISPR sgRNA ^iBAR library was designed and constructed similarly as described in WO2020125762 and Zhu et al. ( “Guide RNAs with embedded barcodes boost CRISPR-pooled screens, ” Genome Biol. 2019; 20: 20) , the contents of each of which are incorporated herein by reference in their entirety. Briefly, 19, 114 annotated protein-coding genes were retrieved from UCSC human genome. sgRNAs targeting each gene were designed using the DeepRank algorithm (see Zhu et al. ) , and four 6-bp iBARs (iBAR ₆s) were randomly assigned to each sgRNA ( “sgRNA ^iBAR” ) . The internal barcode sequence was designed to be placed in the tetra loop of the gRNA scaffold outside of the Cas9-sgRNA ribonucleoprotein complex, which did not affect the activity of its upstream guide sequence. DNA oligonucleotides encoding the sgRNA ^iBAR were designed and array synthesized, then PCR amplified. PCR products were cloned into lentiviral sgRNA ^iBAR-expressing backbone modified in house based on pLenti-sgRNA-Lib (addgene #53121) to obtain sgRNA ^iBAR library plasmids, which encodes 156,848 sgRNAs ^iBAR covering 19,114 human genes (2 sets of sgRNA ^iBAR for each gene targeting 2 different target sites, each set of sgRNA ^iBAR contains 4 sgRNAs ^iBAR) . sgRNA ^iBAR library lentiviruses were then obtained using standard protocol.

sgRNA ^iBAR library lentiviruses were added to activate T cells at an MOI of 3 and gently mixed. The cell mixture was cultured overnight in a 37℃, 5%CO ₂ incubator for infection. The supematant was discarded the next day, equal amount of T cell complete medium supplemented with puromycin was added to the T cells, then cultured overnight in a 37℃, 5%CO ₂ incubator. T cells not successfully infected were then removed, resulting in sgRNA ^iBAR T cell library carrying sgRNAs targeting each of the 19,114 annotated functional genes.

4. Construction of Cas9 ⁺ beta-2-microglobulin KO (B2M ^-) sgRNA ^iBAR T cell library

T cells infected by sgRNA ^iBAR library lentiviruses were transferred to a 50 mL centrifugation tube, placed onto a magnetic rack, let stand for 10 minutes. The supernatant was then transferred to a new 50 mL centrifugation tube, placed onto a magnetic rack, let stand for 5 minutes, to remove as many

as possible. The supernatant containing T cells was transferred to a clean 50 mL centrifugation tube, centrifuged at 400 g 5 minutes, resuspended with 20 mL DPBS, washed twice, then centrifuged at 400 g 5 minutes. The supernatant was discarded, T cells were resuspended with 600 μL

Reduced-Serum Medium and counted cell number (6.60×10 ⁷ T cells) . These T cells were separated into three 1.5 mL Eppendorf tubes and placed on ice. 16 μtg Cas9 mRNA and 16 μg sgRNA specifically targeting B2M (designed and made in house) were added into each tube, gently mixed, then cell mixture was each transferred into a 4 mm BTX electroporation cuvette for electrotransformation. T cells post-electrotransformation were transferred into a T150 cell culture flask, supplemented with T cell complete medium to adjust cell density to 1 × 10 ⁶ cells/mL, then cultured in a 37℃, 5%CO ₂ incubator. Passages of cells were conducted every two days. 96 hours post-electrotransformation, target genes (sgRNA ^iBAR targeted human genes) and B2M were considered to be effectively knocked-out (KO efficiency of B2M was examined to be about 91%) , resulting in Cas9 ⁺ B2M ^-sgRNA ^iBAR T cell library ready for screening.

5. Screening of Cas9 ⁺ B2M ^-sgRNA ^iBAR T cell library treated with NK cells

NK cells were added to the Cas9 ⁺ B2M ^-sgRNA ^iBAR T cell library to examine NK cell killing efficacy. The killing efficacy depends on the ratio of NK cells to B2M ^-T cell library and total incubation time. Hence, four test groups were set up with different treatment intensities and screening schemes (Trials 3-6; see FIG. 2) . A control group was set up without NK cell treatment, Cas9 ⁺ B2M ^-sgRNA ^iBAR T cells were cultured in T cell complete medium and passaged every two days. Two biological replicates were set up for each group. In order to ensure the abundance of sgRNA ^iBAR in the T cell library, 3.56× 10 ⁷ T cells (averagely about 100 fold coverage for each sgRNA ^iBAR, or averagely about 800 fold coverage for each hit gene) were used in each replicate.

In the test groups, after the Cas9 ⁺ B2M ^-sgRNA ^iBAR T cell library and NK cells were incubated for a period of time, all cells were collected and stained with propidium iodide (PI) dye (indicating dead cells) and anti-B2M antibody, then sorted for PI-negative and B2M-negative or deficient cells (i.e., alive Cas9 ⁺ B2M ^-sgRNA ^iBAR T cells) with FACS. For CRISPR screening, multiple rounds of NK cell treatment can help enrich target sgRNA ^iBAR T cells and enhance signal-to-noise ratio. Since activated T cells can only be cultured in vitro for a limited period of time, if sorted alive Cas9 ⁺ B2M ^-sgRNA ^iBAR T cells are in suitable conditions after a first round of NK cell treatment, a second round of NK cell treatment can be conducted, then stained and FACS sorted for PI-negative and B2M-negative or deficient cells again (i.e., enriched alive Cas9 ⁺ B2M ^-sgRNA ^iBAR T cells; see

Trials

3 and 6 in FIG. 2) . Common targets under different screening conditions can be identified.

5.1 NK cell treatment

Suitable amount of NK cells were added to the Cas9 ⁺ B2M ^-sgRNA ^iBAR T cell library according to different screening schemes (FIG. 2) , then co-cultured in a 37℃, 5%CO ₂ incubator.

5.2 FACS sorting of target Cas9 ⁺ B2M ^-sgRNA ^iBAR T cells

Test group cells were collected, centrifuged at 300 g 10 minutes, supernatant was discarded. Cells were resuspended with 500 μL PBS buffer, added with PE anti-human β2-microglobulin antibody (5 μL antibody per 1 × 10 ⁷ cells) , and let stand for 15 minutes in the dark at room temperature. Then 2 mL PBS was added to the cell mixture, centrifuged at 400 g 5 minutes. Supernatant was removed, cells were resuspended with 1.5 mL buffer (PBS + 1%FBS + 10×PS) . 150 μL PI dye was added into the cell suspension and gently mixed, then cells were sorted by FACS for PI-negative and PE-negative cells (i.e., alive Cas9 ⁺ B2M ^-sgRNA ^iBAR T cells) .

6. Target gene analysis

FACS sorted PI-negative and B2M-negative or deficient cells (i.e., alive Cas9 ⁺ B2M-sgRNA ^iBAR T cells) were used for genome extraction. sgRNA ^iBAR encoding fragments were amplified from the extracted genome, purified, and prepared for NGS sequencing. MAGeCK ^iBAR algorithm was used for sequencing data analysis (see Zhu et al., “Guide RNAs with embedded barcodes boost CRISPR-pooled screens, ” Genome Biol. 2019; 20: 20; the content of which is incorporated herein by reference in its entirety) , which contains three main parts: analysis preparation, statistical tests, and rank aggregation. Briefly, each sgRNAs ^iBAR targeted gene was scored and ranked based on the enrichment or depletion degree of each gene between the test group and the control group, in order to determine if such gene was a candidate gene with high confidence. See FIG. 5 for target gene identification workflow. Top ranking candidates (dark grey dots above the dotted horizontal line) from each Trial are shown in FIGs. 3A-3B, with candidate genes whose deletion result in sensitive phenotype to NK cell killing identified from the negative screen, and candidate genes whose deletion result in resistant phenotype to NK cell killing identified from the positive screen. Top ranking candidates with FDR≤0.15 in each Trial were mapped. These top ranking candidates were found to be involved in autoimmune responses (e.g., TAAC2, HES1, LILRB4, KLHL24, ARNTL, LRRC69, PACS2, CSK, and MYB, etc. ) , tumor malignant transformation (e.g., CJD2, FANCB, TPM3, TFG, SMAD6, PTPN14, or MEF12, etc. ) , or tumor metastasis (e.g., STON1, PLS1, SIX1, PIK3R6, PDE4C, SRRM3, SSPO, TLN1, PIH1D2, or SLC35C2, etc. ) . FIGs. 4A-4B show venn diagrams of top ranking candidates in various Trials with FDR≤0.15.

7. Results

Candidate genes that showed up in the negative screens of at least two Trials with FDR≤0.05 were categorized as T cell modulating genes whose deletion result in sensitive phenotype to NK cell killing (Table 1) . Candidate genes that showed up in the positive screen of any Trial with FDR≤0.05, or showed up in the positive screens of at least two Trials with FDR≤0.15, were categorized as T cell modulating genes whose deletion result in resistant phenotype to NK cell killing (Table 2) . Of these, Phosphofurin Acidic Cluster Sorting Protein-2 (PACS-2) was identified as a T cell modulating gene that confers resistance to NK cell killing after deletion. This is consistent with PACS-2’s proapoptotic effector role in Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL) -mediated apoptosis (Werneburg et al., J Biol Chem. 2012; 287 (29) : 24427-24437) . Another example is PTEN, which was identified as a T cell modulating gene that confers sensitivity to NK cell killing after deletion. This is consistent with PTEN’s role in cell proliferation, transcription regulation, and ubiquitination.

Results obtained here, particularly genes whose deletion were found to confer T cell resistance to NK cell killing, demonstrate valuable targets in allogeneic T cell therapy to avoid host rejection.

Table 1. T cell modulating genes sensitive to NK cell killing after deletion

Table 2. T cell modulating genes resistant to NK cell killing after deletion

Gene	Gene ID	Gene full name
TACC2	10579	Transforming acidic coiled-coil containing protein 2
HES1	3280	HES family bHLH transcription factor 1
STON1	11037	Stonin 1
GJD2	57369	Gap junction protein delta 2
LILRB4	11006	Leukocyte immunoglobulin like receptor B4
PLS1	5357	Plastin 1
KLHL24	54800	Kelch like family member 24
FANCB	2187	FA complementation group B
ARNTL	406	Aryl hydrocarbon receptor nuclear translocator like
AMY2A	279	Amylase alpha 2A
SIX1	6495	SIX homeobox 1
USP17L13	100287238	Ubiquitin specific peptidase 17 like family member 13
PIK3R6	146850	Phosphoinositide-3-kinase regulatory subunit 6
ATP6V1H	51606	ATPase H+transporting V1 subunit H
TPM3	7170	Tropomyosin 3
OR5H14	403273	Olfactory receptor family 5 subfamily H member 14
TFG	10342	Trafficking from ER to golgi regulator
SMAD6	4091	SMAD family member 6
PDE4C	5143	Phosphodiesterase 4C
SRRM3	222183	Serine/arginine repetitive matrix 3
LRRC69	100130742	Leucine rich repeat containing 69
KCNJ13	3769	Potassium inwardly rectifying channel subfamily J member 13
C8orf34	116328	Chromosome 8 open reading frame 34
PACS2	23241	Phosphofurin acidic cluster sorting protein 2

Claims

A method of identifying a target gene in a T cell that modulates the activity of the T cell, comprising:

a) providing a T cell library comprising a plurality of T cells, wherein each of the plurality of T cells has a mutation at a hit gene ( “hit gene mutation” ) in the genome, wherein the hit gene in at least two of the plurality of T cells are different from each other;

b) subjecting the T cell library to treatment with NK cells;

c) obtaining a T cell from the T cell library that is sensitive or resistant to the killing of the NK cells; and

d) identifying the hit gene in the T cell obtained from step c) , thereby identifying the target gene in the T cell that modulates the activity of the T cell.
The method of claim 1, wherein the T cell library is generated by subjecting an initial population of T cells to genome-wide gene editing.
The method of claim 1 or 2, wherein the T cell library is generated by contacting an initial population of T cells with i) a single-guide RNA ( “sgRNA” ) library comprising a plurality of sgRNA constructs, wherein each sgRNA construct comprises or encodes an sgRNA, and wherein each sgRNA comprises a guide sequence that is complementary to a target site in the hit gene in the genome; and optionally ii) a Cas component comprising a Cas protein or a nucleic acid encoding the Cas protein, under a condition that allows introduction of the sgRNA constructs and the optional Cas component into the initial population of T cells.
The method of claim 3, wherein the Cas protein is Cas9.
The method of claim 4, wherein each sgRNA comprises the guide sequence fused to a second sequence, wherein the second sequence comprises a repeat-anti-repeat stem loop that interacts with the Cas9.
The method of claim 5, wherein the second sequence of each sgRNA further comprises a stem loop 1, a stem loop 2, and/or a stem loop 3.
The method of any one of claims 3-6, wherein each sgRNA further comprises an internal barcode (iBAR) sequence ( “sgRNA ^iBAR” ) , wherein each sgRNA ^iBAR is operable with the Cas protein to modify the hit gene.
The method of claim 7, wherein the iBAR sequence of each sgRNA ^iBAR is inserted in the loop region of the repeat-anti-repeat stem loop.
The method of claim 7, wherein each sgRNA ^iBAR comprises in the 5’-to-3’ direction a first stem sequence and a second stem sequence, wherein the first stem sequence hybridizes with the second stem sequence to form a double-stranded RNA (dsRNA) region that interacts with the Cas protein, and wherein the iBAR sequence is disposed between the 3’ end of the first stem sequence and the 5’ end of the second stem sequence.
The method of any one of claims 3-9, wherein each guide sequence comprises about 17 to about 23 nucleotides.
The method of any one of claims 7-10, wherein each iBAR sequence comprises about 1 to about 50 nucleotides.
The method of any one of claims 7-11, wherein the sgRNA library comprising a plurality of sgRNA ^iBAR constructs ( “sgRNA ^iBAR library” ) comprises a plurality of sets of sgRNA ^iBAR constructs, wherein each set of sgRNA ^iBAR constructs comprise three or more sgRNA ^iBAR constructs each comprising or encoding an sgRNA ^iBAR, wherein the guide sequences for the three or more sgRNA ^iBAR constructs are the same, wherein the iBAR sequence for each of the three or more sgRNA ^iBAR constructs is different from each other, and wherein the guide sequence of each set of sgRNA ^iBAR constructs is complementary to a different target site in the genome.
The method of claim 12, wherein each set of sgRNA ^iBAR constructs comprises four sgRNA ^iBAR constructs, and wherein the iBAR sequence for each of the four sgRNA ^iBAR constructs is different from each other.
The method of claim 12 or 13, wherein the sgRNA ^iBAR library comprises at least about 100 sets of sgRNA ^iBAR constructs.
The method of any one of claims 12-14, wherein the iBAR sequences for at least two sets of sgRNA ^iBARconstructs are the same.
The method of any one of claims 3-15, wherein the sgRNA library comprising a plurality of sgRNA constructs comprises or encodes sgRNAs with guide sequences complementary to target sites of every annotated gene in the genome.
The method of any one of claims 3-16, wherein at least about 95% of the sgRNA constructs in the sgRNA library are introduced into the initial population of T cells.
The method of any one of claims 12-17, wherein the T cell library has at least about 100-fold coverage for each sgRNA ^iBAR.
The method of any one of claims 3-18, wherein the T cell library has at least about 400-fold coverage for each sgRNA.
The method of any one of claims 3-16, wherein the sgRNA library comprises at least about 150,000 sgRNA constructs.
The method of any one of claims 2-20, wherein the initial population of T cells express a Cas protein.
The method of any one of claims 3-21, further comprising contacting the initial population of T cells or the T cell library with i) an sgRNA construct comprising or encoding an sgRNA which comprises a guide sequence that is complementary to a target site in the B2M gene ( “B2M sgRNA” ) ; and optionally ii) a Cas component comprising a Cas protein or a nucleic acid encoding the Cas protein, under a condition that allows introduction of the B2M sgRNA construct and the optional Cas component into the initial population of T cells or the T cell library.
The method of any one of claims 2-21, wherein the T cells in the initial population of T cells comprise a B2M mutation.
The method of any one of claims 3-23, wherein each sgRNA construct in the sgRNA library and/or the B2M sgRNA construct is an RNA.
The method of any one of claims 3-23, wherein each sgRNA construct in the sgRNA library and/or the B2M sgRNA construct is a plasmid.
The method of any one of claims 3-23, wherein each sgRNA construct in the sgRNA library and/or the B2M sgRNA construct is a viral vector.
The method of claim 26, wherein the viral vector is a lentiviral vector.
The method of any one of claims 3-23, wherein each sgRNA construct in the sgRNA library and/or the B2M sgRNA construct is a virus.
The method of claim 28, wherein the virus is a lentivirus.
The method of any one of claims 26-29, wherein the sgRNA library and/or the B2M sgRNA construct is contacted with the initial population of T cells at a multiplicity of infection (MOI) of at least about2.
The method of any one of claims 1-30, wherein the treatment with NK cells comprises:

i) an initial treatment step comprising contacting the T cell library with the NK cells;

ii) an optional first enrichment step comprising sorting the mixture of treated cells to obtain a first T cell subpopulation that is sensitive or resistant to the killing of the NK cells;

iii) an optional first recovery step comprising culturing the first T cell subpopulation; and

iv) an optional second treatment step comprising contacting the first T cell subpopulation with the NK cells.
The method of claim 31, wherein the initial treatment step comprises contacting the T cell library with the NK cells for at least about 48 hours.
The method of claim 31 or 32, wherein the initial treatment step comprises contacting the T cell library with the NK cells for at least about 5 days.
The method of any one of claims 31-33, wherein the method comprises a first enrichment step.
The method of claim 34, wherein the first enrichment step comprises sorting the mixture of treated cells that are B2M-negative or deficient and alive, thus obtaining the first T cell subpopulation that is resistant to the killing of the NK cells ( “first alive enrichment” ) .
The method of claim 34, wherein the first enrichment step comprises sorting the mixture of treated cells that are B2M-negative or deficient and dead, thus obtaining the first T cell subpopulation that is sensitive to the killing of the NK cells ( “first dead enrichment” ) .
The method of claim 35 or 36, further comprising staining the mixture of treated cells with an anti-B2M antibody before sorting.
The method of any one of claims 35-37, further comprising staining the mixture of treated cells with propidium iodide (PI) before sorting, wherein PI staining indicates cell death.
The method of any one of claims 31-35, 37, and 38, wherein the method comprises a first recovery step.
The method of claim 39, wherein the first recovery step comprises culturing the first T cell subpopulation for at least about 24 hours.
The method of any one of claims 31-35 and 37-40, wherein the method comprises a second treatment step.
The method of claim 41, wherein the second treatment step comprises contacting the first T cell subpopulation with the NK cells for at least about 48 hours.
The method of any one of claims 1-42, wherein the ratio of the NK cells and the T cells in the T cell library in the initial treatment step is about 0.1∶1 to about 20∶1.
The method of any one of claims 1-43, wherein the ratio of the NK cells and the T cells in the T cell library in the initial treatment step is about 0.5∶1.
The method of any one of claims 1-43, wherein the ratio of the NK cells and the T cells in the T cell library in the initial treatment step is about 1∶1.
The method of any one of claims 1-35 and 37-45, wherein the ratio of the NK cells and the T cells in the first T cell subpopulation in the second treatment step is about 0.1∶1 to about 20∶1.
The method of any one of claims 1-35 and 37-46, wherein the ratio of the NK cells and the T cells in the first T cell subpopulation in the second treatment step is about 0.3∶1.
The method of any one of claims 1-47, wherein obtaining the T cell from the T cell library that is sensitive or resistant to the killing of the NK cells comprises:

i) a sorting step comprising sorting the cells obtained from step b) to obtain a second T cell subpopulation that is sensitive or resistant to the killing of the NK cells; and

ii) an optional second recovery step comprising culturing the second T cell subpopulation before harvesting the cells.
The method of claim 48, wherein the sorting step comprises sorting the cells obtained from step b) that are B2M-negative or deficient and alive, thus obtaining the second T cell subpopulation that is resistant to the killing of the NK cells ( “harvest alive sorting” ) .
The method of claim 48, wherein the sorting step comprises sorting the cells obtained from step b) that are B2M-negative or deficient and dead, thus obtaining the second T cell subpopulation that is sensitive to the killing of the NK cells ( “harvest dead sorting” ) .
The method of claim 49 or 50, further comprising staining the cells obtained from step b) with an anti-B2M antibody before sorting.
The method of any one of claims 49-51, further comprising staining the cells obtained from step b) with PI before sorting, wherein PI staining indicates cell death.
The method of any one of claims 48, 49, 51, and 52, wherein the method comprises a second recovery step.
The method of claim 53, wherein the second recovery step comprises culturing the second T cell subpopulation for at least about 24 hours.
The method of any one of claims 1-32, wherein steps b) and c) comprise:

i) an initial treatment step comprising contacting the T cell library with the NK cells for about 72 hours at a ratio of NK cells to T cells of about 0.5∶1;

ii) an enrichment step comprising sorting the mixture of treated cells that are B2M-negative or deficient and alive, thus obtaining a first T cell subpopulation that is resistant to the killing of the NK cells;

iii) a recovery step comprising culturing the first T cell subpopulation for about 48 hours;

iv) a second treatment step comprising contacting the first T cell subpopulation post-recovery with the NK cells for about 96 hours at a ratio of NK cells to T cells of about 0.3∶1; and

v) a sorting step comprising sorting the final mixture of treated cells that are B2M-negative or deficient and alive, thus obtaining a second T cell subpopulation that is resistant to the killing of the NK cells.
The method of any one of claims 1-32, wherein steps b) and c) comprise:

i) a treatment step comprising contacting the T cell library with the NK cells for about 10 days at a ratio of NK cells to T cells of about 0.5∶1; and

ii) a sorting step comprising sorting the mixture of treated cells that are B2M-negative or deficient and alive, thus obtaining a T cell subpopulation that is resistant to the killing of the NK cells.
The method of any one of claims 1-32, wherein steps b) and c) comprise:

i) a treatment step comprising contacting the T cell library with the NK cells for about 48 hours at a ratio of NK cells to T cells of about 1∶1;

ii) a sorting step comprising sorting the mixture of treated cells that are B2M-negative or deficient and alive, thus obtaining a T cell subpopulation that is resistant to the killing of the NK cells; and

iii) a recovery step comprising culturing the T cell subpopulation for about 48 hours before harvesting the cells.
The method of any one of claims 1-32, wherein steps b) and c) comprise:

i) a treatment step comprising contacting the T cell library with the NK cells for about 48 hours at a ratio of NK cells to T cells of about 1∶1;

ii) an enrichment step comprising sorting the mixture of treated cells that are B2M-negative or deficient and alive, thus obtaining a first T cell subpopulation that is resistant to the killing of the NK cells;

iii) a recovery step comprising culturing the first T cell subpopulation for about 48 hours; and

iv) a sorting step comprising sorting the first T cell subpopulation post-recovery that are B2M-negative or deficient and alive, thus obtaining a second T cell subpopulation that is resistant to the killing of the NK cells.
The method of any one of claims 1-58, wherein identifying the hit gene in the T cell obtained from step c) comprises:

i) identifying a sequence comprising the hit gene mutation in the T cell obtained from step c) ; and

ii) identifying the hit gene corresponding to the sequence comprising the hit gene mutation.
The method of any one of claims 3-58, wherein identifying the hit gene in the T cell obtained from step c) comprises:

i) identifying the sgRNA sequence in the T cell obtained from step c) ; and

ii) identifying the hit gene corresponding to the guide sequence of the sgRNA.
The method of claim 59 or 60, wherein the hit gene mutation or the sgRNA sequence is identified by DNA sequencing or RNA sequencing.
The method of any one of claims 59-61, wherein the hit gene mutation or the sgRNA sequence is identified by next-generation sequencing (NGS) .
The method of any one of claims 59, 61, and 62, wherein identifying the target gene comprises:

i) obtaining sequences comprising the hit gene mutations in the final T cell subpopulation obtained from step c) ;

ii) ranking the sequences comprising the hit gene mutations based on sequence counts; and

iii) identifying the hit gene corresponding to a sequence comprising the hit gene mutation ranked above a predetermined threshold level.
The method of any one of claims 60-62, wherein identifying the target gene comprises:

i) obtaining sgRNA sequences in the final T cell subpopulation obtained from step c) ;

ii) ranking the corresponding guide sequences of the sgRNA sequences based on sequence counts; and

iii) identifying the hit gene corresponding to a guide sequence ranked above a predetermined threshold level.
The method of any one of claims 60-62 and 64, wherein the sgRNA is an sgRNA ^iBAR, and wherein identifying the target gene comprises:

i) obtaining sgRNA ^iBAR sequences in the final T cell subpopulation obtained from step c) ;

ii) ranking the corresponding guide sequences of the sgRNA ^iBAR sequences based on sequence counts, wherein the ranking comprises adjusting the rank of each guide sequence based on data consistency among the iBAR sequences in the sgRNA ^iBAR sequences corresponding to the guide sequence; and

iii) identifying the hit gene corresponding to a guide sequence ranked above a predetermined threshold level.
The method of any one of claims 63-65, which is a positive screening.
The method of any one of claims 63-65, which is a negative screening.
The method of any one of claims 63-67, wherein the sequence counts are subject to median ratio normalization followed by mean-variance modeling.
The method of claim 68, wherein the variance of each guide sequence is adjusted based on data consistency among the iBAR sequences in the sgRNA ^iBAR sequences corresponding to the guide sequence.
The method of any one of claims 63-69, wherein the sequence counts obtained from the final T cell subpopulation obtained from step c) are compared to corresponding sequence counts obtained from a subpopulation of control T cells to provide fold changes.
The method of claim 70, wherein the subpopulation of control T cells are obtained from a same T cell library cultured in the same condition without subjected to treatment with NK cells, and optionally subjected to the same obtaining method in step c) .
The method of claim 70 or 71, wherein the data consistency among the iBAR sequences in the sgRNA ^iBAR sequences corresponding to each guide sequence is determined based on the direction of the fold change of each iBAR sequence, wherein the variance of the guide sequence is increased if the fold changes of the iBAR sequences are in different directions with respect to each other.
The method of any one of claims 59-62, further comprising culturing a same T cell library under the same condition without subjected to treatment with NK cells, and optionally subjected to the same obtaining method in step c) to obtain a subpopulation of control T cells, wherein the presence of identifying the hit gene corresponding to the sequence comprising the hit gene mutation or the guide sequence of the sgRNA from the subpopulation of control T cells but absence from the T cell obtained from step c) from the T cell library subjected to treatment with NK cells identifies the hit gene as the target gene.
The method of any one of claims 1-32 and 61-72, wherein the method comprises subjecting the T cell library from step a) to at least two of the four separate Trials before step d) :

(I) Trial I:

i) an initial treatment step comprising contacting the T cell library with the NK cells for about 72 hours at a ratio of NK cells to T cells of about 0.5∶1;

ii) an enrichment step comprising sorting the mixture of treated cells that are B2M-negative or deficient and alive, thus obtaining a first T cell subpopulation that is resistant to the killing of the NK cells;

iii) a recovery step comprising culturing the first T cell subpopulation for about 48 hours;

iv) a second treatment step comprising contacting the first T cell subpopulation post-recovery with the NK cells for about 96 hours at a ratio of NK cells to T cells of about 0.3∶1; and

v) a sorting step comprising sorting the final mixture of treated cells that are B2M-negative or deficient and alive, thus obtaining a second T cell subpopulation that is resistant to the killing of the NK cells;

(II) Trial II:

i) a treatment step comprising contacting the T cell library with the NK cells for about 10 days at a ratio of NK cells to T cells of about 0.5∶1; and

ii) a sorting step comprising sorting the mixture of treated cells that are B2M-negative or deficient and alive, thus obtaining a T cell subpopulation that is resistant to the killing of the NK cells;

(III) Trial III:

i) a treatment step comprising contacting the T cell library with the NK cells for about 48 hours at a ratio of NK cells to T cells of about 1∶1;

ii) a sorting step comprising sorting the mixture of treated cells that are B2M-negative or deficient and alive, thus obtaining a T cell subpopulation that is resistant to the killing of the NK cells; and

iii) a recovery step comprising culturing the T cell subpopulation for about 48 hours before harvesting the cells; and

(IV) Trial IV:

i) a treatment step comprising contacting the T cell library with the NK cells for about 48 hours at a ratio of NK cells to T cells of about 1∶1;

ii) an enrichment step comprising sorting the mixture of treated cells that are B2M-negative or deficient and alive, thus obtaining a first T cell subpopulation that is resistant to the killing of the NK cells;

iii) a recovery step comprising culturing the first T cell subpopulation for about 48 hours; and

iv) a sorting step comprising sorting the first T cell subpopulation post-recovery that are B2M-negative or deficient and alive, thus obtaining a second T cell subpopulation that is resistant to the killing of the NK cells.
The method of claim 74, wherein identifying the target gene comprises identifying the hit genes from the at least two of the four separate Trials, wherein:

i) the hit genes that are identified as depleted from the final T cell subpopulation in at least one Trial with FDR≤0.01, in at least two Trials with FDR≤0.05 are identified as target genes whose mutation makes the T cells sensitive to NK cell killing; and/or

ii) the hit genes that are identified as enriched from the final T cell subpopulation in at least one Trial with FDR≤0.05, or in at least two Trials with FDR≤0.15, are identified as target genes whose mutation makes the T cells resistant to NK cell killing.
The method of any one of claims 1-32 and 61-72, wherein the method comprises subjecting the T cell library from step a) to at least two separate different treatments with NK cells in step b) , and obtaining the T cells that are sensitive or resistant to the killing of the NK cells from each treatment in step c) .
The method of claim 76, wherein identifying the target gene comprises identifying the hit genes in the T cells obtained from the at least two separate different treatments with NK cells, wherein:

i) the hit genes that are identified as depleted from the final T cell subpopulation resistant to the killing of the NK cells in at least one treatment with NK cells with FDR≤0.01, or in at least two separate different treatments with NK cells with FDR≤0.05 are identified as target genes whose mutation make the T cells sensitive to NK cell killing;

ii) the hit genes that are identified as enriched from the final T cell subpopulation resistant to the killing of the NK cells in at least one treatment with NK cells with FDR≤0.05, or in at least two separate different treatments with NK cells with FDR≤0.15, are identified as target genes whose mutation make the T cells resistant to NK cell killing;

iii) the hit genes that are identified as depleted from the final T cell subpopulation sensitive to the killing of the NK cells in at least one treatment with NK cells with FDR≤0.05, or in at least two separate different treatments with NK cells with FDR≤0.15, are identified as target genes whose mutation make the T cells resistant to NK cell killing; and/or

iv) the hit genes that are identified as enriched from the final T cell subpopulation sensitive to the killing of the NK cells in at least one treatment with NK cells with FDR≤0.01, or in at least two separate different treatments with NK cells with FDR≤0.05 are identified as target genes whose mutation make the T cells sensitive to NK cell killing.
The method of any one of claims 1-77, further comprising validating the target gene by:

a) modifying a T cell by creating a mutation in the target gene in the T cell; and

b) determining the sensitivity or resistance of the modified T cell to the killing of NK cells.
The method of claim 78, further comprising creating a mutation in B2M in the T cell.
The method of any one of claims 2-79, wherein T cells in the the initial population of T cells express a chimeric antigen receptor (CAR) .
A method of generating a modified T cell, comprising inactivating a target gene identified by the method of any one of claims 1-80 in a host T cell.
The method of claim 81, wherein the host T cell further comprises a mutation in B2M.
The method of claim 81 or 82, wherein the host T cell expresses a CAR.
The method of claim 81 or 82, further comprising introducing into the host T cell a nucleic acid encoding a CAR.
The method of any one of claims 81-84, wherein the host T cell is an allogeneic T cell.
A modified T cell comprising a mutation in a target gene, wherein the target gene is selected from the group consisting of TACC2, HES1, STON1, GJD2, LILRB4, PLS1, KLHL24, FANCB, ARNTL, AMY2A, SIX1, USP17L13, PIK3R6, ATP6V1H, TPM3, OR5H14, TFG, SMAD6, PDE4C, SRRM3, LRRC69, KCNJ13, C8orf34, and PACS2.
The modified T cell of claim 86, wherein the modified T cell further comprises a mutation in B2M.
The modified T cell of claim 86 or 87, wherein the target gene is PSCS2.
The modified T cell of any one of claims 86-88, wherein the modified T cell further expresses a CAR.
The modified T cell of any one of claims 86-88, wherein the modified T cell is allogeneic.
An sgRNA library comprising one or more sgRNA constructs, wherein each sgRNA construct comprises or encodes an sgRNA, and wherein each sgRNA comprises a guide sequence that is complementary to a target site in a target gene selected from the group consisting of TACC2, HES1, STON1, GJD2, LILRB4, PLS1, KLHL24, FANCB, ARNTL, AMY2A, SIX1, USP17L13, PIK3R6, ATP6V1H, TPM3, OR5H14, TFG, SMAD6, PDE4C, SRRM3, LRRC69, KCNJ13, C8orf34, and PACS2.
The sgRNA library of claim 91, wherein the sgRNA library further comprises an sgRNA construct comprising or encoding an sgRNA whose guide sequence is complementary to a target site in B2M.
A kit for generating a modified T cell resistant to the killing of NK cells, comprising the sgRNA library of claim 91 or 92.
The kit of claim 93, further comprising a Cas protein or a nucleic acid encoding the Cas protein.
The kit of claim 93 or 94, further comprising an isolated nucleic acid encoding a CAR.