A multiplexed RNA-regulated platform for the manipulation of primary immune cells

Abstract

The present disclosure provides a versatile and multifunctional platform for transcriptome control, using the RNA-guided RNA targeting activity of VI-D CRISPR effectors with RNA-guided RNA endonuclease activity, combined with a guide array that expresses multiple guide RNAs. This system can be used to perform quantitative, reversible, and massively multiplexed gene knockdown in primary human T cells, and multiplexed suppression of exhaustion-related genes in T cells. This system can be used to enhance the anti-tumor activity of dysfunctional CAR T cells. TIFF2025529871000026.tif62128

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 400,578, filed August 24, 2022, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0002] STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT This invention was made with government support under Grant No. 1R21CA270609-01 awarded by the National Cancer Institute. The government has certain rights in this invention. [Background technology]

[0003] background Adoptive T-cell therapies, including chimeric antigen receptor (CAR) T-cell therapy, have revolutionized the treatment of refractory B-cell and plasma cell malignancies in patients and have shown promising curative potential in other aggressive preclinical cancer models. However, despite these successes, significant barriers to progress remain, including high rates of primary and acquired resistance, resulting in very limited clinical efficacy in solid tumors. 1~5 Recent studies have implicated intrinsic mechanisms of T cell dysfunction, such as T cell exhaustion and metabolic dysregulation, in these failed states. 1、6~10 .

[0004] Concurrent advances in CRISPR / Cas9-based methods and technologies have led to the next generation of engineered CAR T cells, offering potential solutions to these challenges and opportunities to create new therapeutic modalities. 3、11 For example, Cas9 can be used to introduce pooled, systematic genetic perturbations, often on a genome-wide scale, into primary human T cells to screen for negative regulators of T cell activity in various cancer-related contexts. 12~19Screening hits can then be specifically deleted by Cas9 knockout or base editing strategies to prevent dysfunction or enhance antitumor activities, including T cell expansion and persistence, solid tumor infiltration, cytokine secretion, and cytotoxicity. 12~16 , 19~24 Alternatively, donor-specific protein components, such as TRAC (TCR) and B2M (HLA), can be knocked out to generate allogeneic "low-hanging fruit" T cells that do not induce immune rejection or graft-versus-host disease. 3、11、25~28 .

[0005] The intersection of CRISPR engineering and adoptive T cell therapy holds great promise for the future of cancer treatment, but Cas9-based systems are limited in their safety, efficacy, and potential for widespread application. Both Cas9 nuclease and base editors introduce binary, irreversible changes into the T cell genome. 29、30 These methods completely abolish gene function and may have adverse effects on T cell health and activity. 21、31~35 Furthermore, Cas9 nuclease is not compatible with safe and efficient large-scale multiplexing of gene knockouts—the introduction of multiple double-strand breaks (DSBs) can lead to chromosomal translocations / shortenings. 27、36、37 and p53-related genotoxicity 11、38 Although the Cas9 base editor reduces the risks associated with DSBs by creating nicks in genomic DNA, its large-scale multiplexing is limited by the tradeoff between on-target editing efficiency and extensive off-target editing due to its promiscuous deaminase activity. 3、27、39~41 Taken together, Cas9-based tools are entirely incapable of safely and effectively perturbing the complex multigene programs (e.g., those that drive T cell metabolism, signaling, and exhaustion), yet such perturbations are likely required to create optimized next-generation adoptive T cell therapies.

[0006] To overcome the limitations imposed by current state-of-the-art gene editing technologies in primary human T cells, the present disclosure provides MEGA (Multiplexed Effector Guide Array), a multifunctional platform for transcriptome control using CRISPR / Cas13d, a recently described small class 2 VI-D CRISPR effector with RNA-guided RNA endonuclease activity. 42、43 Unlike Cas9, Cas13d does not bind, target, or cleave DNA. 42 More precisely, Cas13d forms a complex with CRISPR-associated RNA (crRNA) that contains a programmable ~23nt spacer that directs ribonucleoproteins to sequence-specific RNA transcripts for targeted degradation without PAM restriction. Furthermore, Cas13d has the unique ability to process poly-crRNA guide arrays into individual crRNAs, facilitating efficient simultaneous targeting of multiple RNA transcripts in a single cell. 42、44 Finally, Cas13d is approximately two-thirds the size of wild-type Cas9 (fusion Cas9 variants are even larger), making it highly suitable for T cell manufacturing.

[0007] This disclosure describes compositions and methods for massively multiplexed, quantitative, and reversible perturbation of the cellular transcriptome. To address established unmet needs, this disclosure describes the deployment of MEGA in a wide range of applications, ranging from modulating CAR signaling to enhancing antitumor activity in dysfunctional high-affinity GD2-targeting (HA-28z) CAR T cells, a well-characterized in vitro model that recapitulates key molecular, phenotypic, and functional features of T cell exhaustion. 7、45~47 The present disclosure also describes the use of MEGA to target a unique set of genes implicated in metabolic reprogramming and disrupt entire metabolic pathways to alleviate adenosine-mediated immunosuppression of CAR T cells. Summary of the Invention

[0008] A brief overview The present disclosure provides compositions comprising genetically modified T cells expressing (i) a class 2 VI-D CRISPR effector with RNA-guided RNA endonuclease activity and (ii) a guide array comprising a plurality of CRISPR-associated RNA (crRNA) molecules; systems for multiplexed transcriptome control; fusion proteins comprising a class 2 VI-D CRISPR effector with RNA-guided RNA endonuclease activity destabilization domain (DD); nucleic acids, such as plasmids and vectors, encoding the class 2 VI-D CRISPR effector with RNA-guided RNA endonuclease activity, guide array, CAR, and / or fusion protein of the present disclosure; and guide arrays comprising nucleic acids (polynucleotides) comprising direct repeat sequences and spacer sequences. The present disclosure also provides methods for producing modified T cells expressing class 2 VI-D CRISPR effector and guide arrays of the present disclosure, methods for regulating gene expression in T cells, methods for screening to identify regulators of T cell activity, methods for increasing proliferation of T cells, methods for treating cancer or tumors in a subject or patient in need of treatment, and methods for increasing the anti-tumor activity of T cells.

[0009] In one aspect, the present disclosure provides a genetically modified T cell comprising: (i) a class 2 VI-D CRISPR effector with RNA-guided RNA endonuclease activity; and (ii) A guide array comprising multiple CRISPR-associated RNA (crRNA) molecules, each of which independently comprises a direct repeat sequence and a spacer sequence that binds to a target RNA, and wherein the crRNA molecules bind to different target mRNAs or different regions of the same target mRNA.

[0010] In some embodiments, the genetically modified T cell comprises a nucleic acid encoding (i), a nucleic acid encoding (ii), or a nucleic acid encoding both (i) and (ii), wherein the nucleic acid encoding (i) or (ii), or both (i) and (ii), is stably integrated into the genome of the T cell.

[0011] In some embodiments, the Class 2 VI-D CRISPR effector with RNA-guided RNA endonuclease activity comprises a fusion protein comprising a destabilization domain (DD).

[0012] In some embodiments, the Class 2 VI-D CRISPR effector with RNA-guided RNA endonuclease activity is a Cas13 nuclease selected from the group consisting of Cas13a, Cas13b, 13bt1, Cas13bt2, Cas13c, Cas13d, RfxCas13d, Cas13e, and functional variants thereof.

[0013] In some embodiments, the guide array is a multicistronic array containing multiple crRNA molecules. In some embodiments, the guide array contains 2-10 crRNA molecules. In some embodiments, the guide array contains more than 10 crRNA molecules.

[0014] In some embodiments, the guide array comprises crRNA molecules that bind to mRNAs encoding proteins associated with T cell exhaustion. In some embodiments, the proteins associated with T cell exhaustion are selected from the group consisting of TOX, TOX2, NR4A1, NR4A2, NR4A3, TET2, IRF4, JUNB, BATF3, DHX37, FLI1, ZC3H12A, SOCS1, TCEB2, PDCD1 (PD-1), HAVCR2 (TIM3), LAG3, CTLA4, TIGIT, FAS, TRAC, CBLB, RASA2, PTPN2, CD5, ENTPD1, ​​CD46, B2M, and combinations thereof.

[0015] In some embodiments, the T cells or primary T cells further comprise a chimeric antigen receptor (CAR). In some embodiments, the CAR binds to an antigen expressed by a tumor. In some embodiments, the CAR binds to Her-2, B7-H3, GPC2, GD2, CD19, CD20, CD22, MAGE, BAGE, CAGE, GAGE, HAGE, LAGE, PAGE, PRAME, NY-ESO-1, NY-SEO-1, tyrosinase, Melan-A / MART, gpl00, TRP-1, TRP-2, CD30, EGFR, EGFRvIII, FAP, CD33 , CD123, PD-L1, IGF1R, CD4, CSPG4, B7-H4, NKG2D, CS1, CD138, EpCAM, EBNA3C, GPA7, CD244, CA-125, ETA, CEA, CD52, MUC5AC, c-Met, FAB, WT-1, PSMA, AFP, BCMA, mesothelin, GPC3, MUC1, and CTAG1B.

[0016] In some embodiments, intracellular signaling by CAR upregulates T cell exhaustion markers in control T cells. In some embodiments, the control T cells do not contain (i) a class 2 VI-D CRISPR effector with RNA-guided RNA endonuclease activity; and (ii) a guide array comprising multiple CRISPR-associated RNA (crRNA) molecules, wherein each crRNA molecule independently comprises a direct repeat sequence and a spacer sequence that binds to a target RNA, and the crRNA molecules bind to different target mRNAs or different regions of the same target mRNA.

[0017] In some embodiments, the exhaustion marker is selected from the group consisting of LAG3, PDCD1 (PD-1), HAVCR2 (TIM3), CTLA4, and ENTPD1, ​​and combinations thereof.

[0018] In some embodiments, the guide array comprises crRNA molecules that bind to mRNAs encoding LAG3, PDCD1 (PD-1), HAVCR2 (TIM3), CTLA4, and ENTPD1, ​​or combinations thereof. In some embodiments, the guide array comprises crRNA molecules that bind to mRNAs expressed by CARs.

[0019] In some embodiments, the T cells are selected from the group consisting of human T cells and primary human T cells.

[0020] In another aspect, the present disclosure provides a nucleic acid encoding (i) a class 2 VI-D CRISPR effector with RNA-guided RNA endonuclease activity; and (ii) a guide array comprising a plurality of CRISPR-associated RNA (crRNA) molecules, wherein the crRNA molecules independently comprise a direct repeat sequence and a spacer sequence that binds to a target RNA, and the crRNA molecules bind to different target mRNAs or different regions of the same target mRNA. In some embodiments, the class 2 VI-D CRISPR effector with RNA-guided RNA endonuclease activity is a Cas13 nuclease selected from the group consisting of Cas13a, Cas13b, 13bt1, Cas13bt2, Cas13c, Cas13d, RfxCas13d, Cas13e, and functional variants thereof. In some embodiments, the guide array is a multicistronic array comprising a plurality of crRNA molecules. In some embodiments, the guide array comprises 2 to 10 or more crRNA molecules. In some embodiments, the guide array comprises crRNA molecules that bind to mRNAs encoding proteins associated with T cell exhaustion. In some embodiments, the proteins associated with T cell exhaustion are selected from the group consisting of TOX, TOX2, NR4A1, NR4A2, NR4A3, TET2, IRF4, JUNB, BATF3, DHX37, FLI1, ZC3H12A, SOCS1, TCEB2, PDCD1 (PD-1), HAVCR2 (TIM3), LAG3, CTLA4, TIGIT, FAS, TRAC, CBLB, RASA2, PTPN2, CD5, ENTPD1, ​​CD46, B2M, and combinations thereof.

[0021] In another aspect, the present disclosure provides a system for multiplexed transcriptome control, comprising: (i) an expression cassette comprising a nucleic acid sequence encoding a class 2 VI-D CRISPR effector with RNA-guided RNA endonuclease activity; and (ii) an expression cassette comprising a nucleic acid sequence encoding a guide array comprising multiple crRNA molecules, wherein the crRNA molecules comprise direct repeat sequences and spacer sequences that bind to target mRNAs, and the crRNA molecules bind to different target mRNAs or different regions of the same target mRNA.

[0022] In some embodiments, the system further comprises (iii) T cells or primary T cells.

[0023] In another aspect, the present disclosure provides a method for producing modified T cells, comprising: (i) transducing T cells with an expression vector comprising a nucleic acid sequence encoding a class 2 VI-D CRISPR effector with RNA-guided RNA endonuclease activity; and (ii) transducing T cells with an expression vector comprising a nucleic acid sequence encoding a guide array comprising a plurality of CRISPR-associated RNA (crRNA) molecules, wherein the crRNA molecules independently comprise a direct repeat sequence and a spacer sequence that binds to a target mRNA, and the crRNA molecules bind to different target mRNAs or different regions of the same target mRNA, thereby producing modified T cells.

[0024] In some embodiments, the method further comprises (iii) transducing the T cells with an expression vector comprising a nucleic acid sequence encoding the CAR.

[0025] In some embodiments of the method, step (i) is performed before step (ii), step (iii) is performed before step (ii), or steps (i) and (iii) are performed before step (ii).

[0026] In some embodiments, one or more of the expression vectors of steps (i), (ii), and / or (iii) are stably integrated into the genome of the T cell.

[0027] In some embodiments of the method, expression and intracellular signaling by the CAR upregulates T cell exhaustion markers in control T cells that do not contain the expression vectors of steps (i) and (ii).

[0028] In another aspect, the present disclosure provides a fusion protein comprising a Class 2 VI-D CRISPR effector with RNA-guided RNA endonuclease activity covalently linked to a destabilization domain (DD) polypeptide.

[0029] In some embodiments, the Class 2 VI-D CRISPR effector with RNA-guided RNA endonuclease activity is a Cas13 nuclease selected from the group consisting of Cas13a, Cas13b, 13bt1, Cas13bt2, Cas13c, Cas13d, RfxCas13d, Cas13e, and functional variants thereof.

[0030] In some embodiments, the DD comprises an E. coli dihydrofolate reductase DD linked to the C-terminus of a Class 2 VI-D CRISPR effector with RNA-guided RNA endonuclease activity.

[0031] In some aspects, the present disclosure provides nucleic acids encoding the fusion proteins described herein.

[0032] In another aspect, the present disclosure provides a method for manufacturing a semiconductor device comprising: (a) transducing T cells with (i) an expression vector comprising a nucleic acid sequence encoding a fusion protein described herein; and (ii) an expression vector comprising a nucleic acid sequence encoding a guide array comprising a crRNA molecule, the crRNA molecule comprising a direct repeat sequence and a spacer sequence that binds to a target mRNA expressed by a target gene; and (b) contacting the T cell with a compound that binds to and stabilizes the DD, wherein expression of the target gene is decreased in the presence of the compound compared to expression of the target gene in the absence of the compound, thereby controlling gene expression in the T cell. The present invention provides a method for regulating gene expression in T cells, comprising:

[0033] In some embodiments, the fusion protein is degraded in the T cell in the absence of the compound.

[0034] In some embodiments, expression of the target gene increases after removal of the compound, thereby reversibly regulating expression of the target gene.

[0035] In some embodiments, the guide array comprises multiple crRNA molecules that bind to different target mRNAs or different regions of the same target mRNA.

[0036] In some embodiments, the expression of the target gene is regulated in a dose-dependent manner by the compound. In some embodiments, the compound is trimethoprim (TMP).

[0037] In another aspect, the present disclosure provides a method for screening to identify regulators of T cell activity, comprising the steps of expressing in a T cell or population of T cells: (i) a class 2 VI-D CRISPR effector having RNA-guided RNA endonuclease activity and (ii) a library of guide arrays, where each guide array comprises one or more CRISPR-associated RNA (crRNA) molecules, where the crRNA molecules comprise a direct repeat sequence and a spacer sequence that binds to a target RNA, and where the one or more crRNA molecules bind to different target mRNAs or different regions of the same target mRNA; culturing the T cells to produce a clonal population of expanded T cells; and determining whether the guide array is enriched or depleted in the clonal population of expanded T cells, where if the guide array is enriched, the target mRNA encodes a negative regulator of T cell activity, or if the guide array is depleted, the target mRNA encodes a positive regulator of T cell activity.

[0038] In some embodiments, the T cell activity is T cell proliferation, increased cytokine secretion, or increased tumor cell killing.

[0039] In some embodiments, T cells comprising enriched guide arrays have an effector memory phenotype, and T cells comprising depleted guide arrays have a central memory phenotype or a stem cell memory phenotype.

[0040] In some embodiments of the method, each guide array comprises a crRNA molecule that binds to an mRNA encoding a protein associated with T cell exhaustion. In some embodiments, the protein associated with T cell exhaustion is selected from the group consisting of TOX, TOX2, NR4A1, NR4A2, NR4A3, TET2, IRF4, JUNB, BATF3, DHX37, FLI1, ZC3H12A, SOCS1, TCEB2, PDCD1 (PD-1), HAVCR2 (TIM3), LAG3, CTLA4, TIGIT, FAS, TRAC, CBLB, RASA2, PTPN2, CD5, ENTPD1, ​​CD46, B2M, and combinations thereof.

[0041] In some embodiments, the library of guide arrays comprises one or more individual guide arrays, including multicistronic arrays containing multiple crRNA molecules. In some embodiments, an individual guide array comprises 2-10 crRNA molecules. In some embodiments, an individual guide array comprises a single pair of crRNA molecules.

[0042] In some embodiments, determining whether a guide array is enriched or depleted in a clonal population of expanded T cells comprises sequencing the guide RNAs present in the T cells.

[0043] In another aspect, the present disclosure provides a method for increasing proliferation of T cells, comprising the steps of: (i) an expression vector comprising a nucleic acid sequence encoding a class 2 VI-D CRISPR effector with RNA-guided RNA endonuclease activity; and (ii) An expression vector comprising a nucleic acid sequence encoding a guide array comprising one or more crRNA molecules, wherein the crRNA molecules independently comprise a direct repeat sequence and a spacer sequence that binds to a target mRNA, and the crRNA molecules bind to different target mRNAs or different regions of the same target mRNA. Transducing T cells by Here, proliferation of the T cells is increased relative to control T cells expressing a non-targeting control guide array comprising one or more crRNA molecules that do not bind to the target mRNA of (ii).

[0044] In some embodiments, the method further comprises (iii) transducing the T cells with an expression vector comprising a nucleic acid sequence encoding the CAR.

[0045] In another aspect, the present disclosure provides a method for treating a tumor in a subject, the method comprising administering to a subject genetically modified T cells of the present disclosure, wherein the modified T cells kill tumor cells in the subject, thereby treating the tumor.

[0046] In another aspect, the present disclosure provides a method for enhancing the anti-tumor activity of T cells, the method comprising contacting tumor cells with the genetically modified T cells of the present disclosure, wherein contacting the tumor cells with the genetically modified T cells increases the expression of anti-tumor cytokines or kills the tumor cells, thereby enhancing the anti-tumor activity compared to control T cells. In some embodiments, the control T cells do not comprise the expression vector of (i) or (ii) above, or both of (i) and (ii).

[0047] In some embodiments, the method is an in vitro method. In some embodiments, the method is an in vivo method.

[0048] In another aspect, the present disclosure provides a guide array comprising a plurality of CRISPR-associated RNA (crRNA) molecules, wherein the crRNA molecules comprise a direct repeat sequence and a spacer sequence that binds to a target RNA, and the crRNA molecules bind to different target mRNAs or different regions of the same target mRNA.

[0049] In any of the embodiments of the present disclosure, the Class 2 VI-D CRISPR effector with RNA-guided RNA endonuclease activity is a Cas13 nuclease selected from the group consisting of Cas13a, Cas13b, 13bt1, Cas13bt2, Cas13c, Cas13d, RfxCas13d, Cas13e, and functional variants thereof.

[0050] In any of the embodiments of the present disclosure, the guide array is a multicistronic array comprising multiple crRNA molecules.

[0051] In any of the embodiments of the present disclosure, the guide array comprises 2 to 10 or more crRNA molecules.

[0052] In any of the embodiments of the present disclosure, the guide array comprises crRNA molecules that bind to mRNAs encoding proteins selected from the group consisting of TOX, TOX2, NR4A1, NR4A2, NR4A3, TET2, IRF4, JUNB, BATF3, DHX37, FLI1, ZC3H12A, SOCS1, TCEB2, PDCD1 (PD-1), HAVCR2 (TIM3), LAG3, CTLA4, TIGIT, FAS, TRAC, CBLB, RASA2, PTPN2, CD5, ENTPD1, ​​CD46, B2M, and combinations thereof.

[0053] In any of the embodiments of the present disclosure, the T cells or primary T cells further comprise a chimeric antigen receptor (CAR).

[0054] In any of the embodiments of the present disclosure, the CAR binds to an antigen expressed by a tumor.

[0055] In any of the embodiments of the present disclosure, the CAR may be any of the following: Her-2, B7-H3, GPC2, GD2, CD19, CD20, CD22, MAGE, BAGE, CAGE, GAGE, HAGE, LAGE, PAGE, PRAME, NY-ESO-1, NY-SEO-1, tyrosinase, Melan-A / MART, gpl00, TRP-1, TRP-2, CD30, EGFR, EGFRvIII, FAP, CD 33, CD123, PD-L1, IGF1R, CD4, CSPG4, B7-H4, NKG2D, CS1, CD138, EpCAM, EBNA3C, GPA7, CD244, CA-125, ETA, CEA, CD52, MUC5AC, c-Met, FAB, WT-1, PSMA, AFP, BCMA, mesothelin, GPC3, MUC1, and CTAG1B.

[0056] In any of the embodiments of the present disclosure, expression and intracellular signaling by the CAR upregulates T cell exhaustion markers in control T cells compared to T cells containing: (i) an expression cassette comprising a nucleic acid sequence encoding a class 2 VI-D CRISPR effector with RNA-guided RNA endonuclease activity; and (ii) an expression cassette comprising a nucleic acid sequence encoding a guide array comprising multiple crRNA molecules, wherein the crRNA molecules comprise a direct repeat sequence and a spacer sequence that binds to a target mRNA, and wherein the crRNA molecules bind to different target mRNAs or different regions of the same target mRNA.

[0057] In any of the embodiments of the present disclosure, the exhaustion marker is selected from LAG3, PDCD1 (PD-1), or HAVCR2 (TIM3), or a combination thereof.

[0058] In any of the embodiments of the present disclosure, the guide array comprises crRNA molecules that bind to mRNA encoding LAG3, PDCD1 (PD-1), or HAVCR2 (TIM3), or a combination thereof.

[0059] In any of the embodiments of the present disclosure, the guide array comprises a crRNA molecule that binds to the mRNA expressed by the CAR.

[0060] In any of the embodiments of the present disclosure, the T cells are selected from the group consisting of human T cells and primary human T cells. [Brief explanation of the drawings]

[0061] [Figure 1]MEGA HA-28z CAR T cells robustly suppress the upregulation of inhibitory receptors associated with exhaustion. (A) Schematic of the relevant lentiviral constructs in MEGA CAR T cells. CRISPR / Cas13d enables multiplexed RNA targeting with a single guide array to efficiently suppress the upregulation of inhibitory receptors. DR: Direct Repeat. (B) Overview of the optimized workflow for generating MEGA CAR T cells from primary human T cells for phenotypic evaluation. FACS: Fluorescence-activated cell sorting. (C) Overlaid violin and boxplot diagrams showing the surface expression of LAG3, PD-1, and TIM3 for single- and double-guide cases measured by flow cytometry at day 10 from one representative donor. On-target guides are colored (LAG3, yellow; PD-1, red; TIM3, blue); off-target guides are gray. The dashed line indicates the median fluorescence intensity (MFI) of either mock-transduced or exhausted non-targeted cells, used for normalization. The numbers shown indicate the normalized values ​​for each condition (mock MFI = 0; non-targeted MFI = 1). NT: non-targeted guide. (D) Normalized surface expression of LAG3 (L), PD-1 (P), and TIM3 (T) as described in (C) for single-guide and double-guide cases in n = 2-3 donors from independent experiments. The dashed line represents the non-targeted MFI. *p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.0001, ordinary one-way ANOVA and Dunnett's multiple comparison test. Error bars are ± sd. (E) Correlation plot of mRNA transcript expression (measured by RT-qPCR) relative to NT and normalized surface expression of LAG3, PD-1, and TIM3 for single- and double-guide from one representative donor. On-target guides are pink, and off-target guides are black. Colored lines represent best fits from linear regression. Error bars are sem. (F) Volcano diagram representing bulk RNA-Seq data from day 10 MEGA CAR T cells in n=2 donors from independent experiments.Genes with significantly altered expression compared to NT are highlighted (log2fc<-1, padj<0.001). (G) Normalized aggregate surface expression of LAG3, PD-1, and TIM3 for triple-guide arrays in n=2-3 donors from independent experiments. The dashed line represents the non-targeting MFI. ***p<0.0005, ****p<0.0001, ordinary one-way ANOVA and Dunnett's multiple comparison test. Error bars are ±sd. (H) Correlation plot of mRNA transcript expression and normalized surface expression of LAG3, PD-1, and TIM3 for triple-guide arrays compared to NT from one representative donor. The colored line represents the best fit from linear regression. Error bars are sem. (I) Pie chart showing the relative percentage of LAG3+ / -PD-1+ / -TIM3+ / - MEGA CAR T cells for triple guide arrays as measured by flow cytometry on day 10 from one representative donor. Triple positive cells are highlighted in yellow, and triple negative cells are highlighted in blue. [Figure 2]Combinatorial CRISPR screening in dysfunctional CAR T cells identifies paired regulators of proliferation. (A) Overview of the 2D CRISPR screening strategy in dysfunctional CD8+ HA-28z CAR T cells. (B) Volcano plot showing differences in guide array abundance (log2 expression ratio, l2fc) between early (plasmid DNA) and late (day 13) time points for n=2 replicates. Blue dots: significantly depleted arrays (adjusted p<0.05, l2fc<-0.5); red dots: significantly enriched arrays (adjusted p<0.05, l2fc>0.5); open black dots: non-targeting arrays. Adjusted p-values ​​obtained by Wald test performed in DESeq2, adjusted with Benjamini-Hochberg correction. (C) 2D heatmap of guide array enrichment between early and late time points. Top left: targeting double array; top right and bottom left: targeting single array; bottom right: non-targeting array. (D) Guide arrays ranked using a likelihood ratio test performed in DESeq2 with n=2 replicates. The top-ranked arrays with the most significant variation in numbers across all screening time points are colored (blue: depleted, red: enriched). (E) Top: Histogram showing the distribution of l2fcs between early and late time points for all 6,400 guide arrays in the library. Bottom: Rug plot showing l2fc of all 9 guide arrays targeting significantly enriched (red) or depleted (blue) gene pairs overlaid on the l2fc distribution of all 64 non-targeting guides (gray). (F) Fold change expansion of FACS-sorted RfxCas13d+ CD8+ HA-28z CAR T cells compared to non-targeting controls over 15 days of culture in n=3-4 donors from independent experiments. Significantly enriched guide arrays are red; significantly depleted guide arrays are blue; non-targeting control guides are black. The dashed line represents the expansion growth for the non-targeting control. p<0.0001, ordinary one-way ANOVA. Error bars are ±sem. [Figure 3]Paired transcriptome perturbations broadly enhance the antitumor activity of dysfunctional MEGA CAR T cells. (A) Overview of the experimental workflow for generating dysfunctional MEGA CAR T cells expressing the validation array. (B) Schematic detailing the experimental conditions for the cytokine secretion assay. (C, D) Secretion of IFNγ (C) or IL-2 (D) after 24 hours of culture in a 1:1 E:T with (pink dots, stimulated) or without (black dots, baseline) antigen-positive tumor cells. Data are the mean of n = 3 replicate wells from one representative donor. *p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.0001, ordinary one-way ANOVA and Dunnett's multiple comparison test. Error bars are ± sd. (E) Top: Schematic detailing the sequential stimulation assay. MEGA CAR T cells were repeatedly challenged with antigen-positive tumor cells at an E:T ratio of 1:1 every 48–72 hours. Bottom: Kinetics of tumor killing measured using Incucyte live-cell imaging (1:1 E:T). Data are the mean of n=3 replicate wells from one representative donor. Red line: enriched guide array; black line: non-targeting control. *p<0.0001, repeated measures one-way ANOVA. Shaded areas are ±sem. (F) Incucyte live-cell imaging as in Figure 5E, but at a lower E:T ratio of 1:5. (G) Incucyte images at 0 and 48 hours after three rounds of tumor stimulation, as in Figure 3E. Green: Nalm6-GD2 tumor cells; red: MEGA HA-28z CAR T cells. (H) Schematic of the hypothesized mechanism for the enhanced antitumor activity in CBLB+FAS double knockdown CAR T cells (bottom) compared to non-targeting controls (top). [Figure 4]MEGA enables rapid, quantitative, and reversible control of the T cell transcriptome. (A) Schematic detailing exogenous control of RfxCas13d-DD expression with the low-molecular-weight drug trimethoprim (TMP). (B) Violin and box plot overlay showing CD46 surface expression measured by flow cytometry on day 5 from one representative donor. On-target conditions are shown in purple, and off-target conditions are shown in gray. Numbers above the plots indicate the ratio of CD46 expression in paired comparisons. (C) Left: Violin and box plot overlay showing TMP-dependent surface expression of CD46 measured by flow cytometry on day 5 from one representative donor. Right: Dose-response curve showing TMP-dependent changes in CD46 knockdown measured by flow cytometry on day 5. Data are the average of n=3 replicate wells from one representative donor. The linear region of the sigmoidal curve is shown in pink. Error bars are ±sd. (D) Time course showing the kinetics of CD46 knockdown after TMP addition (left, yellow) or removal (right, dark blue) over 72 hours, as measured by flow cytometry. Data are the mean of three replicate wells from one representative donor. Error bars are ±sd. (E) Overlaid violin and boxplots showing surface expression of LAG3, PD-1, and TIM3 in the presence or absence of TMP, as measured by flow cytometry on day 10, from one representative donor. Colored histograms: on-target conditions; gray histograms: off-target conditions. Dashed lines indicate MFI values ​​for either mock-untransduced or exhausted non-targeted cells, used for normalization. Numbers shown indicate normalized values ​​for each condition. (F) Violin and boxplot overlays showing surface expression of LAG3, PD-1, and TIM3 in the presence of increasing concentrations of TMP as measured by flow cytometry on day 10 from one representative donor. (G) Dose-response curves showing TMP-dependent changes in knockdown of LAG3 (yellow), PD-1 (red), or TIM3 (blue) as measured by flow cytometry on day 10.Data are the mean of n=3 replicate wells from one representative donor. Error bars are ±sd. [Figure 5] MEGA enables massively multiplexed knockdown of immune-related endogenous genes in primary human T cells. (A) Overview of an optimized, two-step method for rapid and easy assembly of guide arrays of any length by overlap extension PCR. (B) mRNA transcript levels of LAG3, FAS, CTLA4, PD-1, and TIM3 relative to NT, measured by RT-qPCR at day 10 from n = 2 donors. Numbers within bars represent mean values. ****p < 0.0001, Welch's t-test. Error bars are ± SD. (C) Overlaid violin and boxplot diagrams showing surface expression of LAG3, FAS, CTLA4, PD-1, and TIM3, measured by flow cytometry at day 10 from one representative donor. On-target guide arrays are colored (LAG3, yellow; FAS, green; CTLA4, dark blue; PD-1, red; TIM3, light blue); control is gray. Dashed lines indicate MFI values ​​for either mock or non-targeted cells. (D) Aggregate surface expression of LAG3, FAS, CTLA4, PD-1, and TIM3 compared to NT in n=3 donors from an independent experiment. Dashed lines represent MFI for either mock or non-targeted cells. **p<0.005, ***p<0.0005, ordinary one-way ANOVA and Dunnett's multiple comparison test. Error bars are ±sd. (E) Levels of mRNA transcripts for LAG3, FAS, CD5, CD39, CD46, TRAC, B2M, CTLA4, PD-1, and TIM3 compared to NT from n=2 donors, measured by RT-qPCR on day 10. Numbers within bars represent mean values. *p<0.05, **p<0.005, ***p<0.0005, ****p<0.0001, Welch's t-test. Error bars are ±sd. [Figure 6]Metabolic engineering enhances CAR T cell function by disrupting the entire purinergic signaling cascade. (A) Schematic detailing the pathways involved in purinergic signaling and adenosine-mediated immune suppression. (B) Schematic detailing the predicted impact of disrupting purinergic signaling on metabolite levels and immune activity. (C) mRNA transcript levels of purinergic signaling components A2BR, A2AR, CD73, and CD39 compared to NT, measured by RT-qPCR at day 10 in n=3 technical replicates from one representative donor. Numbers within bars represent mean values. ****p<0.0001, Welch's t-test. Error bars are ±sd. (D, E, F) Concentrations of the metabolites ATP (D), AMP (E), and adenosine (F) in the culture medium of MEGA HA-28z CAR T cells expressing the PURI array (green) or non-targeting guide (gray). Data are the mean of n=2-3 replicate wells from one representative donor. *p<0.05, **p<0.005, unpaired t-test. Error bars are ±sd. (G, H) Time course showing the concentration of metabolites ATP (G) and AMP (H) in the culture medium of MEGA HA-28z CAR T cells after spiking-in with 20 μM ATP. Green line: PURI array; gray line: non-targeting control. Data are the mean of n=3 replicate wells from one representative donor. **p<0.005, ****p<0.0001, repeated measures two-way ANOVA, column factor. Error bars are ±sd. (I, J) Secretion of IFNγ (I) or IL-2 (J) by dysfunctional MEGA CAR T cells after 24 hours of culture with antigen-positive tumor cells (stimulated) or without antigen-positive tumor cells (baseline) at an E:T ratio of 1:1. Data are the mean of n=3 replicate wells from one representative donor. Green bars: PURI array; gray bars: NT control. ****p<0.0001, two-way ANOVA and Bonferroni's multiple comparison test. Error bars are ±sd. (K, L) Kinetics of tumor killing (K) and T-cell proliferation (L) measured using Incucyte live-cell imaging at an E:T ratio of 1:1.Data are means of n=3 replicate wells from one representative donor. Green line: PURI array; gray line: NT control. **p<0.005, repeated measures two-way ANOVA, column factor. Shaded areas are ±sem. [Figure 7] MEGA is a flexible and versatile platform for transcriptome control in primary human T cells using CRISPR / Cas13d. [Figure 8]Figure 8 is related to Figure 1. Characterization and optimization of RfxCas13d expression and activity in primary human T cells. (A) Overview of lentiviral constructs and experimental settings used for GFP reporter suppression experiments. (B) GFP expression measured by flow cytometry on day 5 with increasing GFP virus. Green line: cells transduced with RfxCas13d:GFP and GFP reporter; black dotted line: cells transduced with GFP reporter only. Data are the average of n=3 replicate wells from one representative donor. (C) Overview of lentiviral constructs used to suppress exhaustion markers in HA-28z CAR T cells. (D) Expression of LAG3, PD-1, and TIM3 relative to NT measured by flow cytometry on day 10 in n=3 donors from an independent experiment. Colored bars: on-target guide (LAG3, yellow; PD-1, red; TIM3, blue); gray bars: off-target guide. (E) Histogram showing RfxCas13d expression measured by flow cytometry on day 10 from one representative donor. Black open histogram: mock-untransduced cells; magenta filled histogram: RfxCas13d-transduced cells. (F) Violin and boxplot overlay showing RfxCas13d expression measured by flow cytometry on day 5 from one representative donor. Black open histogram: mock-untransduced cells; magenta filled histogram: cells transduced with various RfxCas13d constructs. (G) Model explaining the context-dependent effect of RfxCas13d activity in LX293T cells on lentiviral titers during viral packaging. (H, I) Violin plots showing the expression of (H) RfxCas13d and (I) HA-28z CARs in co-transduced primary human T cells measured by flow cytometry on day 5 in the presence of increasing viral doses. [Figure 9]Figure 9 is related to Figure 1. Multiplexed suppression of exhaustion markers in MEGA HA-28z CAR T cells. (A) Expression of LAG3, PD-1, and TIM3 mRNA transcripts for single and double guides measured by RT-qPCR at day 10 (see Figure 1E). Data are the mean of n=3 technical replicates from one representative donor. Error bars are ±sd. M: mock, N: non-targeting, L: LAG3, P: PD-1, T: TIM3. (B) Overlaid violin and boxplots showing HA-28z CAR expression for single and double guides measured by flow cytometry at day 5 from one representative donor. Blue histogram: transduced cells, gray histogram: mock non-transduced cells. (C) Violin and boxplot overlay showing the surface expression of LAG3, PD-1, and TIM3 for triple guides measured by flow cytometry at day 10 from one representative donor (see Figure 1F). On-target guides are colored (LAG3, yellow; PD-1, red; TIM3, blue); controls are gray. Dashed lines indicate MFI values ​​for either mock-untransduced or exhausted non-targeted cells, used for normalization. Numbers indicate normalized values ​​for each condition. (D) Expression of LAG3, PD-1, and TIM3 mRNA transcripts for triple guides measured by RT-qPCR at day 10 (see Figure 1H). Data are the mean of n=3 technical replicates from one representative donor. Error bars are ±s.d. (E) Violin and boxplot overlay showing triple-guide HA-28z CAR expression at day 5 as measured by flow cytometry from one representative donor. Blue histograms: transduced cells; gray histograms: mock non-transduced cells. (F, G) Gating strategy used in Figure 1I to identify the LAG3+ / -PD-1+ / -TIM3+ / - population in unsorted MEGA HA-28z CAR T cells expressing (F) non-targeting control or (G) triple-guide array.Data are from one representative donor measured by flow cytometry on day 10. From left to right: live cell (lymphocyte) gate, singlet gate, RfxCas13d+ gate, LAG3+ / -PD-1+ / - gate, TIM3+ / - gate. [Figure 10] Figure 10 is related to Figure 2. Further characterization of Cas13-based CRISPR screening in HA-28z CAR T cells. (A) Plot showing the distribution of guide arrays in plasmid DNA preparations of assembled custom libraries, as measured by NGS. The number of guide arrays is ranked by abundance. Dashed lines represent the 90th and 10th percentiles. Blue numbers indicate a common metric for quantifying library coverage and bias. (B) Histograms showing the expression of RfxCas13d (left) and HA-28z CAR (right) over the screening period. Colored histograms: screening cells (RfxCas13d: magenta, CAR: blue); uncolored histograms: mock-transduced cells. (C) Plot showing the distribution of guide arrays over the screening period in two replicates. (D) Plot showing the correlation of guide array abundance between the two replicates. Left: early time point, right: late time point. (E) Volcano plot showing the difference in average gene pair abundance between early (plasmid DNA) and late (day 13) time points for n=2 replicates. Blue dots: significantly depleted pairs; red dots: significantly enriched pairs; open black dots: non-targeting control. p-values ​​are from robust rank aggregation performed in MAGeCK. (F) 2D heatmap of gene pair enrichment between early and late time points. Genes are ordered by hierarchical clustering performed in pheatmap. [Figure 11]Figure 11 is related to Figure 2. Validation and phenotypic analysis of 2D CRISPR screening hits. (A) Violin and boxplot overlay showing HA-28z CAR expression (top) and RfxCas13d expression (bottom) for validation arrays measured by flow cytometry at day 5 (pre-FACS) from one representative donor. Dashed lines represent the gate for RfxCas13d+ cells. Red histograms: enriched arrays; blue histograms: depleted arrays; gray histograms: control. (B) Total percentage of RfxCas13d+ cells detailed in (A) for n=3 donors from independent experiments. Red bars: enriched arrays; blue bars: depleted arrays; gray bars: non-targeting. **p<0.005, ****p<0.0001, ordinary one-way ANOVA and Dunnett's multiple comparison test. Error bars are ±s.d. (C) Lines showing expansion of FACS-sorted RfxCas13d+ CD8+ HA-28z CAR T cells over 15 days of culture (see Figure 2F). Each panel corresponds to a new donor from an independent experiment. Red line: enriched array; blue line: depleted array; black line: non-targeting; shaded gray area: AUC of non-targeting control. (D) Fold change in expansion of FACS-sorted RfxCas13d+ HA-28z CAR T cells compared to non-targeting control over 15 days of culture in n=3-4 donors from an independent experiment. Significantly enriched guide arrays are red; significantly depleted guide arrays are blue; non-targeting control guides are black. Dashed lines represent expansion for non-targeting control. *p<0.0001, ordinary one-way ANOVA. Error bars are ±s.e.m. (E) Lines showing proliferation of FACS-sorted RfxCas13d+HA-28z CAR T cells over 15 days of culture in n=4 donors (one panel each) from independent experiments. Red line: enriched array, blue line: depleted array, black line: non-targeting, shaded grey area: AUC of non-targeting control.(F, G) Density plots showing the levels of CD62L and CD45RA on HA-28z CAR T cells expressing non-targeting guide arrays (F) or targeting guide arrays (G) from one representative donor, as measured by flow cytometry on day 10. Numbers in each quadrant indicate the relative frequency of the indicated T cell subset (pink: highest overall frequency). [Figure 12] Figure 12 is related to Figure 3. Evaluation of the antitumor activity of dysfunctional MEGA CAR T cells. (A, B) Secretion of IFNγ (A) or IL-2 (B) by dysfunctional MEGA CAR T cells after 24 hours of culture with antigen-positive tumor cells (pink dots, stimulated) or without antigen-positive tumor cells (black dots, baseline) in a 1:1 E:T system, as in Figure 3B-C. Data are the mean of n = 3 replicate wells. Each panel corresponds to a new donor from an independent experiment. *p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.0001, ordinary one-way ANOVA and Dunnett's multiple comparison test. Error bars are ± sd. (C) Kinetics of tumor killing (1:1 E:T) measured using Incucyte live-cell imaging, as in Figure 3D. Data are the mean of n = 3 replicate wells. Each horizontal row corresponds to a new donor from an independent experiment. Red line: enriched guide array, blue line: depleted guide array, black line: non-targeting control. p<0.0001, repeated measures one-way ANOVA and Dunnett's multiple comparison test. Shaded areas are ±sem. [Figure 13]Figure 13 is related to Figure 4. Control of RfxCas13d activity by small molecule drugs. (A) Density plots depicting RfxCas13d and LAG3 / PD-1 / TIM3 expression in MEGA HA-28z CAR T cells at day 10, as measured by flow cytometry, from one representative donor. Left column: targeting guide; right column: non-targeting control. (B) Violin and box plot overlay showing intracellular RfxCas13d expression at day 5, as measured by FLAG-tag staining and flow cytometry, from one representative donor. Blue histograms: high expression conditions; gray histograms: low expression conditions. (C) Violin and box plot overlay showing surface CD46 over 72 hours, as measured by flow cytometry, from one representative donor (see Figure 4D). Top: TMP addition; bottom: TMP removal. Solid histograms: TMP addition (yellow) or removal (dark blue); dotted, open histograms: non-targeting control. (D) Time course showing the kinetics of CD46 degradation after CHX addition, as measured by flow cytometry. Data are the mean of n=3 replicate wells from one representative donor. Error bars are ±s.d. (E) Bar graphs showing CD46 expression (left), mCherry expression (center), or cell viability (right) with various concentrations of TMP. Colored bars: CD46-targeting guide (CD46 expression: purple, mCherry expression: magenta, cell viability: blue); gray bars: non-targeting guide. Numbers above the bars indicate fold change compared to NT. Data are the mean of n=3 replicate wells from one representative donor. *p<0.05, **p<0.005, ****p<0.0001, multiple unpaired t-test with FDR correction (Benjamini, Krieger, and Yekutieli). Error bars are ±sd. [Figure 14]Figure 14 is related to Figure 5. Gene knockdown is robust regardless of spacer and target transcript abundance. (A) mRNA transcript levels for all target genes in Figure 5 compared to NT (three spacers per transcript) measured by RTqPCR at day 10 in n=3 technical replicates from one donor. *p<0.05, **p<0.005, ***p<0.0005, ****p<0.0001, ordinary one-way ANOVA and Dunnett's multiple comparison test. Error bars are ±sd. (B) Relative transcript expression plotted against transcript abundance as measured by bulk RNA-Seq of HA-28z CAR T cells for each target gene in Figure 5. [Figure 15]Interfering with PI3K / Akt-driven glycolytic activity modulates CAR T cell metabolism and attenuates exhaustion. (A) Schematic showing aerobic glycolysis as a metabolic driver of T cell effector differentiation and eventual exhaustion. A 4-gene guide array (GLY) perturbs the upstream glycolytic genes HK1, HK2, AKT1, and AKT2. (B) mRNA transcript levels of HK1, HK2, AKT1, and AKT2 relative to NT measured by RT-qPCR at day 10 in n=3 technical replicates from one donor representing two independent experiments. Numbers within bars represent mean values. **p<0.01, ***p<0.001, ****p<0.0001, ordinary one-way ANOVA and Dunnett's multiple comparison test. Error bars are ±sd. (C) Two-dimensional visualization of clustered high-dimensional mass cytometry (CyTOF) data from day 10 MEGA HA-28z CAR T cells expressing either a GLY array (n = 9,871 cells) or a non-targeting (NT) guide (n = 8,025 cells) using t-distributed stochastic neighbor embedding (t-SNE). Dots represent clusters of cells with similar protein expression profiles, as determined by unsupervised FlowSOM clustering. The dot locations represent cluster centers. The dot sizes represent the number of cells represented by a particular cluster. The dot coloring represents the enrichment (log2 expression ratio counts) of cells in each cluster, distinguished by GLY (dark green) or NT (yellow) after barcode removal. Contours represent kernel density estimation in the two-dimensional latent space. (D) t-SNE visualization of the cell clusters described in (C), colored based on normalized marker expression (% max). High-expressing cell clusters are colored dark purple, and low-expressing cell clusters are colored bright orange. (E) Transcriptome-wide quantification of bulk RNA-seq data from MEGA HA-28z CAR T cells at day 10 using the targeting GLY array (y-axis) or NT control (x-axis), averaged over n=2 donors from independent experiments. Each dot represents a single gene.Gene-level expression was determined by aggregating transcripts per million (TPM) and p-values ​​across all detected transcripts for each gene. Differentially expressed genes with an adjusted p-value <0.2 and absolute log2 expression ratio >0.5 are highlighted (red: upregulated in GLY; blue: downregulated in GLY). The size of the dot is proportional to statistical significance (-log10 adjusted p-value). The green line indicates where gene expression is equal in both conditions (y = x). (F) Gene set enrichment analysis (GSEA) results, including all hallmark gene sets and the previously described gene signature for NK-like T cell dysfunction. The top five most significantly enriched gene sets are shown. Genes were ranked based on the following ranking metric: -log10(adjusted p-value) × sign(log2 expression ratio GLY / NT). The displayed genes contributed most to the normalized enrichment score (NES). (G) Extracellular pH measurements of MEGA HA-28z CAR T cell culture supernatants after 48 hours of culture. Cells were seeded at 1 x 106 cells / ml in triplicate wells. Data are representative of n = 3 donors in independent experiments. ***p<0.001, unpaired t-test. Error bars are ±sd. (H, I) Secretion of IL-2 (H) or IFNγ (I) by dysfunctional MEGA HA-28z CAR T cells after 24 hours of culture with antigen-positive tumor cells in a 1:1 E:T setting. Data are the mean of n = 3 replicate wells from one representative donor. Dark green bars: GLY array; yellow bars: NT control. ***p<0.001, unpaired t-test. Error bars are ±sd. (J) Kinetics of tumor killing in a repeated stimulation assay (1:1 E:T) measured using Incucyte live-cell imaging. Data are the mean of n=3 replicate wells from one representative donor. Black line: mock T cells, yellow line: non-targeting control, dark green line: GLY array. ****p<0.0001, repeated measures two-way ANOVA. Shaded areas are ±sem.(K) Schematic outlining the generation of Cas9-gene-edited HA-28z CAR T cells by RNP electroporation. On day 3 of culture, HA-28z CAR T cells were electroporated with either Cas9 complexed with a control guide (AAVS1) targeting the AAVS1 safe harbor locus or Cas9 complexed with a mixture of four guides (4KO) targeting HK1, HK2, AKT1, and AKT2. (L) Editing efficiency of the 4-gene knockout (4KO, dark blue bars) compared to the control (AAVS1, cyan bars) as determined by TIDE analysis of PCR amplicons from the indicated genomic loci. n = 2 donors in independent experiments. ****p<0.0001, two-way ANOVA and Bonferroni's multiple comparison test. (M) Fold change in expansion of HA-28z CAR T cells with knockdown (left) or knockout (right) of HK1, HK2, AKT1, and AKT2 over 10 days of culture in n=3-4 donors from independent experiments. Left: GLY array (dark green) vs. NT control (yellow). Right: 4KO (dark blue) vs. AAVS1 control (cyan). ***p<0.001, unpaired t-test. Error bars are ±sd. (N) Tumor killing by HA-28z CAR T cells at different effector:target ratios. Data represent tumor intensity at the endpoint (t=48 h) normalized to initial tumor intensity (t=0 h). Gray: mock; dark green: Cas13 GLY array; yellow: Cas13 NT control; dark blue: Cas9 4KO; cyan: Cas9 AAVS1 control. **p<0.01, ****p<0.0001, two-way ANOVA and FDR-corrected multiple comparison test (Benjamini, Krieger and Yekutieli). (O) Transcriptome-wide quantification of bulk RNA-seq data from day 10 HA-28z CAR T cells gene-edited with Cas9 4KO (y-axis) or Cas9 AAVS1 control (x-axis), averaged over n=2 donors from independent experiments. Each dot represents a single gene.Gene-level expression was determined by aggregating transcripts per million (TPM) and p-values ​​across all detected transcripts for each gene. Differentially expressed genes with an adjusted p-value <0.2 and absolute log2 expression ratio >0.5 are highlighted (red: upregulated in 4KO; blue: downregulated in 4KO). The size of the dot is proportional to statistical significance (-log10 adjusted p-value). Green lines indicate areas of equal gene expression in both conditions (y = x). (P) Gene set enrichment analysis (GSEA) results, including all hallmark gene sets and the previously described gene signature for NK-like T cell dysfunction. The top five most significantly enriched gene sets are shown. Genes were ranked based on the following ranking metric: -log10(adjusted p-value) × sign(log2 expression ratio 4KO / AAVS1). The displayed genes contributed most to the normalized enrichment score (NES). (Q) Area-proportional Euler plot showing significantly differentially expressed genes identified from (E) and (O). Top: downregulated genes; bottom: upregulated genes. The Cas13 gene set (n = 188 down, n = 123 up) represents genes significantly different between GLY and NT conditions, and the Cas9 gene set (n = 87 down, n = 92 up) represents genes significantly different between 4KO and AAVS1 conditions. (R) GSEA results for Cas13 (GLY vs. NT) plotted against GSEA results for Cas9 (4KO vs. AAVS1). Dots represent the overall GSEA ranking index for each gene set: -log10(adjusted p-value) × sign(NES). Gene sets included in (F) and (P) are displayed and color-coded by type: green dots are reduced in Cas13 and Cas9; blue dots are reduced in Cas9 only; magenta dots are elevated in Cas13 only; orange dots are elevated in Cas9 only. [Figure 16]RfxCas13d exhibits no side activity in primary human MEGA T cells. (A) Schematic showing the experimental workflow for assessing RfxCas13d RNA and protein side activity resulting from on-target B2M cleavage in primary human T cells from n = 2 donors. (B) Dot plots showing protein expression levels of mCherry-P2A-RfxCas13d relative to either B2M (on-target), CD46 (off-target), or CD3 (off-target) in MEGA T cells expressing either the B2M-targeting guide (left column) or a non-targeting (NT) control (right column). Data are measured by flow cytometry on day 10 from one representative donor. The numbers shown indicate the percentage of cells in each quadrant, respectively. (C) Mean fluorescence intensity (MFI) of mCherry in MEGA T cells expressing either the B2M targeting guide (purple bars) or the non-targeting control (gray bars). Data are the mean of n=2 donors. Error bars are ±s.d. p=0.0596 (not significant), paired t-test. (D) Cell viability of MEGA T cells expressing either the B2M targeting guide (purple bars) or the non-targeting control (gray bars). Data are the mean of n=2 donors. Error bars are ±s.d. p=0.8949 (not significant), paired t-test. (E) Cell number (total yield) of FACS-sorted MEGA T cells expressing either the B2M targeting guide (purple bars) or the non-targeting control (gray bars). Data are the mean of n=2 donors. Error bars are ±s.d. p=0.5410 (not significant), paired t-test. (F) Total RNA (total yield) extracted from FACS-sorted MEGA T cells expressing either the B2M targeting guide (purple bars) or the non-targeting control (gray bars). Data are means from n=2 donors. Error bars are ±sd. p=0.8820 (not significant), paired t-test.(G) Principal component analysis (PCA) of bulk RNA-seq data generated from FACS-sorted MEGA T cells expressing either the B2M-targeting guide (purple dots) or the non-targeting control (gray dots) from n = 2 donors (DN). (H) Transcriptome-wide quantification of bulk RNA-seq data from day 5 MEGA T cells using the targeting B2M guide (y-axis) or the NT control guide (x-axis), averaged over n = 2 donors. Each dot represents a single gene. Gene-level expression was determined by aggregating transcripts per million (TPM) values ​​and p-values ​​across all detected transcripts for each gene. Differentially expressed genes with adjusted p-values ​​< 0.1 and absolute log2 expression ratios > 1 are highlighted (red: upregulated with B2M; blue: downregulated with B2M). Targeted B2M transcripts are highlighted in purple. Mitochondrial RNAs are highlighted in orange. The size of the dot is proportional to statistical significance (-log10 adjusted p-value). The green line indicates where gene expression is equal in both conditions (y=x). [Figure 17]MEGA CAR T cells with disrupted glycolytic metabolism exhibit enhanced antitumor efficacy in vivo. (A) Schematic detailing the experimental procedure for in vivo tumor challenge of MEGA HA-28z CAR T cells. On day 0, NSG mice were inoculated with 1 x 10 antigen-positive Nalm6-GD2 (GFP+Luc+) cells. One week later, mice were injected with 1) mock-untransduced T cells, 2) MEGA HA-28z CAR T cells expressing a non-targeting control guide, or 3) MEGA HA-28z CAR T cells expressing the GLY (HK1 / HK2 / AKT1 / AKT2) array. (B) Quantification of tumor burden in Nalm6-GD2 tumor-bearing mice by bioluminescence imaging (BLI) on day 12. Gray bars: non-targeting control; dark gray bars: GLY array; light gray bars: mock. Each dot represents a tumor BLI measurement per mouse. Data are the mean of n=5 mice per condition (error bars are ±sem). (C) Quantification of tumor burden in Nalm6-GD2 tumor-bearing mice by bioluminescence imaging (BLI) over 12 days. Dark gray line: MEGA T cells expressing the GLY array; gray line: MEGA HA-28z CAR T cells expressing a non-targeting control guide; light gray line: mock untransduced T cells. n=5 mice per condition. [Figure 18]Hypermultiplexed knockdown of 10 genes in MEGA HA-28z CAR T cells broadly and robustly reprograms the transcriptome at the single-cell level. (A) Uniform manifold approximation and projection (UMAP) two-dimensional visualization of single-cell RNA sequencing data (scRNA-seq) from MEGA HA-28z CAR T cells expressing either a 10-gene guide array (SURF2, blue dots, n = 113 cells) or a non-targeting control (NT, gray dots, n = 307 cells). (B) UMAP visualization of the same scRNA-seq data as in (A), colored based on normalized gene expression. Single-cell expression data for the top 12 genes most significantly enriched in SURF2 cells compared to NT are shown. (C) UMAP visualization of the same scRNA-seq data as in (A), colored based on normalized gene expression. Single-cell expression data for the top 12 genes most significantly depleted in SURF2 cells compared to NT are shown. DETAILED DESCRIPTION OF THE INVENTION

[0062] Detailed Description Introduction The present disclosure provides compositions and methods for multiplexed transcriptome control in cells, which provide a versatile and multifunctional platform for programmable and scalable control of cellular transcriptome.The compositions and methods of the present disclosure provide advantages over existing CRISPR gene editing technology, which has limited safety, efficacy and scope.The compositions and methods of the present disclosure enable quantitative, reversible and massively multiplexed gene knockdown in cells, without targeting or cutting genomic DNA.

[0063] These compositions and methods use a class 2 CRISPR / Cas system, which uses a single RNA-guided protein effector with RNA-guided RNA endonuclease activity. In some embodiments, the class 2 CRISPR / Cas system includes a VI-D CRISPR effector. Unlike Cas9, the VI-D CRISPR effector does not bind to, target, or cut DNA. 42 More precisely, VI-D CRISPR effectors form complexes with CRISPR-associated RNAs (crRNAs) that contain programmable spacer sequences that direct ribonucleoproteins to specific RNA transcripts for targeted degradation.

[0064] Type VI-D CRISPR effector proteins are thought to be guided to their target RNAs by a single crRNA, which contains a direct repeat stem-loop sequence and a spacer sequence (guide RNA) that binds to the target sequence via RNA-RNA hybridization. 49 The direct repeat sequence used is determined by the particular class 2 VI-D CRISPR effector protein co-expressed in the cell. For example, the direct repeats in guide RNAs complexed with Cas13d are highly conserved in length and secondary structure.

[0065] Representative, non-limiting examples of VI-D type CRISPR effectors of the present disclosure include the Cas13 family, including Cas13a, Cas13b, 13bt1, Cas13bt2, Cas13c, Cas13d, RfxCas13d, and Cas13e, and their functional variants. In some embodiments, the VI-D type CRISPR effector is Cas13d. Cas13d has the unique ability to process poly-crRNA guide arrays into separate crRNAs, facilitating the efficient simultaneous targeting of multiple RNA transcripts in a single cell. 42、44Finally, Cas13d is approximately two-thirds the size of wild-type Cas9 (Cas9 fusion variants are even larger), making it highly suitable for T cell manufacturing.

[0066] general principle The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA, which are within the skill of one in the art. See Sambrook, Fritsch, and Maniatis, Molecular Cloning: A Laboratory Manual, 2nd edition (1989), Current Protocols in Molecular Biology (F.M. Ausubel, et al. eds., (1987), the series Methods in Enzymology (Academic Press, Inc.), PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames, and G.R. Taylor eds. (1995), Antibodies, A Laboratory Manual, and Animal Cell Culture (R.I. Freshney, ed. (1987)).

[0067] Non-commercially available oligonucleotides can be chemically synthesized by the solid-phase phosphoramidite triester method first described by Beaucage and Caruthers, Tetrahedron Lett. 22:1859-1862 (1981), using an automated synthesizer, as described, for example, in Van Devanter et al., Nucleic Acids Res. 12:6159-6168 (1984). Purification of oligonucleotides is carried out using any art-recognized strategy, such as native acrylamide gel electrophoresis or anion-exchange high-performance liquid chromatography (HPLC), as described in Pearson and Reanier, J. Chrom. 255:137-149 (1983).

[0068] definition Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. In addition, any method or material similar or equivalent to the methods or materials described herein can be used in the practice of this disclosure. For the purposes of this disclosure, the following terms are defined:

[0069] As used herein, the terms "a," "an," or "the" not only include aspects containing one member, but also aspects containing multiple members. For example, the singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells, reference to "the agent" includes reference to one or more agents known to those of skill in the art, and so forth.

[0070] The term "about" in connection with a reference numerical value can include a range of values ​​from that value plus or minus 10%. For example, the amount "about 10" includes amounts from 9 to 11, including the reference numbers 9, 10, and 11. The term "about" in connection with a reference numerical value can also include a range of values ​​from that value plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%.

[0071] The terms "nucleic acid," "nucleotide," or "polynucleotide" refer to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and polymers thereof, in either single-, double-, or multi-stranded form. This term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and / or pyrimidine bases or other natural, chemically modified, biochemically modified, non-natural, synthetic, or derivatized nucleotide bases. In some embodiments, nucleic acids can comprise mixtures of DNA, RNA, and their analogs. Unless otherwise specified, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, single nucleotide polymorphisms (SNPs), and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0072] The term "guide array" refers to a nucleic acid molecule comprising one or more, two, three, or more sequences encoding RNA molecules (e.g., CRISPR-associated RNAs or "crRNAs"), where the RNA comprises a direct repeat stem-loop sequence and a spacer sequence (e.g., guide RNA) that can bind to an RNA target sequence via RNA-RNA hybridization. The term includes DNA molecules or vectors comprising one or more, two, three, or more sequences encoding different crRNA molecules, including polycistronic and multicistronic DNA molecules encoding different crRNA molecules.

[0073] The term "encode" or "encodes" refers to a nucleic acid sequence comprising an open reading frame that can be transcribed by an RNA polymerase and translated into a polypeptide or protein of the present disclosure (e.g., a Class 2 VI-D CRISPR effector). The term also includes nucleic acid sequences that are transcribed to produce RNA that is not translated into a polypeptide or protein, such as a nucleic acid sequence encoding a guide array of the present disclosure.

[0074] As used herein, the term "non-targeting control guide" is a crRNA with a spacer that is randomly generated and compared to the human transcriptome using BLAST to confirm that it does not match any known human RNA transcript.

[0075] The term "gene" refers to a segment of DNA involved in producing a polypeptide chain. This DNA segment may include regions preceding and following the coding region (leader and trailer) that are involved in the transcription / translation and regulation of transcription / translation of the gene product, as well as intervening sequences (introns) between individual coding segments (exons).

[0076] The term "cassette" refers to a combination of genetic sequence elements that can be introduced as a single element and function together to achieve a desired result. Cassettes typically contain polynucleotides in a combination not found in nature. Cassettes can be inserted into a vector, such as an expression vector.

[0077] The term "operably linked" means that two or more genetic sequence elements, such as a polynucleotide coding sequence and a promoter sequence, are positioned in a polynucleotide, cassette, or vector in relative positions that allow for proper biological function of those elements, e.g., a promoter that directs transcription of the coding sequence.

[0078] The term "inducible promoter" refers to a promoter that responds to environmental factors and / or external stimuli that can be artificially controlled to alter the expression or expression level of a polynucleotide sequence, or to a combination of elements, e.g., an exogenous promoter and an additional element, such as a transactivator, operably linked to another promoter. An inducible promoter can respond to abiotic factors, such as oxygen levels, or to chemical or biological molecules. In some embodiments, the chemical or biological molecule can be a molecule that does not naturally occur in humans.

[0079] The terms "vector" and "expression vector" refer to a recombinantly or synthetically produced nucleic acid construct containing a series of designated nucleic acid elements that enable transcription of a particular polynucleotide sequence in a host cell. An expression vector may be part of a plasmid, a viral genome, or a nucleic acid fragment. Typically, an expression vector contains a polynucleotide to be transcribed operably linked to a promoter. The term "promoter" is used herein to refer to a series of nucleic acid control sequences that direct transcription of a nucleic acid. As used herein, a promoter includes necessary nucleic acid sequences near the transcription start site, such as a TATA element in the case of a polymerase II type promoter. A promoter also optionally includes distal enhancer or repressor elements, which can be located as far as several thousand base pairs from the transcription start site. Other elements that may be present in an expression vector include elements that promote transcription (e.g., enhancers) and elements that terminate transcription (e.g., terminators).

[0080] "Recombinant" refers to a genetically modified polynucleotide, polypeptide, cell, tissue, or organism. For example, a recombinant polynucleotide (or a copy or complement of a recombinant polynucleotide) is one that has been engineered using well-known methods. A recombinant expression cassette comprising a promoter operably linked to a second polynucleotide (e.g., a coding sequence) can contain a promoter that is heterologous to the second polynucleotide as a result of human manipulation (e.g., by the methods described in Sambrook et al., Molecular Cloning - A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, (1989) or Current Protocols in Molecular Biology Volumes 1-3, John Wiley & Sons, Inc. (1994-1998)). A recombinant expression cassette (or expression vector) typically contains polynucleotides in a combination not found in nature. For example, human-engineered restriction sites or plasmid vector sequences can flank the promoter or separate it from other sequences. Recombinant proteins are those expressed from recombinant polynucleotides, and recombinant cells, tissues, and organisms are those that contain recombinant sequences (polynucleotides and / or polypeptides).

[0081] The terms "subject," "individual," and "patient" are used interchangeably herein and refer to a vertebrate, including a mammal or a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. Tissues, cells, and their progeny of biological entities obtained in vivo or cultured in vitro are also encompassed.

[0082] "Percent similarity" or "percent identity" in the context of polynucleotide or peptide sequences is determined by comparing two optimally aligned sequences over a comparison window, where the portion of the sequence in the comparison window may contain additions or deletions (i.e., gaps) due to optimal alignment of the two sequences, whereas the reference sequence contains no additions or deletions. The percentage is calculated by determining the number of positions where the same nucleotide or amino acid occurs in both sequences to determine the number of matched positions, dividing this number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to derive the percentage of similarity or identity (e.g., sequence similarity).

[0083] Polynucleotides or peptides are considered to be substantially similar if they contain a sequence having at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% or more similarity to a reference sequence when compared and aligned for maximum correspondence over a comparison window or designated region, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. With respect to polynucleotide sequences, this definition also refers to the complement of a test sequence.

[0084] When comparing sequences, typically, one sequence serves as a reference sequence, and test sequences are compared to it.When using sequence comparison algorithm, test sequences and reference sequences are input into computer, and if necessary, subsequence coordinates are designated, and sequence algorithm program parameters are designated.Default program parameters can be used, or alternative parameters can be designated.Then, sequence comparison algorithm calculates the sequence similarity percentage of test sequences and reference sequences based on program parameters.For nucleic acid and protein sequence comparison, the BLAST and BLAST 2.0 algorithms and default parameters discussed below are used.

[0085] Methods for aligning sequences for comparison are well known in the art. Optimal sequence alignment for comparison can be performed, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA, included in the Wisconsin Genetics Software Package (Genetics Computer Group, 575 Science Dr., Madison, WI)), or by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology (Ausubel et al., eds. 1995 Supplement)).

[0086] Further examples of algorithms suitable for determining percent sequence similarity are the BLAST algorithm and the BLAST 2.0 algorithm, which are described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. (1977) Nucleic Acids Res. 25: 3389-3402, respectively. Software for performing BLAST analysis is publicly available at the National Center for Biotechnology Information website ncbi.nlm.nih.gov. The algorithm first involves identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in a query sequence that, when aligned with words of the same length in a database sequence, match or meet a certain positive threshold score T. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. These word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always greater than 0) and N (penalty score for mismatching residues; always less than 0). The BLASTN program (for nucleotide sequences) uses as defaults a word size (W) of 28, an expectation (E) of 10, M=1, N=-2, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see, e.g., Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0087] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Nat'l. Acad. Sci. USA, 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability when comparing the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

[0088] As used herein, the term "administering" includes oral administration to a subject, topical contact, administration as a suppository, intravenous, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal, or subcutaneous administration. Administration can be by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration can include, for example, intravenous, intramuscular, intraarteriolar, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Other delivery modes include, but are not limited to, the use of liposome formulations, intravenous infusion, and transdermal patches.

[0089] The term "treating" refers to an approach to obtain beneficial or desired results, including, but not limited to, therapeutic benefit and / or prophylactic benefit. Therapeutic benefit refers to any therapeutically relevant improvement of, or any therapeutically relevant effect on, one or more diseases, conditions, or symptoms being treated. For prophylactic benefit, the compositions of the present disclosure can be administered to subjects at risk of developing a particular disease, condition, or symptom, or to subjects who are experiencing one or more physiological symptoms of the disease, even if the disease, condition, or symptom may not yet be apparent.

[0090] The term "effective amount" or "sufficient amount" refers to the amount of an agent that is sufficient to produce beneficial or desired results. A therapeutically effective amount can vary depending on one or more of the subject and disease state to be treated, the subject's weight and age, the severity of the disease state, and the mode of administration, which can be easily determined by those skilled in the art. The specific amount can vary depending on one or more of the specific agent selected, the host cell type, the location of the host cell in the subject, the administration regimen to be followed, whether it is administered in combination with other compounds, the timing of administration, and the physical delivery system used.

[0091] The term "pharmaceutically acceptable carrier" refers to a substance that aids in the administration of an active agent to a cell, organism, or subject. The term also refers to a carrier or excipient that can be included in the compositions of the present disclosure and that does not cause significant adverse toxicological effects in patients. Non-limiting examples of pharmaceutically acceptable carriers include water, NaCl, saline, lactated Ringer's solution, normal sucrose, normal glucose, cell culture media, and the like. Those skilled in the art will recognize that other pharmaceutical carriers are useful in the present disclosure.

[0092] Detailed Description of the Embodiments I. Genetically modified T cells Provided herein are genetically modified T cells that express (i) a class 2 VI-D CRISPR effector with RNA-guided RNA endonuclease activity and (ii) a guide array comprising multiple CRISPR-associated RNA (crRNA) molecules. In some embodiments, the crRNA molecules independently comprise a direct repeat sequence and a spacer sequence that binds to a target RNA. In some embodiments, the crRNA molecules bind to different target mRNAs. In some embodiments, the crRNA molecules bind to different regions of the same target mRNA.

[0093] In some embodiments, the modified T cell comprises a nucleic acid encoding a class 2 VI-D CRISPR effector with RNA-guided RNA endonuclease activity and / or a nucleic acid encoding or expressing a guide array comprising a plurality of CRISPR-associated RNA (crRNA) molecules. In some embodiments, the nucleic acid encoding (i), or the nucleic acid encoding or expressing (ii), or the nucleic acid encoding or expressing both (i) and (ii), is stably integrated into the genome of the T cell. In some embodiments, the nucleic acid encoding (i), or the nucleic acid encoding or expressing (ii), or the nucleic acid encoding or expressing both (i) and (ii) is not integrated into the genome of the T cell (e.g., transient transfection of the nucleic acid).

[0094] For a given Class 2 VI-D CRISPR effector, the direct repeat sequence typically comprises the same sequence in all crRNA molecules in the guide array. Thus, a guide array comprising N different crRNA molecules can comprise crRNA molecules with the same or substantially the same direct repeat sequence and N different spacer sequences. In some embodiments, the guide array comprises different spacer sequences that bind to different or distinct target mRNAs expressed by different target genes, e.g., 2 to N different target genes. In some embodiments, the guide array comprises different spacer sequences that bind to different sequences in the same target mRNA expressed by a given target gene (e.g., mRNA expressed by a TOX gene). The spacer sequences can bind to distinct, different, or non-overlapping sequences present in the same target mRNA, or to partially overlapping sequences in the same target mRNA. In some embodiments, the guide array comprises a combination of i) spacer sequences that bind to different or distinct target mRNAs expressed by different target genes and ii) spacer sequences that bind to different sequences in the same target mRNA expressed by a given target gene.

[0095] In some embodiments, the guide array is a multicistronic array containing multiple crRNA molecules. The guide array can contain two or more crRNA molecules. In some embodiments, the guide array contains 2-10 crRNA molecules. In some embodiments, the guide array contains more than 10 crRNA molecules. It will be understood that the number of crRNA molecules in the guide array is a function of the transcription elongation activity and / or processivity of the promoter used in the nucleic acid comprising the multicistronic array. In embodiments where the promoter has increased transcription elongation activity and / or processivity, longer guide arrays containing, for example, more than 10 crRNA molecules, are expected to be transcribed from the multicistronic array. In some embodiments, the promoter is an RNA polymerase (Pol) III promoter, such as the U6 promoter and the H1 promoter, or modified versions thereof. In some embodiments, the promoter is an RNA polymerase II promoter.

[0096] In some embodiments, the Class 2 VI-D CRISPR effector with RNA-guided RNA endonuclease activity is a Cas13 nuclease selected from the group consisting of Cas13a, Cas13b, 13bt1, Cas13bt2, Cas13c, Cas13d, RfxCas13d, Cas13e, and functional variants or orthologs thereof. In some embodiments, the Cas13 nuclease comprises an amino acid sequence having at least (e.g., at least the values ​​described below) about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence similarity or sequence identity to the amino acid sequence of a nuclease selected from the group consisting of Cas13a, Cas13b, 13bt1, Cas13bt2, Cas13c, Cas13d, RfxCas13d, and Cas13e. In some embodiments, the Cas13 nuclease comprises an amino acid sequence having at least (e.g., at least as much as described below) about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO:1.

[0097] In some embodiments, the Class 2 VI-D CRISPR effector with RNA-guided RNA endonuclease activity comprises a fusion protein comprising a destabilization domain (DD).

[0098] In some embodiments, the guide array comprises crRNA molecules that bind to mRNAs encoding proteins associated with T cell exhaustion. In some embodiments, the proteins associated with T cell exhaustion are selected from the group consisting of TOX, TOX2, NR4A1, NR4A2, NR4A3, TET2, IRF4, JUNB, BATF3, DHX37, FLI1, ZC3H12A, SOCS1, TCEB2, PDCD1 (PD-1), HAVCR2 (TIM3), LAG3, CTLA4, TIGIT, FAS, TRAC, CBLB, RASA2, PTPN2, CD5, ENTPD1, ​​CD46, B2M, and combinations thereof.

[0099] In some embodiments, the guide array comprises crRNA molecules that bind to mRNAs encoding proteins selected from the group consisting of CCNC, CDK8, CDK19, MED12, MED12L, MED13, MED13L, MED15, MED16, MED19, MED24, MED26, and combinations thereof.

[0100] II CAR-T cells In some embodiments, the engineered T cells further comprise a chimeric antigen receptor (CAR). CAR T cells express a receptor that binds to an antigen on a target cell, such as a tumor cell, and activate the function of natural T cells to target and kill the target cell. The therapeutic receptor of first-generation CAR T cells comprises an extracellular antigen-binding domain and an intracellular T cell activation domain. The therapeutic receptor of second-generation CAR T cells comprises both a costimulatory domain and a T cell activation domain on its intracellular side. The costimulatory domain improves the therapeutic response of T cells by increasing T cell proliferation or cytotoxicity. In some embodiments, the CAR comprises an antigen-binding scFv or nanobody. In some embodiments, the CAR comprises intramembrane signaling domains CD28 and CD3ζ (CD247). In some embodiments, the CAR comprises intramembrane signaling domains 4-1BB (CD137) and CD3ζ.

[0101] In some embodiments, the CAR binds to an antigen expressed by a tumor. In some embodiments, the CAR binds to an antigen expressed by a tumor, such as Her-2, B7-H3, GPC2, GD2, CD19, CD20, CD22, MAGE, BAGE, CAGE, GAGE, HAGE, LAGE, PAGE, PRAME, NY-ESO-1, NY-SEO-1, tyrosinase, Melan-A / MART, gpl00, TRP-1, TRP-2, CD30, EGFR, EGFRvIII, FAP, CD33 , CD123, PD-L1, IGF1R, CD4, CSPG4, B7-H4, NKG2D, CS1, CD138, EpCAM, EBNA3C, GPA7, CD244, CA-125, ETA, CEA, CD52, MUC5AC, c-Met, FAB, WT-1, PSMA, AFP, BCMA, mesothelin, GPC3, MUC1, and CTAG1B.

[0102] As is known in the art, prolonged intracellular signaling by CARs can lead to T cell exhaustion, resulting in dysfunctional CAR T cells. Similarly, prolonged intracellular signaling by endogenous T cell receptors (TCRs) can also lead to T cell exhaustion, resulting in reduced anti-tumor activity. T cell exhaustion is characterized by increased expression of exhaustion-related genes. The modified T cells of the present disclosure can be used to reduce the expression of genes whose expression increases during T cell exhaustion, thereby enhancing the anti-tumor activity of the modified T cells. Thus, in some embodiments, intracellular signaling by CARs or endogenous TCRs upregulates T cell exhaustion markers in control T cells compared to the modified T cells of the present disclosure. In some embodiments, the control T cells are T cells that do not express (i) a class 2 VI-D CRISPR effector with RNA-guided RNA endonuclease activity; and / or (ii) a guide array comprising multiple CRISPR-associated RNA (crRNA) molecules that bind to target RNAs expressed by exhaustion genes. In some embodiments, the control T cells are CAR T cells that do not express (i) a class 2 VI-D CRISPR effector with RNA-guided RNA endonuclease activity; and / or (ii) a guide array comprising multiple CRISPR-associated RNA (crRNA) molecules that bind to a target RNA expressed by an exhausted gene. In some embodiments, the control T cells are transfected with or express a non-targeting control guide array comprising crRNA molecules that do not bind to a target RNA.

[0103] In some embodiments, the exhaustion markers are selected from LAG3, PDCD1 (PD-1), HAVCR2 (TIM3), CTLA4, and ENTPD1, ​​or a combination thereof. In some embodiments, the guide array comprises crRNA molecules that bind to mRNA encoding LAG3, PDCD1 (PD-1), HAVCR2 (TIM3), CTLA4, and ENTPD1, ​​or a combination thereof.

[0104] In some embodiments, the modified T cells of the present disclosure can comprise a "safety switch" to downregulate the expression of a CAR expressed by a CAR T cell in a subject or patient undergoing CAR T cell therapy. Thus, in some embodiments, the guide array comprises a crRNA molecule that binds to the mRNA expressed by the CAR, thereby reducing CAR expression. In some embodiments, the crRNA molecule binds to a region of the mRNA that encodes the signaling elements of the CAR, such as the intramembrane signaling domains 4-1BB, CD28, and CD3ζ, or endogenous TCRs, such as ZAP70 and LCK.

[0105] In some embodiments, the T cells are primary T cells isolated from a subject. In some embodiments, the T cells are human T cells. In some embodiments, the T cells are primary human T cells.

[0106] III. A system for multiplexed transcriptome control Also provided herein are systems for multiplexed transcriptome control.These systems include: (i) an expression cassette comprising the nucleic acid sequence encoding the class 2 VI-D CRISPR effector with RNA-guided RNA endonuclease activity of the present disclosure; and (ii) an expression cassette comprising the nucleic acid sequence encoding the guide array of the present disclosure.

[0107] In some embodiments, the crRNA molecule comprises a direct repeat sequence and a spacer sequence that binds to the target mRNA. In some embodiments, the crRNA molecule binds to different target mRNAs or different regions of the same target mRNA.

[0108] In some embodiments, the Class 2 VI-D CRISPR effector with RNA-guided RNA endonuclease activity is a Cas13 nuclease selected from the group consisting of Cas13a, Cas13b, 13bt1, Cas13bt2, Cas13c, Cas13d, RfxCas13d, Cas13e, and functional variants or orthologs thereof. In some embodiments, the Cas13 nuclease comprises an amino acid sequence having at least (e.g., at least the values ​​described below) about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence similarity or sequence identity to the amino acid sequence of a nuclease selected from the group consisting of Cas13a, Cas13b, 13bt1, Cas13bt2, Cas13c, Cas13d, RfxCas13d, and Cas13e. In some embodiments, the Cas13 nuclease comprises an amino acid sequence having at least (e.g., at least as much as described below) about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO:1.

[0109] In some embodiments, the Class 2 VI-D CRISPR effector with RNA-guided RNA endonuclease activity comprises a fusion protein comprising a destabilization domain (DD).

[0110] In some embodiments, the guide array comprises crRNA molecules that bind to mRNAs encoding proteins associated with T cell exhaustion. In some embodiments, the proteins associated with T cell exhaustion are selected from the group consisting of TOX, TOX2, NR4A1, NR4A2, NR4A3, TET2, IRF4, JUNB, BATF3, DHX37, FLI1, ZC3H12A, SOCS1, TCEB2, PDCD1 (PD-1), HAVCR2 (TIM3), LAG3, CTLA4, TIGIT, FAS, TRAC, CBLB, RASA2, PTPN2, CD5, ENTPD1, ​​CD46, B2M, and combinations thereof.

[0111] In some embodiments, these systems can further comprise T cells or primary T cells. In some embodiments, the T cells are primary T cells isolated from a subject. In some embodiments, the T cells are human T cells. In some embodiments, the T cells are primary human T cells. In some embodiments, the T cells are transformed or transduced with the expression cassette of the system. In some embodiments, the T cells are transformed or transduced with the expression cassettes of (i) and (ii) above, so that the T cells express the class 2 VI-D CRISPR effector with RNA-guided RNA endonuclease activity of the present disclosure and the guide array of the present disclosure.

[0112] In some embodiments, the T cells are engineered T cells comprising a chimeric antigen receptor (CAR). In some embodiments, the CAR binds to an antigen expressed by a tumor. In some embodiments, the CAR binds to Her-2, B7-H3, GPC2, GD2, CD19, CD20, CD22, MAGE, BAGE, CAGE, GAGE, HAGE, LAGE, PAGE, PRAME, NY-ESO-1, NY-SEO-1, tyrosinase, Melan-A / MART, gpl00, TRP-1, TRP-2, CD30, EGFR, EGFRvIII, FAP, CD33 , CD123, PD-L1, IGF1R, CD4, CSPG4, B7-H4, NKG2D, CS1, CD138, EpCAM, EBNA3C, GPA7, CD244, CA-125, ETA, CEA, CD52, MUC5AC, c-Met, FAB, WT-1, PSMA, AFP, BCMA, mesothelin, GPC3, MUC1, and CTAG1B.

[0113] In some embodiments, intracellular signaling by CAR or endogenous TCR upregulates T cell exhaustion markers in control T cells. In some embodiments, the control T cells are T cells that do not express (i) a class 2 VI-D CRISPR effector with RNA-guided RNA endonuclease activity; and / or (ii) a guide array comprising multiple CRISPR-associated RNA (crRNA) molecules. In some embodiments, the control T cells are CAR T cells that do not express (i) a class 2 VI-D CRISPR effector with RNA-guided RNA endonuclease activity; and / or (ii) a guide array comprising multiple CRISPR-associated RNA (crRNA) molecules. In some embodiments, the control T cells are transfected with or express a non-targeting control guide array comprising crRNA molecules that do not bind to target RNA.

[0114] In some embodiments, the exhaustion markers are selected from LAG3, PDCD1 (PD-1), HAVCR2 (TIM3), CTLA4, and ENTPD1, ​​or a combination thereof. In some embodiments, the guide array comprises crRNA molecules that bind to mRNA encoding LAG3, PDCD1 (PD-1), HAVCR2 (TIM3), CTLA4, and ENTPD1, ​​or a combination thereof.

[0115] In some embodiments, the T cells contain a "safety switch" to downregulate the expression of the CAR expressed by the CAR T cells in subjects or patients receiving CAR T cell therapy. Thus, in some embodiments, the guide array contains a crRNA molecule that binds to the mRNA expressed by the CAR, thereby reducing the expression of the CAR. In some embodiments, the crRNA molecule binds to regions of the mRNA that encode the signaling elements of the CAR, such as the intramembrane signaling domains 4-1BB, CD28, and CD3ζ, or endogenous TCRs, such as ZAP70 and LCK.

[0116] In some embodiments, the T cells are primary T cells isolated from a subject. In some embodiments, the T cells are human T cells. In some embodiments, the T cells are primary human T cells.

[0117] IV Fusion Protein In some aspects, the class 2 VI-D CRISPR effector with RNA-guided RNA endonuclease activity comprises a fusion protein comprising a destabilization domain (DD). In some embodiments, the DD comprises the E. coli dihydrofolate reductase DD linked to the C-terminus of the class 2 VI-D CRISPR effector with RNA-guided RNA endonuclease activity.

[0118] In some embodiments, the Class 2 VI-D CRISPR effector with RNA-guided RNA endonuclease activity is a Cas13 nuclease selected from the group consisting of Cas13a, Cas13b, 13bt1, Cas13bt2, Cas13c, Cas13d, RfxCas13d, Cas13e, and functional variants or orthologs thereof. In some embodiments, the Cas13 nuclease comprises an amino acid sequence having at least (e.g., at least the values ​​described below) about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence similarity or sequence identity to the amino acid sequence of a nuclease selected from the group consisting of Cas13a, Cas13b, 13bt1, Cas13bt2, Cas13c, Cas13d, RfxCas13d, and Cas13e. In some embodiments, the Cas13 nuclease comprises an amino acid sequence having at least (e.g., at least as much as described below) about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO:1.

[0119] In some embodiments, the DD domain comprises an amino acid sequence having at least (e.g., at least as much as described below) about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence similarity or identity to SEQ ID NO: 5.

[0120] In some embodiments, the fusion protein comprises a linker sequence connecting the Class 2 VI-D CRISPR effector and the DD. In some embodiments, the linker sequence is Includes TIFF2025529871000002.tif4128.

[0121] V. Nucleic Acids and Vectors Also provided are nucleic acids encoding the class 2 VI-D CRISPR effector, guide array, CAR, and / or fusion protein of the present disclosure with RNA-guided RNA endonuclease activity.These nucleic acids can include plasmids and vectors that contain the nucleic acids encoding i) any class 2 VI-D CRISPR effector with RNA-guided RNA endonuclease activity disclosed herein, ii) any CAR disclosed herein, iii) guide array disclosed herein, and / or iv) fusion protein of the present disclosure.In some embodiments, the class 2 VI-D CRISPR effector with RNA-guided RNA endonuclease activity is a Cas13 nuclease selected from the group consisting of Cas13a, Cas13b, 13bt1, Cas13bt2, Cas13c, Cas13d, RfxCas13d, Cas13e, and functional variants thereof.In some embodiments, the guide array is a multicistronic array that contains multiple crRNA molecules. In some embodiments, the guide array comprises 2 to 10 or more crRNA molecules. In some embodiments, the guide array comprises crRNA molecules that bind to mRNAs encoding proteins associated with T cell exhaustion. In some embodiments, the proteins associated with T cell exhaustion are selected from the group consisting of TOX, TOX2, NR4A1, NR4A2, NR4A3, TET2, IRF4, JUNB, BATF3, DHX37, FLI1, ZC3H12A, SOCS1, TCEB2, PDCD1 (PD-1), HAVCR2 (TIM3), LAG3, CTLA4, TIGIT, FAS, TRAC, CBLB, RASA2, PTPN2, CD5, ENTPD1, ​​CD46, B2M, and combinations thereof.

[0122] In some embodiments, the vector is an expression vector or comprises an expression cassette. In some embodiments, the vector comprises sequences that control transcription, translation, and / or RNA stability, such as an enhancer, 5'UTR, promoter, polyA sequence, 3'UTR, and / or nuclear localization sequence. It will be understood that sequences that control transcription, translation, and / or RNA stability can be operably linked to other sequences in the vector. For example, the promoter can be operably linked to an open reading frame encoding a class 2 VI-D CRISPR effector, CAR, and / or fusion protein with RNA-guided RNA endonuclease activity of the present disclosure. In some embodiments, the promoter is operably linked to a nucleic acid that expresses the guide array disclosed herein. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an inducible promoter.

[0123] In some embodiments, the vector is a viral vector.The viral vector can be any viral vector suitable for delivering nucleic acid to cells.The vector can be, for example, an adeno-associated virus (AAV) vector, an adenovirus vector, a retrovirus vector, a lentivirus vector, or a herpes simplex virus (HSV) vector.In some embodiments, the vector is a lentivirus vector.

[0124] VI Guide Array Also provided are guide arrays. The guide arrays comprise nucleic acids (polynucleotides) comprising direct repeat sequences and spacer sequences. The guide arrays are typically expressed from a plasmid or vector comprising a promoter operably linked to the guide array. The direct repeat sequences can form a stem-loop structure, while the spacer sequences comprise sequences complementary to the target RNA in the cell. The spacer sequences bind to the target RNA by RNA-RNA hybridization. Exemplary, non-limiting spacer sequences are provided in Tables 1 and 2.

[0125] In some embodiments, the guide array is a multicistronic array containing multiple crRNA molecules. The guide array can contain two or more crRNA molecules. In some embodiments, the guide array contains 2-10 crRNA molecules. In some embodiments, the guide array contains more than 10 crRNA molecules. It will be understood that the number of crRNA molecules in the guide array is a function of the transcription elongation activity and / or processivity of the promoter used in the nucleic acid comprising the multicistronic array. In embodiments where the promoter has increased transcription elongation activity and / or processivity, longer guide arrays containing, for example, more than 10 crRNA molecules, are expected to be transcribed from the multicistronic array. In some embodiments, the promoter is an RNA polymerase (Pol) III promoter, such as the U6 promoter and the H1 promoter, or modified versions thereof. In some embodiments, the promoter is an RNA polymerase II promoter.

[0126] As mentioned above, the direct repeat sequence typically contains the same sequence in all crRNA molecules in a guide array. Thus, a guide array containing N different crRNA molecules can contain crRNA molecules with the same or substantially the same direct repeat sequence and N different spacer sequences. In some embodiments, a guide array contains different spacer sequences that bind to different or distinct target mRNAs expressed by different target genes, e.g., 2 to N different target genes. In some embodiments, a guide array contains different spacer sequences that bind to different sequences in the same target mRNA expressed by a given target gene (e.g., mRNA expressed by a TOX gene). The spacer sequences can bind to distinct, different, or non-overlapping sequences present in the same target mRNA, or to partially overlapping sequences in the same target mRNA. In some embodiments, a guide array contains a combination of i) spacer sequences that bind to different or distinct target mRNAs expressed by different target genes and ii) spacer sequences that bind to different sequences in the same target mRNA expressed by a given target gene.

[0127] In some embodiments, the direct repeat sequence is TIFF2025529871000003.tif4128. In some embodiments, the direct repeat sequence comprises: Includes TIFF2025529871000004.tif4135.

[0128] VII. Methods for Producing Modified T Cells Also provided is a method for producing modified T cells expressing the class 2 VI-D CRISPR effector and guide array of the present disclosure, which method can include transfecting or transducing T cells with one or more expression vectors encoding the class 2 VI-D CRISPR effector with RNA-guided RNA endonuclease activity and / or expressing a guide array comprising one or more CRISPR-associated RNA (crRNA) molecules.

[0129] In some embodiments, T cells are transfected or transduced with two different vectors: a first vector containing a nucleic acid sequence encoding a class 2 VI-D CRISPR effector with RNA-guided RNA endonuclease activity, and a second vector containing a nucleic acid sequence encoding a guide array comprising multiple CRISPR-associated RNA (crRNA) molecules. In some embodiments, co-transduction of T cells with two different vectors increases targeting efficiency compared to transduction with a single vector expressing both the class 2 VI-D CRISPR effector and the guide array. In some embodiments, T cells are transduced with a first vector expressing a class 2 VI-D CRISPR effector prior to transduction with a second vector containing a guide array. In some embodiments, T cells are transduced with a first vector expressing a class 2 VI-D CRISPR effector approximately 12 to 36 hours prior to transduction with a second vector containing a guide array.

[0130] In some embodiments, the guide array is a multicistronic array containing multiple crRNA molecules. The guide array can contain two or more crRNA molecules. In some embodiments, the guide array contains 2-10 crRNA molecules. In some embodiments, the guide array contains more than 10 crRNA molecules. It will be understood that the number of crRNA molecules in the guide array is a function of the transcription elongation activity and / or processivity of the promoter used in the nucleic acid comprising the multicistronic array. In embodiments where the promoter has increased transcription elongation activity and / or processivity, longer guide arrays containing, for example, more than 10 crRNA molecules, are expected to be transcribed from the multicistronic array. In some embodiments, the promoter is an RNA polymerase (Pol) III promoter, such as the U6 promoter and the H1 promoter, or modified versions thereof. In some embodiments, the promoter is an RNA polymerase II promoter.

[0131] In some embodiments, the Class 2 VI-D CRISPR effector with RNA-guided RNA endonuclease activity is a Cas13 nuclease selected from the group consisting of Cas13a, Cas13b, 13bt1, Cas13bt2, Cas13c, Cas13d, RfxCas13d, Cas13e, and functional variants or orthologs thereof. In some embodiments, the Cas13 nuclease comprises an amino acid sequence having at least (e.g., at least the values ​​described below) about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence similarity or sequence identity to the amino acid sequence of a nuclease selected from the group consisting of Cas13a, Cas13b, 13bt1, Cas13bt2, Cas13c, Cas13d, RfxCas13d, and Cas13e. In some embodiments, the Cas13 nuclease comprises an amino acid sequence having at least (e.g., at least as much as described below) about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO:1.

[0132] In some embodiments, the guide array comprises crRNA molecules that bind to mRNAs encoding proteins associated with T cell exhaustion. In some embodiments, the proteins associated with T cell exhaustion are selected from the group consisting of TOX, TOX2, NR4A1, NR4A2, NR4A3, TET2, IRF4, JUNB, BATF3, DHX37, FLI1, ZC3H12A, SOCS1, TCEB2, PDCD1 (PD-1), HAVCR2 (TIM3), LAG3, CTLA4, TIGIT, FAS, TRAC, CBLB, RASA2, PTPN2, CD5, ENTPD1, ​​CD46, B2M, and combinations thereof.

[0133] In some embodiments, the T cells are further or optionally transduced with a third vector comprising a nucleic acid sequence encoding a CAR. The T cells can be transduced with a first vector encoding a Class 2 VI-D CRISPR effector of the present disclosure and a third vector encoding a CAR at about the same time, e.g., simultaneously or contemporaneously, or within minutes or hours (e.g., 1-8 hours) of each other. In some embodiments, the T cells are transduced with the first and third vectors, and then 12-36 hours later, transformed with a second vector comprising a guide array.

[0134] In some embodiments, the first, second, and / or third vector is stably integrated into the genome of the host T cell. In some embodiments, the first, second, and / or third vector is not integrated into the genome of the T cell (e.g., transient transfection of the vector).

[0135] In some embodiments, the CAR binds to an antigen expressed by a tumor. In some embodiments, the CAR binds to an antigen expressed by a tumor, such as Her-2, B7-H3, GPC2, GD2, CD19, CD20, CD22, MAGE, BAGE, CAGE, GAGE, HAGE, LAGE, PAGE, PRAME, NY-ESO-1, NY-SEO-1, tyrosinase, Melan-A / MART, gpl00, TRP-1, TRP-2, CD30, EGFR, EGFRvIII, FAP, CD33 , CD123, PD-L1, IGF1R, CD4, CSPG4, B7-H4, NKG2D, CS1, CD138, EpCAM, EBNA3C, GPA7, CD244, CA-125, ETA, CEA, CD52, MUC5AC, c-Met, FAB, WT-1, PSMA, AFP, BCMA, mesothelin, GPC3, MUC1, and CTAG1B.

[0136] In some embodiments, the CAR binds to a tumor antigen described herein.

[0137] In some embodiments, intracellular signaling by CAR or endogenous TCR upregulates T cell exhaustion markers in control T cells. In some embodiments, the control T cells are T cells that do not express (i) a class 2 VI-D CRISPR effector with RNA-guided RNA endonuclease activity; and / or (ii) a guide array comprising a plurality of CRISPR-associated RNA (crRNA) molecules that bind to the RNA expressed by exhaustion genes. In some embodiments, the control T cells are CAR T cells that do not express (i) a class 2 VI-D CRISPR effector with RNA-guided RNA endonuclease activity; and / or (ii) a guide array comprising a plurality of CRISPR-associated RNA (crRNA) molecules that bind to the RNA expressed by exhaustion genes. In some embodiments, the control T cells are transfected with or express a non-targeting control guide array comprising crRNA molecules that do not bind to target RNA.

[0138] In some embodiments, the exhaustion markers are selected from LAG3, PDCD1 (PD-1), HAVCR2 (TIM3), CTLA4, and ENTPD1, ​​or a combination thereof. In some embodiments, the guide array comprises crRNA molecules that bind to mRNA encoding LAG3, PDCD1 (PD-1), HAVCR2 (TIM3), CTLA4, and ENTPD1, ​​or a combination thereof.

[0139] In some embodiments, the T cells contain a "safety switch" to downregulate the expression of the CAR expressed by the CAR T cells in subjects or patients receiving CAR T cell therapy. Thus, in some embodiments, the guide array contains a crRNA molecule that binds to the mRNA expressed by the CAR, thereby reducing the expression of the CAR. In some embodiments, the crRNA molecule binds to regions of the mRNA that encode the signaling elements of the CAR, such as the intramembrane signaling domains 4-1BB, CD28, and CD3ζ, or endogenous TCRs, such as ZAP70 and LCK.

[0140] VIII. Methods for Regulating Gene Expression in T Cells In some aspects, the present disclosure provides a method for controlling gene expression in T cells. This method offers the advantage of adjustable and reversible control of the T cell transcriptome. In some embodiments, T cells express a guide array comprising a fusion protein of the present disclosure and a crRNA molecule, wherein the crRNA molecule comprises a direct repeat sequence and a spacer sequence that binds to a target mRNA. In some embodiments, the fusion protein comprises a class 2 VI-D CRISPR effector with RNA-guided RNA endonuclease activity and a destabilization domain (DD). In some embodiments, the DD comprises an E. coli dihydrofolate reductase DD linked to the C-terminus of a class 2 VI-D CRISPR effector with RNA-guided RNA endonuclease activity.

[0141] In steady state, the fusion protein of class 2 VI-D CRISPR effector and DD is rapidly degraded by proteasome due to disordered DD.Therefore, in some embodiments, the fusion protein is degraded in T cells in the absence of compound.In the presence of suitable compound, the tertiary structure of DD is stabilized, allowing the fusion protein of class 2 VI-D CRISPR effector and DD to bind to and target RNA.Furthermore, the present inventors have found that T cells expressing fusion protein and guide RNA can conditionally suppress target gene expression in the presence of compound, while in the absence of compound, target gene expression is not inhibited.Therefore, in some embodiments, the expression of target gene is reduced in the presence of compound compared to the expression of target gene in the absence of compound.

[0142] The present inventors have also found that target gene expression in T cells can be regulated by controlling the dose of a compound that stabilizes the DD of a fusion protein. The regulation of target gene expression was observed to exhibit a sigmoidal dose-response curve in the range of about 1 to 100 nM, as described in the Examples and Figure 4. Thus, in some embodiments, expression of one or more target genes is regulated by the compound in a dose-dependent manner.

[0143] The present inventors have further found that the suppression of target gene expression in T cells expressing a fusion protein and a guide RNA that binds to a target mRNA can be relieved by removing from the culture medium a compound that stabilizes the DD of the fusion protein, resulting in a rapid increase in target gene expression upon removal of the compound from the culture medium. Thus, in some embodiments, target gene expression increases in T cells after removal of the compound, thereby reversibly controlling target gene expression.

[0144] In some embodiments, the method comprises: (a) transducing T cells with (i) an expression vector comprising a nucleic acid sequence encoding a fusion protein of the present disclosure; and (ii) an expression vector comprising a nucleic acid sequence encoding a guide array comprising a crRNA molecule, the crRNA molecule comprising a direct repeat sequence and a spacer sequence that binds to a target mRNA; and (b) contacting the T cells with a compound that binds to and stabilizes a DD, wherein expression of a target gene is decreased in the presence of the compound compared to expression of the target gene in the absence of the compound.

[0145] In some embodiments, the compound is trimethoprim (TMP).

[0146] In some embodiments, the Class 2 VI-D CRISPR effector with RNA-guided RNA endonuclease activity is a Cas13 nuclease selected from the group consisting of Cas13a, Cas13b, 13bt1, Cas13bt2, Cas13c, Cas13d, RfxCas13d, Cas13e, and functional variants or orthologs thereof. In some embodiments, the Cas13 nuclease comprises an amino acid sequence having at least (e.g., at least the values ​​described below) about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence similarity or sequence identity to the amino acid sequence of a nuclease selected from the group consisting of Cas13a, Cas13b, 13bt1, Cas13bt2, Cas13c, Cas13d, RfxCas13d, and Cas13e. In some embodiments, the Cas13 nuclease comprises an amino acid sequence having at least (e.g., at least as much as described below) about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO:1.

[0147] In some embodiments, the guide array comprises one or more crRNA molecules that bind to different target mRNAs or different regions of the same target mRNA. In some embodiments, the method can control the expression of multiple target genes in T cells. In some embodiments, the method can simultaneously suppress the expression of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more target genes. In some embodiments, the method can simultaneously suppress the expression of one or more target genes associated with exhaustion in T cells. In some embodiments, the method is capable of simultaneously suppressing expression of proteins selected from the group consisting of TOX, TOX2, NR4A1, NR4A2, NR4A3, TET2, IRF4, JUNB, BATF3, DHX37, FLI1, ZC3H12A, SOCS1, TCEB2, PDCD1 (PD-1), HAVCR2 (TIM3), LAG3, CTLA4, TIGIT, FAS, TRAC, CBLB, RASA2, PTPN2, CD5, ENTPD1, ​​CD46, B2M, and combinations thereof.

[0148] IX. Methods for screening to identify regulators of T cell activity. In another aspect, the present disclosure provides a method for screening to identify regulators of T cell activity. This method allows systematic gene perturbation in primary T cells. In some embodiments, the method comprises the following steps: In T cells or populations of T cells (i) a class 2 VI-D CRISPR effector with RNA-guided RNA endonuclease activity; and (ii) A library of guide arrays, each guide array comprising one or more CRISPR-associated RNA (crRNA) molecules, each crRNA molecule comprising a direct repeat sequence and a spacer sequence that binds to a target RNA, and each crRNA molecule binding to different target mRNAs or different regions of the same target mRNA. expressing the culturing the T cells to produce a clonal population of expanded T cells; and Determining whether the guide array is enriched or depleted in the expanded clonal population of T cells. wherein, when the guide array is enriched, the target mRNA encodes a negative regulator of T cell activity, or when the guide array is depleted, the target mRNA encodes a positive regulator of T cell activity.

[0149] In some embodiments, the T cell activity is T cell proliferation, increased cytokine secretion, or increased tumor cell killing.

[0150] In some embodiments, T cells comprising enriched guide arrays have an effector memory phenotype, and T cells comprising depleted guide arrays have a central memory phenotype or a stem cell memory phenotype. In some embodiments, determining whether a guide array is enriched or depleted in a clonal population of expanded T cells comprises sequencing guide RNAs present in the T cells.

[0151] In some embodiments, each guide array in the library comprises a crRNA molecule that binds to an mRNA encoding a protein associated with T cell exhaustion. In some embodiments, the protein associated with T cell exhaustion is selected from the group consisting of TOX, TOX2, NR4A1, NR4A2, NR4A3, TET2, IRF4, JUNB, BATF3, DHX37, FLI1, ZC3H12A, SOCS1, TCEB2, PDCD1 (PD-1), HAVCR2 (TIM3), LAG3, CTLA4, TIGIT, FAS, TRAC, CBLB, RASA2, PTPN2, and combinations thereof.

[0152] In some embodiments, the library of guide arrays comprises one or more individual guide arrays, including multicistronic arrays containing multiple crRNA molecules. In some embodiments, an individual guide array comprises 2-10 crRNA molecules. In some embodiments, an individual guide array comprises a single pair of crRNA molecules.

[0153] In some embodiments, the Class 2 VI-D CRISPR effector with RNA-guided RNA endonuclease activity is a Cas13 nuclease selected from the group consisting of Cas13a, Cas13b, 13bt1, Cas13bt2, Cas13c, Cas13d, RfxCas13d, Cas13e, and functional variants thereof. In some embodiments, the Cas13 nuclease comprises an amino acid sequence having at least (e.g., at least the values ​​described below) about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence similarity or sequence identity to the amino acid sequence of a nuclease selected from the group consisting of Cas13a, Cas13b, 13bt1, Cas13bt2, Cas13c, Cas13d, RfxCas13d, and Cas13e. In some embodiments, the Cas13 nuclease comprises an amino acid sequence having at least (e.g., at least as much as described below) about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO:1.

[0154] In some embodiments, the T cells further express a chimeric antigen receptor (CAR). Expression of a CAR can result in tonic signaling that leads to both T cell expansion and subsequent dysfunction.

[0155] In some embodiments, the CAR binds to an antigen expressed by a tumor. In some embodiments, the CAR binds to an antigen expressed by a tumor, such as Her-2, B7-H3, GPC2, GD2, CD19, CD20, CD22, MAGE, BAGE, CAGE, GAGE, HAGE, LAGE, PAGE, PRAME, NY-ESO-1, NY-SEO-1, tyrosinase, Melan-A / MART, gpl00, TRP-1, TRP-2, CD30, EGFR, EGFRvIII, FAP, CD33 , CD123, PD-L1, IGF1R, CD4, CSPG4, B7-H4, NKG2D, CS1, CD138, EpCAM, EBNA3C, GPA7, CD244, CA-125, ETA, CEA, CD52, MUC5AC, c-Met, FAB, WT-1, PSMA, AFP, BCMA, mesothelin, GPC3, MUC1, and CTAG1B.

[0156] Methods for increasing proliferation of XT cells In another aspect, the present disclosure provides a method for increasing T cell proliferation that allows for systematic genetic perturbation in primary T cells to identify novel combinations of putative exhaustion-associated genes that control the proliferation of dysfunctional T cells.

[0157] In some embodiments, the method comprises the steps of: (i) an expression vector comprising a nucleic acid sequence encoding a class 2 VI-D CRISPR effector with RNA-guided RNA endonuclease activity; and (ii) An expression vector comprising a nucleic acid sequence encoding a guide array comprising one or more crRNA molecules, wherein the crRNA molecules independently comprise a direct repeat sequence and a spacer sequence that binds to a target mRNA, and the crRNA molecules bind to different target mRNAs or different regions of the same target mRNA. Transducing T cells by Here, proliferation of the T cells is increased relative to control T cells expressing a non-targeting control guide array comprising one or more crRNA molecules that do not bind to the target mRNA of (ii).

[0158] In some embodiments, the guide array comprises crRNA molecules that bind to mRNAs encoding proteins associated with T cell exhaustion. In some embodiments, the proteins associated with T cell exhaustion are selected from the group consisting of FAS, ZC3H12A, CTLA4, SOCS1, CBLB, PD-1, TOX, TOX2, FLI1, CD5, CD39, CD46, TRAC, B2M, JUNB, IRF4, PDCD1 (PD-1), HAVCR2 (TIM3), DHX37, and combinations thereof.

[0159] In some embodiments, the guide array is or is included in a library of guide arrays, where the library of guide arrays includes one or more individual guide arrays. An individual guide array can include a multicistronic array including one or more crRNA molecules. In some embodiments, the guide array includes a multicistronic array including multiple crRNA molecules. In some embodiments, the guide array includes 2-10 or more crRNA molecules. In some embodiments, an individual guide array includes a pair (two) of crRNA molecules.

[0160] In some embodiments, the Class 2 VI-D CRISPR effector with RNA-guided RNA endonuclease activity is a Cas13 nuclease selected from the group consisting of Cas13a, Cas13b, 13bt1, Cas13bt2, Cas13c, Cas13d, RfxCas13d, Cas13e, and functional variants thereof. In some embodiments, the Cas13 nuclease comprises an amino acid sequence having at least (e.g., at least the values ​​described below) about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence similarity or sequence identity to the amino acid sequence of a nuclease selected from the group consisting of Cas13a, Cas13b, 13bt1, Cas13bt2, Cas13c, Cas13d, RfxCas13d, and Cas13e. In some embodiments, the Cas13 nuclease comprises an amino acid sequence having at least (e.g., at least as much as described below) about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO:1.

[0161] In some embodiments, the method further comprises (iii) transducing the T cell with an expression vector comprising a nucleic acid sequence encoding the CAR. In some embodiments, the CAR binds to an antigen expressed by the tumor. In some embodiments, the CAR binds to an antigen selected from the group consisting of Her-2, B7-H3, GPC2, GD2, CD19, CD20, CD22, MAGE, BAGE, CAGE, GAGE, HAGE, LAGE, PAGE, PRAME, NY-ESO-1, NY-SEO-1, tyrosinase, Melan-A / MART, gplOO, TRP-1, TRP-2, CD30, EGFR, FAP, CD33, CD123, PD-L1, IGF1R, CD4, CSPG4, B7-H4, NKG2D, CS1, CD138, EpCAM, EBNA3C, GPA7, CD244, CA-125, ETA, CEA, CD52, MUC5AC, c-Met, EGFR, FAB, WT-1, PSMA, AFP, CEA, and CTAG1B.

[0162] XI. Methods for Treating Tumors The present disclosure also provides a method for treating cancer or tumor in a subject or patient who needs treatment.In some embodiments, the method comprises administering an effective amount of genetically modified T cells of the present disclosure to the subject, wherein the modified T cells kill tumor cells in the subject, thereby treating the tumor.In some embodiments, the modified T cells express or comprise (i) the class 2 VI-D CRISPR effector with RNA-guided RNA endonuclease activity of the present disclosure, (ii) the guide array of the present disclosure, or (iii) both (i) and (ii).

[0163] In some embodiments, the modified T cells comprise: (i) an expression vector comprising a nucleic acid sequence encoding a class 2 VI-D CRISPR effector with RNA-guided RNA endonuclease activity; and (ii) an expression vector comprising a nucleic acid sequence encoding a guide array comprising one or more crRNA molecules, wherein the crRNA molecules independently comprise a direct repeat sequence and a spacer sequence that binds to a target mRNA, and the crRNA molecules bind to different target mRNAs or different regions of the same target mRNA.

[0164] In some embodiments, the guide array comprises crRNA molecules that bind to mRNAs encoding proteins associated with T cell exhaustion. In some embodiments, the proteins associated with T cell exhaustion are selected from the group consisting of FAS, ZC3H12A, CTLA4, SOCS1, CBLB, PD-1, TOX, TOX2, FLI1, CD5, CD39, CD46, TRAC, B2M, JUNB, IRF4, PDCD1 (PD-1), HAVCR2 (TIM3), DHX37, and combinations thereof.

[0165] In some embodiments, the Class 2 VI-D CRISPR effector with RNA-guided RNA endonuclease activity is a Cas13 nuclease selected from the group consisting of Cas13a, Cas13b, 13bt1, Cas13bt2, Cas13c, Cas13d, RfxCas13d, Cas13e, and functional variants thereof. In some embodiments, the Cas13 nuclease comprises an amino acid sequence having at least (e.g., at least the values ​​described below) about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence similarity or sequence identity to the amino acid sequence of a nuclease selected from the group consisting of Cas13a, Cas13b, 13bt1, Cas13bt2, Cas13c, Cas13d, RfxCas13d, and Cas13e. In some embodiments, the Cas13 nuclease comprises an amino acid sequence having at least (e.g., at least as much as described below) about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO:1.

[0166] In some embodiments of the method, the modified T cell further comprises a modified CAR. In some embodiments, the CAR binds to an antigen expressed by the tumor. In some embodiments, the CAR binds to an antigen selected from the group consisting of Her-2, B7-H3, GPC2, GD2, CD19, CD20, CD22, MAGE, BAGE, CAGE, GAGE, HAGE, LAGE, PAGE, PRAME, NY-ESO-1, NY-SEO-1, tyrosinase, Melan-A / MART, gplOO, TRP-1, TRP-2, CD30, EGFR, FAP, CD33, CD123, PD-L1, IGF1R, CD4, CSPG4, B7-H4, NKG2D, CS1, CD138, EpCAM, EBNA3C, GPA7, CD244, CA-125, ETA, CEA, CD52, MUC5AC, c-Met, EGFR, FAB, WT-1, PSMA, AFP, CEA, and CTAG1B.

[0167] XII. Methods for enhancing antitumor activity of T cells The present disclosure further provides a method for enhancing the anti-tumor activity of T cells. In some embodiments, the method includes contacting tumor cells with the genetically modified T cells of the present disclosure, wherein contacting tumor cells with the genetically modified T cells increases the expression of anti-tumor cytokines or kills the tumor cells. In some embodiments, the modified T cells express or include (i) a class 2 VI-D CRISPR effector with RNA-guided RNA endonuclease activity of the present disclosure, (ii) a guide array of the present disclosure, or (iii) both (i) and (ii). In some embodiments of the method, contacting tumor cells with the genetically modified T cells of the present disclosure enhances the anti-tumor activity compared to control T cells that do not include either (i) or (ii), or both (i) and (ii).

[0168] In some embodiments, the method is an in vitro method, in which tumor cells are contacted with the genetically modified T cells of the present disclosure in culture.In some embodiments, the method is an ex vivo method, in which T cells are isolated from a subject with cancer or tumor, and the T cells are modified during culture to express the class 2 VI-D CRISPR effector with RNA-guided RNA endonuclease activity of the present disclosure and the guide array of the present disclosure.After ex vivo modification, the modified T cells can be administered to a subject (for example, autologous treatment).In some embodiments, the method is an in vivo method, in which an effective amount of the modified T cells of the present disclosure is administered to a subject.

[0169] In some embodiments of the method, the engineered T cell comprises: (i) an expression vector comprising a nucleic acid sequence encoding a class 2 VI-D CRISPR effector with RNA-guided RNA endonuclease activity; and (ii) an expression vector comprising a nucleic acid sequence encoding a guide array comprising one or more crRNA molecules, wherein the crRNA molecules independently comprise a direct repeat sequence and a spacer sequence that binds to a target mRNA, and the crRNA molecules bind to different target mRNAs or different regions of the same target mRNA.

[0170] In some embodiments, the guide array comprises crRNA molecules that bind to mRNAs encoding proteins associated with T cell exhaustion. In some embodiments, the proteins associated with T cell exhaustion are selected from the group consisting of FAS, ZC3H12A, CTLA4, SOCS1, CBLB, PD-1, TOX, TOX2, FLI1, CD5, CD39, CD46, TRAC, B2M, JUNB, IRF4, PDCD1 (PD-1), HAVCR2 (TIM3), DHX37, and combinations thereof.

[0171] In some embodiments, the Class 2 VI-D CRISPR effector with RNA-guided RNA endonuclease activity is a Cas13 nuclease selected from the group consisting of Cas13a, Cas13b, 13bt1, Cas13bt2, Cas13c, Cas13d, RfxCas13d, Cas13e, and functional variants thereof. In some embodiments, the Cas13 nuclease comprises an amino acid sequence having at least (e.g., at least the values ​​described below) about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence similarity or sequence identity to the amino acid sequence of a nuclease selected from the group consisting of Cas13a, Cas13b, 13bt1, Cas13bt2, Cas13c, Cas13d, RfxCas13d, and Cas13e. In some embodiments, the Cas13 nuclease comprises an amino acid sequence having at least (e.g., at least as much as described below) about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO:1.

[0172] In some embodiments of the method, the modified T cell further comprises a modified CAR. In some embodiments, the CAR binds to an antigen expressed by the tumor. In some embodiments, the CAR binds to an antigen selected from the group consisting of Her-2, B7-H3, GPC2, GD2, CD19, CD20, CD22, MAGE, BAGE, CAGE, GAGE, HAGE, LAGE, PAGE, PRAME, NY-ESO-1, NY-SEO-1, tyrosinase, Melan-A / MART, gplOO, TRP-1, TRP-2, CD30, EGFR, FAP, CD33, CD123, PD-L1, IGF1R, CD4, CSPG4, B7-H4, NKG2D, CS1, CD138, EpCAM, EBNA3C, GPA7, CD244, CA-125, ETA, CEA, CD52, MUC5AC, c-Met, EGFR, FAB, WT-1, PSMA, AFP, CEA, and CTAG1B.

[0173] In any of the aspects described herein, the tumor may be selected from the group consisting of acanthoma, acinic cell carcinoma, acoustic neuroma, acral lentiginous melanoma, acral hidradenoma, acute eosinophilic leukemia, acute lymphoblastic leukemia, acute megakaryoblastic leukemia, acute monocytic leukemia, acute myeloblastic leukemia with maturation, acute myeloid dendritic cell leukemia, acute myeloid leukemia, acute promyelocytic leukemia, ameloblastoma, adenocarcinoma, adenoid cystic carcinoma, adenoma, adenoid odontogenic tumor, adrenocortical carcinoma, adult T-cell leukemia, aggressive NK-cell leukemia, AIDS-related cancer, AIDS-related lymphoma, alveolar soft tissue leukemia. tumor, ameloblastic fibroma, anal cancer, anaplastic large cell lymphoma, anaplastic thyroid cancer, angioimmunoblastic T-cell lymphoma, angiomyolipoma, angiosarcoma, appendix cancer, astrocytoma, atypical teratoid rhabdoid tumor, basal cell carcinoma, basal cell-like carcinoma, B-cell leukemia, B-cell lymphoma, Bellini duct carcinoma, biliary tract cancer, bladder cancer, blastoma, bone cancer, bone tumor, brainstem glioma, brain tumor, breast cancer, Brenner tumor, bronchial tumor, bronchioloalveolar carcinoma, pheochromocytoma, Burkitt lymphoma, carcinoma of unknown primary site, carcinoid tumor, cancer, carcinoma in situ, penile cancer, carcinoma of unknown primary site, carcinosarcoma, Castleman disease, Central nervous system embryonal tumors, cerebellar astrocytoma, cerebral astrocytoma, cervical cancer, cholangiocarcinoma, chondroma, chondrosarcoma, chordoma, choriocarcinoma, choroid plexus papilloma, chronic lymphocytic leukemia, chronic monocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disorder, chronic neutrophilic leukemia, clear cell tumor, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, Degos disease, dermatofibrosarcoma protuberans, dermoid cyst, desmoplastic small round cell tumor, diffuse large B-cell lymphoma, dysembryoplastic neuroepithelial tumor, embryonal carcinoma, endodermal sinus tumor, endometrial cancer, endometrioid tumor, enteropathy-associated T-cell lymphoma tumor, ependymoblastoma, ependymoma, epithelioid sarcoma, erythroleukemia, esophageal cancer, esthesioneuroblastoma, Ewing family of tumors, Ewing family of sarcoma, Ewing sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, extramammary Paget's disease, fallopian tube cancer, fetal in utero, fibroma, fibrosarcoma, follicular lymphoma, follicular thyroid cancer, gallbladder cancer, gallbladder cancer, ganglioglioma, ganglioneuroma, gastric cancer, gastric lymphoma, digestive tract cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gastrointestinal stromal tumor, germ cell tumor, germinoma, gestational choriocarcinoma, gestational trophoblastic tumor, giant cell tumor of bone,Glioblastoma multiforme, glioma, gliomatosis cerebri, glomus tumor, glucagonoma, gonadoblastoma, granulosa cell tumor, hairy cell leukemia, head and neck cancer, cardiac cancer, hemangioblastoma, hemangiopericytoma, angiosarcoma, hematologic malignancies, hepatocellular carcinoma, hepatosplenic T-cell lymphoma, hereditary breast and ovarian cancer syndrome, Hodgkin lymphoma, hypopharyngeal cancer, hypothalamic glioma, inflammatory breast cancer, intraocular melanoma, islet cell carcinoma, islet cell tumor, juvenile myelomonocytic leukemia, Kaposi's sarcoma, renal cancer, Klatzkin's tumor, Krukenberg's tumor, laryngeal cancer, Laryngeal cancer, lentigo maligna melanoma, leukemia, leukemia, lip and oral cavity cancer, liposarcoma, lung cancer, luteoma, lymphangioma, lymphangiosarcoma, lymphoepithelioma, lymphocytic leukemia, lymphoma, macroglobulinemia, malignant fibrous histiocytoma, malignant fibrous histiocytoma of bone, malignant glioma, malignant mesothelioma, malignant peripheral nerve sheath tumor, malignant rhabdoid tumor, malignant Triton tumor, MALT lymphoma, mantle cell lymphoma, mast cell leukemia, mediastinal germ cell tumor, mediastinal tumor, medullary thyroid cancer, medulloblastoma, medulloepithelioma, melanoma, melanoma, meningioma, Merkel cell carcinoma, mesothelioma tumor, metastatic squamous cell carcinoma of occult primary, metastatic urothelial carcinoma, mixed Müllerian tumor, monocytic leukemia, mouth cancer, mucinous tumor, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis fungoides, mycosis fungoides, myelodysplasia, myelodysplastic syndrome, myeloid leukemia, myelosarcoma, myeloproliferative disorder, myxoma, nasal cavity cancer, nasopharyngeal carcinoma, nasopharyngeal carcinoma, neoplasm, schwannoma, neuroblastoma, neuroblastoma, neurofibroma, neuroma, nodular melanoma, non-Hodgkin's lymphoma, non-melanoma skin cancer, non-small cell lung cancer, eye tumor, oligoastrocytoma, oligodendroglioma, oncocytoma Itoma, optic nerve sheath meningioma, oral cavity cancer, oral cancer, oropharyngeal cancer, osteosarcoma, osteosarcoma, ovarian cancer, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, Paget's disease of the breast, Pancoast tumor, pancreatic cancer, pancreatic cancer, papillary thyroid cancer, papillomatosis, paraganglioma, sinus cancer, parathyroid cancer, penile cancer, perivascular epithelioid cell tumor, pharyngeal cancer, pheochromocytoma, intermediate pineal parenchymal tumor, pineoblastoma, pituitary cell tumor, pituitary adenoma, pituitary tumor, plasma cell tumor, pleuropulmonary blastoma, polyblastoma, precursor T-lymphoblastic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma,Primary hepatocellular carcinoma, primary liver cancer, primary peritoneal cancer, primitive neuroectodermal tumor, prostate cancer, pseudomyxoma peritonei, rectal cancer, renal cell carcinoma, respiratory cancer involving the NUT gene on chromosome 15, retinoblastoma, rhabdomyoma, rhabdomyosarcoma, Richter transformation, sacrococcygeal teratoma, salivary gland cancer, sarcoma, schwannomatosis, sebaceous gland carcinoma, secondary neoplasms, seminoma, serous tumor, Sertoli-Leydig cell tumor, sex cord-stromal tumor, Sézary syndrome, signet ring cell carcinoma, skin cancer, small round blue cell tumor, small cell carcinoma, small cell lung cancer, small cell lymphoma, small intestine cancer, soft tissue sarcoma, somatostatinoma, sooty warts, spinal cord tumor, spinal cord tumor, splenic marginal zone lymphoma, squamous cell carcinoma, stomach melanoma, superficial spreading melanoma, supratentorial primitive neuroectodermal tumor, superficial epithelial-stromal tumor, synovial sarcoma, T-cell acute lymphoblastic leukemia, T-cell large granular lymphocytic leukemia, T-cell leukemia, T-cell lymphoma, T-cell prolymphocytic leukemia, teratoma, end-stage lymphoid cancer, testicular cancer, thecoma, cancer of the pharynx, thymic carcinoma, thymoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, transitional cell carcinoma, urachal cancer, urethral cancer, genitourinary neoplasms, uterine sarcoma, uveal melanoma, vaginal cancer, Verner-Morrison syndrome, verrucous carcinoma, optic pathway glioma, vulvar cancer, Waldenstrom's macroglobulinemia, Warthin's tumor, Wilms' tumor, and combinations thereof.

[0174] In any of the embodiments described herein, the CAR binds to an antigen expressed by a tumor selected from the group consisting of acanthoma, acinic cell carcinoma, acoustic neuroma, acral lentiginous melanoma, acral hidradenoma, acute eosinophilic leukemia, acute lymphoblastic leukemia, acute megakaryoblastic leukemia, acute monocytic leukemia, acute myeloblastic leukemia with maturation, acute myeloid dendritic cell leukemia, acute myeloid leukemia, acute promyelocytic leukemia, ameloblastoma, adenocarcinoma, adenoid cystic carcinoma, adenoma, adenoid odontogenic tumor, adrenocortical carcinoma, adult T-cell leukemia, aggressive NK-cell leukemia, AIDS-related myeloma, AIDS-related lymphoma, alveolar soft part sarcoma, ameloblastic fibroma, anal cancer, anaplastic large cell lymphoma, anaplastic thyroid cancer, angioimmunoblastic T-cell lymphoma, angiomyolipoma, angiosarcoma, appendix cancer, astrocytoma, atypical teratoid rhabdoid tumor, basal cell carcinoma, basal cell-like carcinoma, B-cell leukemia, B-cell lymphoma, Bellini duct carcinoma, biliary tract cancer, bladder cancer, blastoma, bone cancer, bone tumor, brainstem glioma, brain tumor, breast cancer, Brenner tumor, bronchial tumor, bronchioloalveolar carcinoma, pheochromocytoma, Burkitt lymphoma, cancer of unknown primary site, carcinoid tumor, cancer, carcinoma in situ, penile cancer, primary Cancer of unknown origin, carcinosarcoma, Castleman's disease, central nervous system embryonal tumor, cerebellar astrocytoma, cerebral astrocytoma, cervical cancer, bile duct carcinoma, chondroma, chondrosarcoma, chordoma, choriocarcinoma, choroid plexus papilloma, chronic lymphocytic leukemia, chronic monocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disorder, chronic neutrophilic leukemia, clear cell tumor, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, Degos disease, dermatofibrosarcoma protuberans, dermoid cyst, desmoplastic small round cell tumor, diffuse large B-cell lymphoma, dysembryoplastic neuroepithelial tumor, embryonal carcinoma, endodermal sinus tumor, endometrial cancer, endometrial carcinoma Uterine cancer, endometrioid tumor, enteropathy-associated T-cell lymphoma, ependymoblastoma, ependymoma, epithelioid sarcoma, erythroleukemia, esophageal cancer, esthesioneuroblastoma, Ewing family tumors, Ewing family sarcoma, Ewing sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, extramammary Paget's disease, fallopian tube cancer, fetal in utero, fibroma, fibrosarcoma, follicular lymphoma, follicular thyroid cancer, gallbladder cancer, gallbladder cancer, ganglioglioma, ganglioneuroma, gastric cancer, gastric lymphoma, digestive tract cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gastrointestinal stromal tumor, germ cell tumor, germinoma,Gestational choriocarcinoma, gestational trophoblastic tumor, giant cell tumor of bone, glioblastoma multiforme, glioma, gliomatosis cerebri, glomus tumor, glucagonoma, gonadoblastoma, granulosa cell tumor, hairy cell leukemia, hairy cell leukemia, head and neck cancer, head and neck cancer, cardiac cancer, hemangioblastoma, hemangiopericytoma, angiosarcoma, hematologic malignancies, hepatocellular carcinoma, hepatosplenic T-cell lymphoma, hereditary breast and ovarian cancer syndrome, Hodgkin's lymphoma, Hodgkin's lymphoma, hypopharyngeal cancer, hypothalamic glioma, inflammatory breast cancer, intraocular melanoma, pancreatic islet cell carcinoma, pancreatic islet cell tumor, juvenile myelomonocytic leukemia, Kaposi's sarcoma, renal cancer, Klatzkin tumor, Krukenberg tumor, laryngeal cancer, laryngeal cancer, lentigo maligna melanoma, leukemia, leukemia, lip and oral cavity cancer, liposarcoma, lung cancer, luteoma, lymphangioma, lymphangiosarcoma, lymphoepithelioma, lymphocytic leukemia, lymphoma, macroglobulinemia, malignant fibrous histiocytoma, malignant fibrous histiocytoma of bone, malignant glioma, malignant mesothelioma, malignant peripheral nerve sheath tumor, malignant rhabdoid tumor, malignant Triton tumor, MALT lymphoma, mantle cell lymphoma, mast cell leukemia, mediastinal germ cell tumor, mediastinal tumor, medullary thyroid cancer, medulloblastoma, medulloepithelioma, melanoma , melanoma, meningioma, Merkel cell carcinoma, mesothelioma, mesothelioma, metastatic squamous cell carcinoma of occult primary, metastatic urothelial carcinoma, mixed Müllerian tumor, monocytic leukemia, oral cancer, mucinous tumor, multiple endocrine neoplasia syndrome, multiple myeloma, multiple myeloma, mycosis fungoides, mycosis fungoides, myelodysplasia, myelodysplastic syndrome, myeloid leukemia, myelosarcoma, myeloproliferative disorder, myxoma, nasal cavity cancer, nasopharyngeal carcinoma, nasopharyngeal carcinoma, neoplasm, schwannoma, neuroblastoma, neuroblastoma, neurofibroma, neuroma, nodular melanoma, non-Hodgkin's lymphoma, non-Hodgkin's lymphoma, non-melanoma skin cancer, non-small cell lung cancer, Eye tumors, oligoastrocytoma, oligodendroglioma, oncocytoma, optic nerve sheath meningioma, oral cavity cancer, oral cancer, oropharyngeal cancer, osteosarcoma, osteosarcoma, ovarian cancer, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, Paget's disease of the breast, Pancoast tumor, pancreatic cancer, pancreatic cancer, papillary thyroid cancer, papillomatosis, paraganglioma, sinus cancer, parathyroid cancer, penile cancer, perivascular epithelioid cell tumor, pharyngeal cancer, pheochromocytoma, intermediate pineal parenchymal tumor, pineoblastoma, pituitary cell tumor, pituitary adenoma, pituitary tumor, plasma cell tumor, pleuropulmonary blastoma, polyembryoma, precursor T-lymphoblastic lymphoma,Primary central nervous system lymphoma, primary effusion lymphoma, primary hepatocellular carcinoma, primary liver cancer, primary peritoneal cancer, primitive neuroectodermal tumor, prostate cancer, pseudomyxoma peritonei, rectal cancer, renal cell carcinoma, respiratory cancer involving the NUT gene on chromosome 15, retinoblastoma, rhabdomyoma, rhabdomyosarcoma, Richter transformation, sacrococcygeal teratoma, salivary gland cancer, sarcoma, schwannomatosis, sebaceous gland carcinoma, secondary neoplasms, seminoma, serous tumor, Sertoli-Leydig cell tumor, sex cord-stromal tumor, Sézary syndrome, signet ring cell carcinoma, skin cancer, small round blue cell tumor, small cell carcinoma, small cell lung cancer, small cell lymphoma, small intestine cancer, soft tissue sarcoma, somatostatinoma, sooty warts, spinal cord tumor, spinal cord tumor, splenic marginal zone lymphoma melanoma, squamous cell carcinoma, gastric cancer, superficial spreading melanoma, supratentorial primitive neuroectodermal tumor, superficial epithelial-stromal tumor, synovial sarcoma, T-cell acute lymphoblastic leukemia, T-cell large granular lymphocytic leukemia, T-cell leukemia, T-cell lymphoma, T-cell prolymphocytic leukemia, teratoma, end-stage lymphoid cancer, testicular cancer, thecoma, cancer of the pharynx, thymic carcinoma, thymoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, transitional cell carcinoma, urachal carcinoma, urethral cancer, genitourinary neoplasms, uterine sarcoma, uveal melanoma, vaginal cancer, Verner-Morrison syndrome, verrucous carcinoma, optic pathway glioma, vulvar cancer, Waldenstrom's macroglobulinemia, Warthin's tumor, Wilms' tumor, and combinations thereof. [Example]

[0175] The following examples illustrate representative, non-limiting methods of the present disclosure.

[0176] method Cloning of lentiviral constructs All oligonucleotides were synthesized by IDT or the Stanford Protein and Nucleic Acid (PAN) Facility. To facilitate flexible cloning of the entire guide array and avoid hairpin formation near the Esp31 digestion site, pLentiRNAGuide_002 (Addgene #138151) was modified to remove the direct repeat region (pSLQ4419). The sequences of the high-affinity 14g2a-GD2(E101K) chimeric antibody receptor (HA-28z CAR) and the mCherry-P2A-Ruminococcus flavefaciens Cas13d (RfxCas13d) (Addgene #155305) sequences have been previously described. 7、48 The HA-28z CAR sequence was cloned into the pHR lentiviral backbone (pSLQ5263) with a constitutive SFFV promoter. The controllable RfxCas13d-DD was constructed by fusing the destabilization domain (DD) from Escherichia coli dihydrofolate reductase (DHFR) to the C-terminus of RfxCas13d using a short glycine-serine linker.

[0177] RfxCas13d spacer sequences were generated using the cas13design web tool (see www.cas13design.nygenome.org) as previously described. 50These can be seen in Tables 1 and 2. Guide array constructs were cloned using the following methods: for conventional restriction ligation cloning, forward and reverse oligonucleotides for each spacer were annealed, phosphorylated with T4 PNK (NEB), and ligated into the backbone with T4 DNA ligase (NEB). For In-Fusion HD assembly (Takara), the entire guide array was amplified using PCR primers containing 15-20 nt long 5' and 3' homology regions. The PCR amplicon was then directly inserted into the backbone. For NEBuilder HiFi assembly (NEB), single-stranded oligonucleotides with 20-30 nt long 5' and 3' homology regions were synthesized and directly inserted into the backbone.

[0178] Longer guide arrays (5-plex and 10-plex) were cloned using a two-step overlap extension PCR method. Arrays were synthesized as single-stranded oligo fragments encoding direct repeats flanked by two overlapping spacer regions (see Figure 5A and Table 3). In the first PCR reaction, the oligo pairs were annealed and extended. In the second PCR reaction, the double-stranded fragments were pooled together and amplified for 15 cycles without primers. Then, forward and reverse primers containing 5' and 3' homology regions were spiked in, followed by an additional 15-20 cycles. The PCR amplicons were run on a 2% agarose gel, purified, and inserted into the scaffold using NEBuilder HiFi assembly.

[0179] cell culture Lenti-X 293T cells (Takara) were cultured in 0.22 μm sterile-filtered DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS). Nalm6-GD2 cells were cultured in 0.22 μm sterile-filtered complete medium (CM) (RPMI (Gibco) supplemented with 10% FBS, 100 U / ml penicillin, and 100 μg / ml streptomycin). All cells were cultured in a humidified incubator at 37°C and 5% CO2.

[0180] Isolation and expansion of primary human T cells Buffy coats from anonymous healthy blood donors were purchased from the Stanford Blood Center. Primary human T cells were isolated using the EasySep Human T Cell Isolation Kit (STEMCELL) according to the manufacturer's protocol, using Ficoll-Paque PLUS (GE Healthcare) and SepMate-50 tubes. Isolated T cells were cultured at 4–10 × 10 cells / well in vials containing either FBS (Sigma) or CryoStor (STEMCELL) supplemented with 10% DMSO. 6 The cells were immediately frozen and stored at a concentration of 100 cells / vial.

[0181] Cryopreserved T cells were thawed and activated the same day using human T activator CD3 / CD28 Dynabeads (Gibco) at a 3:1 bead-to-cell ratio. T cells were cultured in human T cell medium (HTCM) (0.22 μm sterile-filtered AIM V (Gibco) supplemented with 5% FBS, 10 mM HEPES, 2 mM GlutaMAX, 100 U / ml penicillin, and 100 μg / ml streptomycin). Human recombinant IL-2 (STEMCELL) was added at 100 U / ml. On day 3 of culture, the Dynabeads were magnetically removed. Cells were expanded every other day by adding HTCM until the overall cell concentration reached 0.5–1 × 10. 6 Viable cell counts were obtained using a Countess 3 automated cell counter (Invitrogen) according to the manufacturer's protocol for trypan blue exclusion.

[0182] For culture conditions requiring trimethoprim (TMP), a freshly thawed 1000 μM concentrated stock solution of TMP (in DMSO) was added to cells to a final concentration of 1 μM, starting on day 3 of culture, unless otherwise noted. To maintain the culture conditions, cells were expanded every other day in HTCM supplemented with 1 μM TMP, unless otherwise noted. To remove TMP, cells were washed twice with FACS buffer (DPBS (Gibco) supplemented with 2% FBS and 1 mM EDTA) and resuspended in fresh, prewarmed HTCM.

[0183] Lentiviral preparations 7.5×10 5 Lenti-X cells were seeded overnight in 6-well plates containing 2 ml of DMEM supplemented with 10% FBS. The following morning, 850 μl of culture medium was removed, and the cells were transfected with 0.55 μg of pMD2.G (Addgene Plasmid No. 12259), 1.28 μg of psPAX2 (Addgene Plasmid No. 12260), and 1.79 μg of transfer plasmid in 426 μl of Opti-MEM (Gibco) using 10.88 μl of TransIT-LT1 (Mirus Bio). After 6 hours, the culture medium was completely removed and replaced with fresh DMEM supplemented with 10% FBS and 1x ViralBoost (Alstem Bio). At 24 hours post-transfection, the viral supernatant was collected and filtered through a 0.45 μm syringe filter (MilliporeSigma). The supernatant was mixed with lentivirus precipitation solution (Alstem Bio) according to the manufacturer's protocol, incubated at 4°C for a minimum of 4 hours, and concentrated 10-100 times in Opti-MEM. The concentrated virus was used fresh or frozen at -80°C for later use. Lentivirus production in 10 cm and 15 cm Petri dishes was scaled up proportionally to the surface area of ​​the culture vessel.

[0184] Immunostaining and flow cytometry In all flow cytometry experiments, mCherry fluorescence was used as a quantitative indicator of RfxCas13d expression. The 1A7 anti-14G2a idiotype antibody used to detect HA-28z CAR surface expression was conjugated in-house using the DyLight 650 antibody labeling kit (Thermo Fisher). The following fluorescent antibodies were used to stain human T cell surface markers: [TIM3-BV421 (clone F38-2E2), LAG3-FITC (clone 11C3C65), PD-1-APC (clone A17188B), FAS-FITC (clone DX2), CTLA4-APC (clone BNI3), CD46-APC (clone TRA-2-10), CD62L-PerCP-Cy5.5 (clone DREG-56), and CD45RAPE-Cy7 (clone HI100)] (BioLegend), [CD8-AF405 (clone 3B5)] (Invitrogen). Approximately 1–2 × 10 5 Cells were resuspended in 100 μl of FACS buffer and stained for 20 minutes at room temperature. Samples investigated for CAR surface expression were stained for 10 minutes on ice to minimize receptor internalization. Prior to flow cytometry, cells were washed twice with FACS buffer. Flow cytometry was performed using a CytoFLEX S (Beckman Coulter). For all samples, a minimum of approximately 1 × 10 cells were present within the final gated population of interest. 4 events were collected.

[0185] Transduction of primary human T cells Primary human T cells were transduced with concentrated lentivirus at 1-10% v / v (unless otherwise noted) 24-48 hours after bead activation. Construct expression was confirmed on day 5 of culture by flow cytometry as previously described. For constructs requiring puromycin selection, a concentrated stock solution of 10 μg / μl puromycin was thawed and added to cells on day 3 of culture (24-48 hours after transduction) to a final concentration of 1 μg / ml. Puromycin selection was complete by day 5 of culture.

[0186] Cell sorting On day 5 of culture, primary human T cells were spun down and diluted to 1-2 x 10 in ice-cold FACS buffer. 7 Cells were resuspended at a concentration of 1000 cells / ml. Cells were sorted on a Sony SH800 sorter (Sony Biotechnology) using a 130 μm tip at low sample pressure in "semi-purity" or "purity" mode. Mock untransduced T cells were used to transduce RfxCas13d. + / - Gating was determined. Live RfxCas13d + Cells were sorted into collection tubes containing cold HTCM. After sorting, cells were spun down and resuspended in fresh pre-warmed HTCM supplemented with 100 U / ml IL-2 to obtain approximately 3-5 x 10 5 After selection, cells were expanded as described above.

[0187] RNA extraction and quantitative reverse transcription PCR (RT-qPCR) Approximately 1–10 × 10 RNEasy Plus kit (QIAGEN) was used according to the manufacturer's protocol, with the addition of 2-mercaptoethanol. 6 Total RNA was extracted from 100 primary human T cells. RNA concentration was measured using a NanoDrop One (Thermo Fisher) and normalized across all cell samples before reverse transcription using an iScript cDNA synthesis kit (Bio-Rad). Diluted cDNA was added to iTaq Universal SYBR Green Supermix (Bio-Rad) and run in a CFX384 Touch (Bio-Rad) using the following PCR conditions: 50°C for 10 minutes, 95°C for 30 seconds, 40x (95°C for 10 seconds, 60°C for 30 seconds). SDHA was used as an internal control, and 2 -ΔΔCt Gene expression was quantified using the method. Primer sequences can be found in Table 3.

[0188] Bulk RNA-seq and differential expression analysis Total RNA was prepared as previously described at approximately 5 x 10 6Bulk RNA-seq was performed on an Illumina NovaSeq 6000 by Novogene (Davis, CA) using 20–30 × 10 RNA samples extracted from primary human T cells on day 10 of culture. 6 The analysis was performed using 150 bp paired-end reads. 81 Differential expression analysis was performed using a custom script in R (version 4.1.2): tximeta 82 Import and batch correct quantification results using DESeq2 83 The log2 expression ratios were analyzed using apeglm 84 The images were reduced using and visualized using ggplot2.

[0189] Pooled double-guide array library design and assembly Based on results from previous CRISPR-Cas9 propagation and functional knockout screens in primary human T cells, we designed a custom curated library of 24 putative exhaustion-associated genes (see Table 3). 50Using the RT-PCR tool, the top three 23-nt guide spacer sequences targeting the CDS of the most diverse human transcript isoforms were generated for each gene, yielding 72 targeting guide spacers. Additionally, eight random 23-nt non-targeting guide spacers that did not align to the human transcriptome (confirmed by nucleotide BLAST) were also generated. A total of 80 guide spacers were generated. Using a custom Python script, RfxCas13d direct repeats were ligated to the spacer sequences, and all paired guide array combinations were output, yielding 80 × 80 = 6,400 double-guide array sequences (see Table 3). As previously described, flanking 5' and 3' homology regions were added to facilitate insertion into the pSLQ4419 backbone. Guide array libraries were synthesized as single-stranded oligo pools (Twist Bioscience). 10 ng of template was amplified using KAPA HiFi HotStart (Roche) under the following PCR conditions: 95°C for 3 min, 8x (98°C for 20 s, 55°C for 15 s, 72°C for 15 s), 72°C for 1 min. Reaction products were recovered using a NucleoSpin PCR Purification Kit (Macherey-Nagel) and run on a 2100 Bioanalyzer (Agilent) by the Stanford PAN Facility for size measurement and quantification (85% double-guide array) and quality control (minimal PCR overamplification). The amplified library was efficiently cloned into pSLQ4419(Esp31) using NEBuilder HiFi DNA assembly with less than 5% background, and the diverse spacer regions consistent with the library's nucleotide distribution were confirmed by Sanger sequencing. Minimal bias. 85 Representativeness of the entire library, with Gini count: 0.146, 90th / 10th percentile of guide array reads: 1.96, see Figure 10A, was further verified by Miseq v3 (600 cycles) paired-end sequencing (Illumina).

[0190] CD8+CAR T cell proliferation screening 8×10 7 Primary human T cells (1:1 CD4 + Against CD8 + The CD8 / CD28 cells (adjusted to a 3:1 ratio) were thawed and activated the same day with CD3 / CD28 Dynabeads at a 3:1 ratio as described above. + T cells were cultured in HTCM supplemented with 100 U / ml IL-2 and CD4 + The cells were cultured with T cells. On day 1 of culture, the cells were co-transduced with the following freshly collected, 100x concentrated lentivirus (prepared as described above using endotoxin-free plasmid DNA): 2% v / v mCherry-P2A-RfxCas13d, 1.5% v / v HA-28z CAR, and 0.2% v / v pooled double-guide array library. On day 3 of culture, the cells were selected with 1 μg / ml puromycin for 48 hours. Approximately 28.9% of the cells survived puromycin selection, corresponding to approximately 83.9% single lentiviral integration (assuming independent transduction events following a Poisson distribution). On day 5, a portion of the cells was stained and measured by flow cytometry for expression of HA-28z CAR and CD8 to confirm the CAR expression. + CD8 + The total percentage of T cells was determined. This percentage was determined by the RfxCas13d + Subsequent FACS analysis of the cells provides information about the CAR + CD8 + This ensured that sufficient numbers of cells were sorted to achieve approximately 1000x library coverage within the T cell subpopulation. After sorting, cells were split into two replicates at approximately 1000x representation and expanded every other day as described above. On days 11, 13, and 15 of culture, a portion of the cells were stained and measured by flow cytometry to identify CD8 + , RfxCas13d + , and CAR + The percentage of cells was determined by flow cytometry. +T cells were magnetically isolated from whole T cell cultures and harvested at approximately 1000x representation using the EasySep Human CD8 Positive Selection Kit (STEMCELL). Proliferation screening was terminated after 15 days of ex vivo expansion.

[0191] Genomic DNA extraction and library preparation Magnetically isolated CD8 using the DNeasy Blood and Tissue Kit (QIAGEN) + Genomic DNA (gDNA) was extracted from T cells. Cells were lysed using Buffer AL and spun at maximum speed for 3 minutes to pellet the positive selection magnetic beads. The cell lysate supernatant was then carefully transferred to a clean tube before proceeding with the manufacturer's protocol for gDNA isolation.

[0192] The double-guide array library was prepared for next-generation sequencing by a two-step PCR protocol. Briefly, PCR1 primers targeted and amplified the guide array sequences integrated by the lentivirus, added Illumina sequencing primer binding sites, and used eight custom-designed staggered sequences to increase the overall sequence diversity of the amplicons for better cluster identification. PCR2 primers added paired-end indexes for sample identification and contained Illumina P5 / P7 sequences that bind to the flow cell. Primer sequences for both PCR1 and PCR2 can be found in Table 3. For each sample collected, a total of approximately 40 μg of gDNA was amplified in PCR1 as follows (6 μg of gDNA was used to generate 1 × 10 6(Approximately corresponding to 100 cells): Up to 2 μg of gDNA template per 50 μl reaction was amplified using the following cycling conditions: 95°C for 3 minutes, 24× (98°C for 20 seconds, 70°C for 15 seconds, 72°C for 15 seconds), 72°C for 1 minute. 200 ng of a plasmid DNA (pDNA) library was also amplified in PCR1. PCR1 products were pooled together for each sample and column purified. Approximately 10 ng of each purified PCR1 product was added as template DNA for PCR2 using the following cycling conditions: 95°C for 3 minutes, 12× (98°C for 20 seconds, 65°C for 15 seconds, 72°C for 15 seconds), 72°C for 1 minute. All reactions were set up using a KAPA HiFi HotStart (Roche). PCR2 products were run on a 2% ultrapure agarose gel (Invitrogen), bands were gel extracted using a Nucleospin gel purification kit (Macherey-Nagel), and DNA was quantified in a Qubit fluorometer (Invitrogen).

[0193] Readout and analysis of screening results Enrichment of the CRISPR guide arrays was read out by pooled Miseq v3 600-cycle paired-end sequencing (Illumina). Samples were accurately identified based on paired-end indexing. Raw fastq files were trimmed of 5' and 3' adapters and filtered based on quality (-q 30) and length using cutadapt. Processed reads were aligned end-to-end using bowtie2. 86 The sequences were aligned to a custom index constructed from a reference library containing all double-guide array combinations using samtools. The number of guide arrays was calculated from the alignment data.

[0194] Screening data were analyzed using the DESeq2 negative binomial distribution model within a custom R script. Correlation between technical replicate pairs was assessed by linear regression of normalized replicate count data. Significant enrichment and depletion of guide arrays were analyzed by pairwise comparison using Wald tests to calculate log2 expression ratios and adjusted p-values ​​for each guide array, as previously described. 87 Based on the log2 expression ratios, log2 expression ratios were determined by comparing the array abundance in harvested cell samples (harvested on days 11, 13, or 15 of culture) to the array abundance in the original plasmid DNA preparation as the starting time point. A likelihood ratio test was performed by comparing the full model (~ time point) with the reduced model (~1). These results were used to rank guide arrays based on how significantly the (reduced) time point variable affected the count variation between samples. Significant gene-level enrichment and depletion were determined using a robust rank aggregation of guide-level Wald test results. 88 Data were visualized using ggplot2, and gene-level hierarchical clustering was performed using pheatmap.

[0195] Cytokine secretion On day 10 of culture, primary human T cells and GFP + Nalm6-GD2 cells were spun down and resuspended in fresh pre-warmed CM. 1 × 10 5 GFP + Nalm6-GD2 cells were seeded in clear flat-bottom 96-well plates at 1 × 10 5 The cells were co-cultured with primary human HA-28z CAR T cells in a total volume of 200 μl of CM. Triplicate wells per condition were seeded. After 24 hours, plates were spun down and co-culture supernatants were collected for immediate analysis or frozen at -80°C. Cytokine concentrations were measured using IFNγ or IL-2 ELISA Max kits (Biolegend) according to the manufacturer's protocol.

[0196] Tumor killing and serial restimulation In vitro tumor killing assays and serial restimulation assays were performed in an IncuCyte S3 (Sartorius). Primary human T cells and GFP were cultured on day 10 of the culture. + Nalm6-GD2 cells were spun down and resuspended in fresh pre-warmed CM. 5 × 10 4 GFP + Nalm6-GD2 cells were seeded in clear, flat-bottom 96-well plates and co-cultured with primary human HA-28z CAR T cells in a total volume of 200 μl of CM at various effector:target (E:T) ratios as indicated in the figure legends. Triplicate wells were seeded per condition, and four images were acquired per well over a total period of 48–72 h. Total integrated GFP intensity per well was calculated as GFP + Nalm6-GD2 was used as an index to quantify viable cells. All values ​​were normalized to the corresponding initial scan and plotted over time using IncuCyte analysis software. For sequential restimulation experiments, GFP + Nalm6-GD2 cells and HA-28z CAR T cells were co-cultured as previously described. 48–72 hours after the initial stimulation, remaining cells were spun down, resuspended in fresh pre-warmed CM, counted, and then resuspended in an additional 5 × 10 cells at various E:T ratios as previously described. 4 GFP + This process was repeated twice for a total of three stimulations.

[0197] Detection of metabolites in cell culture media On day 10 of culture, 5 x 10 4Primary human HA-28z CAR T cells were seeded in a flat-bottom 96-well plate in a total volume of 200 μl of phenol red-free AIM-V (Gibco) supplemented with 100 U / ml IL-2. ATP (Sigma) was spiked in to a final concentration of 20 μM. Cells were incubated at 37°C and 5% CO2 for 1 hour (unless otherwise noted). Cells were then spun down, and culture supernatants were collected for immediate analysis using ATP (Promega), AMP (Promega), and adenosine (Abcam) detection kits according to the manufacturer's protocol.

[0198] Representativeness of the data Boxplots and violin plots were generated using ggplot2. These were based on a minimum of approximately 1 × 10 per sample. 4 Each figure represents 100 cells. Box plots show the median and the 1st and 3rd quartiles. The whiskers extend from the box up to 1.5 times the distance between the 1st and 3rd quartiles. Unless otherwise noted, all bar graphs show the mean and standard deviation of replicates plotted as points.

[0199] statistical analysis Unless otherwise noted, statistical analysis of significance between groups was performed using either conventional one-way ANOVA or repeated measures one-way ANOVA with correction for multiple comparisons (using Dunnett's test) in GraphPad Prism 9. Linear regression analysis was performed using R or GraphPad Prism 9.

[0200] result Development of Cas13d for optimal expression and activity in primary human T cells The CRISPR / Cas13d system (RfxCas13d) derived from Ruminococcus flavefaciens XPD3002 exhibits excellent catalytic activity and targeting specificity in mammalian cells 42、48We first confirmed the functional activity of lentivirally integrated RfxCas13d in primary human T cells by characterizing crRNA-guided knockdown of a transgenic GFP reporter (Figure 8A-B). We then attempted to target endogenous genes associated with T cell exhaustion in HA-28z CAR T cells. Antigen-independent clustering of HA-28z CARs led to exhaustion-associated reprogramming, including tonic signaling and upregulation of the inhibitory receptors LAG3, PD-1, and TIM3, by day 10 of ex vivo culture. 7 Previous studies have demonstrated the clinical importance of disrupting these classic markers of fatigue. 49 Therefore, we hypothesized that MEGA HA-28z CAR T cells co-expressing RfxCas13d and targeting guide RNA arrays could simultaneously suppress the upregulation of LAG3, PD-1, and TIM3 (Figure 1A). We first designed three single guides per gene. 50Knockdown efficiency was assessed compared to a non-targeting control guide by measuring the corresponding surface expression of LAG3 / PD-1 / TIM3 by flow cytometry at day 10 (Figure 8C-D). Using the bicistronic CRISPR system, we noticed limited knockdown in three different donors due to inefficient lentiviral transduction of RfxCas13d (Figure 8E). Interestingly, we found that all bicistronic configurations resulted in low functional viral titers, either due to RfxCas13d array processing on the lentiviral RNA during packaging (forward orientation) or guide-mediated cleavage of the crRNA (reverse orientation) (Figure 8F-G). Based on these data, we established an optimized workflow for manufacturing MEGA HA-28z CAR T cells. In this workflow, primary human T cells were co-transduced with separate RfxCas13d and HA-28z CAR constructs, followed by transduction with the crRNA construct (Figure 1B, Figure S8H-I).

[0201] MEGA CAR T cells robustly and specifically suppress the upregulation of classical exhaustion markers driven by tonic signaling To evaluate whether the optimized MEGA HA-28z CAR T cells could suppress the upregulation of LAG3, PD-1, and TIM3, we isolated primary T cells from multiple healthy blood donors and co-transduced the cells with the RfxCas13d construct and the HA-28z CAR construct. One day later, we transduced the cells with either 1) a single guide targeting each receptor, 2) a double-guide array targeting all receptor pair combinations, or 3) a non-targeting control guide. This sequential transduction process ensured that the expression of RfxCas13d and CAR was the same across experimental conditions. Then, on day 5, we used co-expressed mCherry as a marker to detect the expression of RfxCas13d. +Cells were sorted, and receptor surface expression was measured using flow cytometry on day 10 (Figure 1B). As previously reported, we noted significant upregulation of LAG3 / PD-1 / TIM3 in HA-28z CAR T cells expressing a non-targeting guide compared to mock-transduced T cells (Figure 1C). Single-guide and double-guide arrays specifically suppressed the upregulation of each targeted receptor to near-baseline levels. These results were consistent across independent experiments using multiple donors (Figure 1D).

[0202] To confirm that protein-level suppression of inhibitory receptors was due to RfxCas13d activity at the transcriptional level, we isolated bulk RNA from MEGA HA-28z CAR T cells at day 10 and quantified the abundance of LAG3, PDCD1 (PD-1), and HAVCR2 (TIM3) mRNA transcripts using quantitative reverse transcription PCR (RT-qPCR) compared to non-targeting controls (Figure 1E, Figure 9A). We observed a strong correlation between transcript abundance and surface protein expression in all samples. Interestingly, we noted a position-dependent effect of the spacer within the double-guide array. The LAG3-targeting spacer showed efficient knockdown regardless of array position, whereas the PD-1- and TIM3-targeting spacers were generally more effective at the 3'-proximal positions. The knockdown efficiency of the LAG3 and TIM3 spacers was also more consistent between donors than the PD-1 spacer, which may be due to a narrower and more variable operating range of PD-1 upregulation in donors or to suboptimal PD-1 spacer sequences.

[0203] To assess potential off-target and / or collateral cleavage effects across the transcriptome, we performed bulk RNA-Seq on RNA extracted from MEGA HA-28z CAR T cells generated from two donors at day 10. Recent studies have addressed whether collateral RfxCas13d activity correlates with on-target transcript cleavage activity in mammalian cells. 51~53 Therefore, we chose to analyze the LAG3 targeting guide, TIM3 targeting guide, and LAG3 + TIM3 targeting guide, as they showed the greatest knockdown efficiency in donors. We identified significant on-target knockdown of LAG3 and / or HAVCR2 transcripts compared to non-targeting controls in all samples, and no significant (adjusted p-value < 0.001) off-target activity was detected (Figure 1F). We observed no activation of the interferon pathway in response to crRNA expression or target RNA cleavage. 54 Notably, we also observed no off-target protein-level effects, as CAR surface expression was uniform across all targeted and non-targeted samples (Figure 9B).

[0204] We next constructed triple-guide arrays encoding all different permutations of the LAG3 / PD-1 / TIM3 targeting spacer to simultaneously knockdown all three inhibitory receptors in MEGA HA-28z CAR T cells. All triple-guide arrays were able to suppress the upregulation of exhaustion markers in two to three different donors, as measured by flow cytometry (Figure 1G, Figure 9C). Transcript abundance strongly correlated with surface protein expression (Figure 1H, Figure 9D), and CAR expression was uniform across all samples, as expected (Figure 9E). Certain spacer arrangements showed more consistent triple knockdown efficiency than others, a pattern consistent with the positional dependency observed in double-guide array experiments. To confirm whether triple knockdown occurred at the single-cell level, we performed triple-guide arrays encoding LAG3 / PD-1 / TIM3 targeting spacers. + / - PD-1 + / - TIM3 + / - Flow cytometry data were gated on day 10 for all cell combinations (Figure 1I, Figure 9F-G). We observed a decrease in the triple-positive population from 35.01% in the non-targeting control to 5.46% in cells expressing the optimal triple-guide array (LPT), accompanied by an increase in the triple-negative population from 15.80% to 55.09%. This demonstrates that MEGA can achieve highly multiplexed gene knockdown in primary human T cells.

[0205] MEGA facilitates two-dimensional genetic screening to identify novel pairs of growth regulators in models of CD8+ CAR T cell dysfunction Although CRISPR / Cas9 screens have identified individual genes that suppress T cell antitumor activity, little is known about the complex network of genetic interactions and broader genetic programs that drive T cell dysfunction and exhaustion. Combinatorial CRISPR screens offer a powerful method for perturbing multiple genes per cell and deciphering these interactions, but such studies have not previously been performed in primary human T cells. 55、56 .

[0206] The programmable targeting specificity of the MEGA platform, when combined with efficient multiplexing, makes it highly suitable for combinatorial CRISPR screening, and the compact guide arrays allow for easy deconvolution of perturbation information in individual cells. We hypothesized that co-expression of RfxCas13d with pooled guide array libraries would enable systematic pairwise gene perturbations in primary human T cells. We applied this technology in a proof-of-concept study to demonstrate the potency of dysfunctional CD8 T cells in culture. + We identified novel combinations of putative exhaustion-related genes that control HA-28z CAR T cell proliferation (Figure 2A). We first curated a list of 24 genes based on previous CRISPR-Cas9 knockout studies in primary human CD8+ T cells (see Table 3). We then designed and assembled a custom library containing 5,184 double-guide arrays targeting all 576 paired combinations of these genes (Figure 10A, Methods, Table 3). Each targeting guide was also paired with a set of eight randomly generated non-targeting guides to simulate an additional 1,152 "single" guide perturbations. Finally, we included 64 non-targeting guide pairs as controls, for a total of 6,400 different guide arrays. We utilized a heuristic screening approach to eliminate the need for prior functional validation of targeting guides.

[0207] Briefly, we transduced T cells isolated from a single healthy donor with RfxCas13d, HA-28z CAR, and a pooled double-guide array library (Figure 2A, Figure 10B, see Methods). On day 5, cells were sorted based on RfxCas13d expression and split into two replicates to account for technical variations in culture conditions and downstream processing. When cells were cultured in parallel for two weeks, the tonic signaling of HA-28z drove both T cell expansion and subsequent dysfunction. CD8 + T cells were magnetically isolated from the bulk population at sampling times on days 11, 13, and 15 of culture. We quantified guide array abundance by PCR amplification and deep sequencing of guide array cassettes. Unlike multiplexed Cas9 screening performed in cancer cell lines, which requires multiple reads and complex barcoding schemes to accurately identify guide pairs, 55 Our approach, by directly sequencing the compact RfxCas13d guide arrays in a single read, readily identified pairs without barcoding. Library coverage was confirmed by highly correlated replicates and non-zero counts for all guide arrays across all samples (Figure 10C-D).

[0208] We performed pairwise statistical tests to determine whether guide arrays were significantly enriched (targeting pairs of negative regulators of proliferation) or depleted (targeting gene pairs essential for proliferation) between the early and late screening time points (Figure 2B-C). We identified numerous guide arrays that consistently selected positively or negatively in proliferating HA-28z CAR T cells across all replicates. As expected, non-targeting control arrays were neither enriched nor depleted. The effects on proliferation caused by single perturbations (those paired with non-targeting spacers) were typically weaker than double perturbations, with the exception of guides targeting IRF4, JUNB, and CBLB, which had observable dominant-negative effects. We were initially surprised by the robust depletion of these guides due to our library design. However, previous studies suggest that despite their established role in driving T cell dysfunction, the transcription factors IRF4 and JUNB are also important for T cell expansion and survival. 7、31、35、57 CBLB (negative regulator of T cell signaling) in the setting of CAR T dysfunction 14 The function of genes such as CBLB-1 and CBLB-2 spacers requires further investigation—we note that the CBLB-1 spacer appears to have a dominant-negative effect, while the CBLB-2 and CBLB-3 spacers were enriched in certain guide combinations. Highly enriched double arrays often contain FAS (such as FAS in combination with either ZC3H12A, CTLA4, or SOCS1), which is supported by previous studies implicating FAS as a negative regulator of CAR T cell activity through activation-induced cell death. 58、59We also noticed positional effects on the guide array, consistent with our previous results, allowing us to sample a larger range of perturbations that could alter the relative levels of repression of the two target genes. When we pooled the guide array data, we observed comparable results for a broader range of gene-level interactions (Figure 9E-F). Finally, we ranked the guide arrays based on the most significant number of variations across all screening time points, as tested by a likelihood ratio test, and identified the top hits consistent with the pairwise analysis (Figure 2D).

[0209] We selected a set of top-ranked guide array hits for functional validation based on both guide-level and gene-level data (Figure 2E). We generated MEGA HA-28z CAR T cells from 3-4 different blood donors and individually perturbed either one of the seven significantly enriched gene pairs or one of the two significantly depleted gene pairs. We tracked T cell expansion in culture up to day 15 and measured normalized CD8 expression across all donors compared to non-targeting controls. + The fold change in expansion of CD8 was calculated (Fig. 2F, Fig. 11A). + In both T cells and bulk T cells, T cell expansion in culture was controlled, recapitulating the screening results (Figure 2F, Figures 11A-C). Thus, we also demonstrated that the significantly smaller RfxCas13d expression in bulk T cells (despite uniform CAR expression) was expressed in the depleted array by day 5. +Focusing on the CAR T cell population, we suggested that dysregulation of proliferation was rapid, occurring within 48 hours after transduction (Figure 11D-E). To investigate whether the difference in proliferation could be explained by alterations in T cell differentiation, we measured surface CD62L / CD45RA expression at day 10 (Figure 11F-G). The effector memory phenotype was more abundant in CAR T cells expressing enriched guide arrays (+12% when targeting CBLB+FAS), whereas depleted guide arrays suppressed effector differentiation and biased cells toward central memory (+26.4% when targeting TOX2+JUNB) and stem cell memory (+19.3% when targeting PDCD1+IRF4) phenotypes.

[0210] Paired transcriptome perturbations enhance the antitumor activity of dysfunctional CAR T cells Encouraged by our validation experiments, we next investigated whether the top paired screening hits could improve not only the proliferative capacity but also the antitumor activity of dysfunctional CAR T cells. We performed a small-scale secondary screen of the top enriched guide arrays using in vitro cytokine secretion and cytotoxicity in response to antigen-positive tumors as functional readouts (Figure 3A). On day 10 of culture, we incubated MEGA HA-28z CAR T cells with Nalm6 leukemia cells expressing GD2 (Nalm6-GD2) and measured IFN-γ and IL-2 levels in the coculture supernatants 24 hours later (Figures 3B-D, 12A-B). The top guide arrays robustly enhanced IFN-γ secretion in tonic-signaling CAR T cells, regardless of antigen stimulation, whereas IL-2 secretion was only modestly improved upon antigen stimulation. To assess the cytotoxicity of MEGA CAR T cells, we used live-cell imaging to monitor the abundance of Nalm6-GD2 in cocultures over 48–72 hours. The top-ranked guide array significantly improved the tumor-killing ability of dysfunctional CAR T cells compared with non-targeting controls across multiple donors and effector:target ratios (Figures 3E–F, 12C). This was particularly evident upon repeated antigen stimulation in serial rechallenge assays—paired knockdown of CBLB and FAS enhanced robust and long-lasting anti-tumor responses by exhausted CAR T cells compared with non-targeting controls, which failed to control tumor growth (Figures 3E–G). We hypothesize that dual targeting of CBLB and FAS improves CAR T cell activity by potentially synergistically disrupting two independently acting pathways known to negatively regulate T cell activation. 14、59、60 (Figure 3H).

[0211] MEGA enables rapid, tunable, and reversible perturbation of the T cell transcriptome In previous experiments, we noted that transcript knockdown in cells expressing targeting crRNAs was entirely dependent on RfxCas13d expression (Figure 13A). Based on this data, we were interested in whether modulating the level of RfxCas13d might enable tunable and reversible control of the T cell transcriptome. To complement the fast timescale over which RfxCas13d-mediated RNA degradation occurs, we fused a destabilization domain (DD) from Escherichia coli dihydrofolate reductase to the C-terminus of RfxCas13d (Figure 4A). At steady state, the controllable RfxCas13d-DD is rapidly degraded by the proteasome due to the unregulated DD. 61 In the presence of trimethoprim (TMP), an FDA-approved small molecule antibiotic, the tertiary structure of the DD is stabilized, allowing RfxCas13d-DD to bind and target RNA.

[0212] We first confirmed that RfxCas13d-DD protein expression was regulated by the presence of TMP in primary human T cells by staining for FLAG-tagged RfxCas13d in cells (Figure 13B). To characterize the activity and range of action of RfxCas13d-DD for endogenous gene repression, we generated MEGA T cells expressing RfxCas13d-DD and either a guide targeting CD46 (a ubiquitous surface protein) or a non-targeting guide. After co-transduction, we cultured MEGA T cells for 48 hours in the presence or absence of TMP and measured CD46 surface expression on day 5. MEGA T cells conditionally repressed CD46 in a TMP-dependent manner, fully retaining functional activity in the presence of the drug and showing minimal leaky activity in its absence, without altering the targeting specificity of the guide (Figure 4B).

[0213] Next, we investigated whether gene repression could be reversed in MEGA T cells. After transduction of RfxCas13d-DD with a CD46-targeting or non-targeting guide, we cultured MEGA T cells in the presence or absence of TMP. On day 5, TMP was removed or added, respectively. We then used flow cytometry to track surface CD46 expression over 72 hours, whereby we observed rapid kinetics of both induction and remission of CD46 downregulation, further confirming that gene repression was completely reversed to baseline (pre-induction) levels upon TMP removal (Figure 4C, Figure 13C). Remission of RfxCas13d activity was faster than induction, and the observed time 1 / 2 were 21.36 h after TMP removal or 23.71 h after TMP addition, respectively. To further characterize the rate of target suppression upon induction with TMP, we measured the kinetics of CD46 proteolysis in primary human T cells. We instantaneously blocked protein translation with cycloheximide (CHX) and performed flow cytometry tracking assays, and CD46 was remarkably stable, not reaching half-maximal expression before T cell apoptosis (Figure 13D). By fitting our data to an exponential decay curve, we determined that t 1 / 2 = 20.74 hours, suggesting that gene repression occurs almost immediately after TMP addition.

[0214] Having established precise, reversible binary (ON / OFF) gene regulation, we next sought to quantitatively modulate CD46 surface expression by controlling the dose of TMP administered to MEGA T cells. We performed TMP titration experiments and characterized the CD46 dose-response using flow cytometry (Figure 4D-E). We observed a sigmoidal response curve and found that TMP-dependent regulation of CD46 surface expression was highly sensitive, exhibiting a linear dose-response pattern (observed Hill coefficient = -1.425) in the range of approximately 1 to 100 nM.

[0215] Using our inducible RfxCas13d-DD, we have demonstrated that (as reported by others) 51~53 We further investigated whether there were any secondary effects in our system that affected either off-target protein expression or cell viability. Increasing TMP concentrations increased RfxCas13d-DD on-target cleavage activity, as measured by a decrease in CD46 expression, but mCherry expression and cell viability were unaffected (Figure 13E).

[0216] The ability of RfxCas13d to cleave and process guide arrays is determined by a HEPN-2 domain located at the C-terminus of the protein 44 To assess whether our C-terminal RfxCas13d-DD fusions could still process guide arrays for drug-controllable multiplexed knockdown, we extended this system to our method for suppressing inhibitory receptor upregulation in dysfunctional CAR T cells. We expanded MEGA HA-28z CAR T cells expressing a triple-guide array targeting LAG3 / PD-1 / TIM3 with varying levels of TMP in culture for 10 days and measured the surface expression of exhaustion markers by flow cytometry (Figure 4F). RfxCas13d-DD retained native array processing capabilities, enabling simultaneous suppression of all targeted exhaustion markers in a dose-dependent manner with knockdown efficiency comparable to that of the constitutive RfxCas13d system (Figure 1G).

[0217] Dynamic control of CAR-proximal signaling regulates MEGA CAR T cell activity and function Recent work from our group and others has shown that the safety and efficacy of CAR T cells can be improved by creating novel CAR designs that allow for artificial (exogenous) control of activity at the protein level. 46、62、63Taking a receptor-independent approach to controlling CAR activation, we decided to utilize our inducible system to target the proximal signaling proteins LCK and ZAP70, which transmit antigen recognition signals to downstream T cell activation circuits, using a multiplexed guide array (termed the "PROX array") (Figure 4G). By gradually varying the level of double knockdown of LCK and ZAP70 using various TMP dosages, we speculated that we could adjust the upper limit of CAR activation given a strong input signal, as in the case of an electronic amplitude limiter. Indeed, when stimulated with antigen-positive Nalm6-GD2 tumor cells, MEGA HA-28z CAR T cells showed a dose-dependent decrease in IL-2 secretion with increasing amounts of TMP in culture (Figure 4H).

[0218] Next, we evaluated the efficiency of tonic signal attenuation by measuring the expression of the T cell activation marker CD69 on MEGA HA-28z CAR T cells after 10 days of culture. The percentage of CD69+ cells in cells expressing the PROX array decreased linearly with increasing TMP levels (approximately 1–100 nM), whereas that in cells expressing the non-targeting control remained unchanged (Figure 4I). To examine off-target effects, we also quantified CD3 expression, which was not significantly altered by the addition of TMP in either group (Figure 4J). Finally, we measured the levels of PD-1, TIM3, and LAG3 compared with non-targeting and mock controls and observed a drug-dependent decrease in the surface phenotype of exhausted T cells (Figure 4K). Together, these data demonstrate the ability of MEGA to fine-tune the amount of CAR signaling that can be transmitted to downstream activation pathways by enforcing the individual steady-state levels of LCK and ZAP70.

[0219] Hypermultiplexed knockdown enables robust perturbation of diverse gene sets in primary human T cells We next attempted to extend MEGA to even higher multiplexing and target various gene sets involved in suppressing T cell function in the context of anti-tumor immunity. To do so, we designed a simple two-step cloning scheme that allows for the rapid and accurate assembly of RfxCas13d guide arrays of any length (Figure 5A, see Methods). We used this approach to construct new multiplexed arrays, which were then expressed in MEGA HA-28z CAR T cells to evaluate the knockdown of target gene sets at the RNA level.

[0220] We first targeted lactate metabolism, which has been proposed as a strategy to regulate T cell health and function. 64、65 We screened three different spacers per gene and assembled a three-plex guide array ("LAC array") targeting PDK1 (an inhibitor of pyruvate to acetyl-CoA conversion) and the lactate dehydrogenase isozymes LDHA and LDHB by linking the best spacers together (Figure 5B, Figure 14A). Notably, most of the spacers we tested resulted in significant gene silencing, demonstrating the overall robustness of RfxCas13d activity. We then expanded MEGA HA-28z CAR T cells expressing either the LAC array or a non-targeting control in culture for 10 days. We analyzed the transcriptome data at day 10 by RT-qPCR and noted significant upregulation of LDHA and LDHB in the non-targeting control. On the other hand, cells expressing the LAC array significantly suppressed LDHA, LDHB, and PDK1 to levels below baseline ( Fig. 5C ).

[0221] Previous studies have shown that inhibiting the PI3K / Akt pathway in T cells using small molecule inhibitors can enhance their stemness and antitumor activity. 66Based on these findings, we were interested in whether MEGA could also be used to inhibit PI3K / Akt signaling and downstream glycolytic metabolism in a specific and cell-specific manner. We constructed a 4-plex array ("GLY array") targeting the two major isoforms of AKT (AKT1 and AKT2) and the two major hexokinase isozymes (HK1 and HK2) expressed in human T cells (Figure 5D).

[0222] After expanding MEGA HA-28z CAR T cells in culture for 10 days, we measured RNA levels and confirmed strong upregulation of AKT1, AKT2, and HK2 in tonic-signaling CAR T cells, consistent with increased PI3K activation and glycolysis (Figure 5E). Expression of the GLY array counteracted this upregulation, as demonstrated by significant knockdown of all four target transcripts in the gene set.

[0223] Our group recently discovered that targeting MED12, a core component of the mediator kinase module (CKM), using CRISPR / Cas9 knockout enhances T cell effector function. 19 As an alternative strategy, herein we designed a guide array ("CKM array") targeting all four components of CKM, namely MED12, MED13, CCNC, and CDK8 (Figure 5F). Notably, although the CKM module was upregulated in HA-28z CAR T cells compared to mock-resting cells at day 10 of culture, cells expressing the CKM array showed significant knockdown of all four genes below baseline levels (Figure 5G).

[0224] After successfully demonstrating robust knockdown of various 3- and 4-gene sets, we further scaled up multiplexing to explore the potential limits of the number of genes that can be simultaneously targeted. We decided to target the surfaceome of exhausted CAR T cells because it is rich in unique and functional surface proteins with proven clinical relevance. We constructed a 5-plex array (the "SURF1 array") by adding two new spacers targeting FAS59 and CTLA467 to our previous guide array targeting the exhaustion markers LAG3, PDCD1, and HAVCR2. We generated MEGA HA-28z CAR T cells expressing the SURF1 array and observed that all five target transcripts were significantly knocked down to or below baseline levels at day 10 of culture compared to non-targeting controls (Figure 5H).

[0225] Finally, to stress-test the multiplexing capabilities of our system, we constructed a 10-plex guide array ("SURF2 array") targeting 10 genes: LAG3, FAS, CD568, ENTPD169, CD4670, TRAC, B2M, CTLA4, PDCD1, and HAVCR2. We isolated bulk RNA from day 10 MEGA HA-28z CAR T cells expressing the SURF2 array and quantified transcript abundance for all 10 genes relative to non-targeting controls (Figure 5H). Unusually, 8 of the 10 genes showed significant transcript knockdown without prior optimization of spacer sequence or position. Because RfxCas13d is a shared resource among 10 different crRNAs, we compared it with previous Cas9 retroactivity studies. 40、58As described in [ 1 ], we expected that the SURF2 array would exhibit lower knockdown efficiency than SURF1 due to competition between guides for limited Cas13 protein. Indeed, for all genes targeted in the SURF1 array, we observed slightly lower knockdown efficiency with SURF2. However, we did not observe a correlation between RfxCas13d activity and the abundance of target transcripts in all gene sets perturbed with MEGA (Figure 14B). Collectively, our data demonstrate the ability of MEGA to simultaneously knockdown putative functional gene sets ranging from 3 to 10 targets, providing a powerful approach for investigating protein complexes and pathways that remain unexplored in primary human T cells.

[0226] Interfering with the entire purinergic signaling pathway modulates peripheral metabolic flux and improves effector function in dysfunctional CAR T cells Solid tumors remain challenging for T cell-based therapy, in part due to the accumulation of immunosuppressive metabolites such as adenosine (ADO) within the tumor microenvironment. 71 Although both ATP and ADO are present at high (micromolar) levels in the solid tumor environment, 72 The purinergic pathway rapidly converts inflammatory ATP signals into immunosuppressive ADO signals through a cascade of enzymatic reactions involving four key surface proteins: CD39, CD73, and the adenosine receptors A2AR and A2BR (Figure 6A).

[0227] Having established a robust method for perturbing gene sets, we sought to explore whether disrupting the entire purinergic signaling cascade using our platform would delay ADO production, simultaneously accumulating ATP and enhancing the antitumor activity of dysfunctional CAR T cells. We constructed a four-gene guide array (PURI array) targeting ENTPD1 (CD39), NT5E (CD73), ADORA2A (A2AR), and ADORA2B (A2BR) (Figure 6B). To validate the functionality of the array, we generated MEGA HA-28z CAR T cells expressing either the PURI array or a non-targeting guide and analyzed RNA transcript abundance at day 10 of culture. We confirmed significant knockdown of all four target transcripts as measured by RT-qPCR (Figure 6C). We next performed ATP spike-in experiments to measure extracellular concentrations of ATP, AMP, and ADO to assess whether the PURI array regulated metabolic flux within the pathway (Figure 6D-F). MEGA HA-28z CAR T cells expressing the PURI array accumulated significantly more ATP and produced significantly less AMP and ADO than non-targeting controls. We also measured ATP and AMP levels at regular time intervals to calculate effective rate constants (Figure 6G-H). Expression of the PURI array significantly slowed ATP hydrolysis and AMP production compared to non-targeting controls.

[0228] Encouraged by these results, we set up a coculture assay with Nalm6-GD2 tumor cells to investigate whether disruption of the purinergic pathway could improve effector function in dysfunctional CAR T cells. We observed no significant changes in cytokine secretion from baseline tonic signaling, but antigen-stimulated PURI array-expressing MEGA HA-28z CAR T cells secreted significantly greater amounts of both IFN-γ and IL-2 compared with non-targeting controls (Figures 6I-J). Furthermore, knockdown by the PURI array significantly enhanced in vitro tumor killing and T cell activation and proliferation in response to antigen stimulation (Figures 6K-L).

[0229] MEGA enables metabolic engineering of CAR T cells to improve antitumor activity, modulate T cell differentiation, and reduce exhaustion We used MEGA to knock down four genes: AKT1, AKT2, HK1, and HK2. These genes encode functionally redundant protein isoforms involved in PI3K / Akt-driven aerobic glycolysis, a metabolic process initiated by CAR / TCR activation that leads to T cell effector differentiation and eventual exhaustion / dysfunction (Figure 15A). After disrupting aerobic glycolysis with MEGA, we observed a healthier (less exhausted) surface phenotype in HA-28z CAR T cells (Figures 15B-E). We also noticed a reduction in molecular / metabolic signatures associated with T cell dysfunction (Figures 15F-I). In functional assays, these cells resembled less effectors, were better able to grow in culture, and maintained antitumor activity in repeated challenge assays (Figure 15J). We then performed a direct comparison with state-of-the-art Cas9 knockouts. We introduced Cas9 RNPs targeting the same four genes into HA-28z CAR T cells by electroporation (Figure 15K-L). In contrast to MEGA, Cas9 knockouts reduced the ability of T cells to expand and control tumors in culture (Figure 15M-N). By examining the transcriptome, we found that these cells also downregulated p53 and upregulated DNA damage / G2M checkpoint pathways (Figure 15O-R). This is consistent with previous findings showing that Cas9 is genotoxic. Importantly, we did not observe differential enrichment of these pathways in MEGA CAR T cells (Figure 15O-R).

[0230] MEGA can be used to target and degrade specific transcripts in primary human T cells without off-target side activities Recent studies suggest that on-target RfxCas13d activity induces off-target side activities (nonspecific cleavage of RNA). We conducted experiments to investigate whether such off-target side activities exist in the MEGA system. We examined RfxCas13d RNA and protein levels to assess side activities resulting from on-target B2M cleavage in primary human T cells derived from two donors (Figure 16A). No off-target side activities were observed in primary human MEGA T cells at either the RNA or protein levels of mCherry-P2A-RfxCas13d (Figure 16B-H).

[0231] MEGA can be used to improve the antitumor efficacy of (CAR) T cells in vivo In vitro assays demonstrated that GLY arrays targeting HK1, HK2, AKT1, and AKT2 prevented CAR T cells from acquiring a dysfunctional phenotype and terminal effector differentiation through the disruption of aerobic glycolysis (Figure 15). Next, we performed in vivo tumor challenge experiments in mice to validate our in vitro findings. We prepared these same cells and injected them into mice bearing antigen-positive tumors (Nalm6-GD2) (Figure 17A). As measured by tumor bioluminescence imaging (BLI) over a 12-day period, the GLY array improved the anti-tumor response of MEGA HA-28z CAR T cells in vivo compared to non-targeting controls (Figures 17B-C).

[0232] Hypermultiplexed knockdown of 10 genes in MEGA HA-28z CAR T cells broadly and robustly reprograms the transcriptome at the single-cell level We performed single-cell RNA sequencing (scRNA-seq) of MEGA HA-28z CAR T cells expressing either the SURF2 array (Figure 5) or a non-targeting guide to investigate the impact of 10-gene knockdown on global gene expression in single cells. We performed unsupervised clustering of these cells according to their global gene expression signatures and visualized the resulting clusters in a two-dimensional UMAP plot, in which we observed clear separation of cells by sample ID (Figures 18A-C). This indicates that 1) SURF2 cells were similar to each other, suggesting robust and uniform perturbation by MEGA; and 2) SURF2 cells were significantly different from non-targeting control cells, suggesting that perturbation of the 10 genes resulted in widespread and global transcriptome changes. When we visualize the gene expression levels of the most significantly altered genes (SURF2 vs. NT) on a UMAP plot, we see that SURF2 increases the expression of genes related to glycolysis and mitophagy while simultaneously decreasing the expression of mitochondrial genes (Figure 18B-C).

[0233] Consideration Multiplexed silencing of endogenous genes has been a major focus of recent studies seeking to advance T cell therapy or develop similar “off-the-shelf” modalities. 3、25、27Despite the constantly expanding list of promising gene targets, scaling up multiplexed perturbations to more than three genes quickly becomes difficult with current methods. Therefore, addressing larger gene sets, such as metabolic pathways (which often contain several protein isoforms with overlapping or compensatory functions) or functional heteromeric protein complexes, has been essentially impossible. Herein, we demonstrate the simultaneous suppression of up to 10 endogenous genes with a single guide array without the induction of DNA double-strand breaks. We also demonstrate the robust knockdown of multiple protein isoforms, multiunit protein complexes, signaling cascades, and entire immunosuppressive metabolic pathways to reshape the chemical composition of the tumor microenvironment. We believe that MEGA will pave the way for further metabolic pathway discovery and manipulation studies in primary human T cells. This is particularly appealing, as these studies have previously only been possible in microorganisms and plants.

[0234] Unlike Cas9-based tools, MEGA does not incur significant tradeoffs with increased complexity, such as increased genotoxicity risk or reduced editing specificity. Our results also demonstrate that MEGA does not cause observable off-target effects or secondary cleavage effects that may impede T cell survival. While higher multiplexing (10 or more genes) may require optimization of spacer position or sequence to robustly suppress all target genes, we describe a rapid and easy assembly method for arbitrarily long guide arrays to accelerate this process.

[0235] By applying MEGA to an established model of primary human CAR T cell dysfunction, we demonstrate that MEGA does not interfere with CAR expression or signaling, and can coexist with T cell antitumor functionality. Tonic signaling MEGA CAR T cells suppress the active transcription of these genes due to a cell-intrinsic exhaustion program. 7、46 Upregulation and epigenetics 47Despite upregulation, the expression of multiple inhibitory receptors can be robustly suppressed. Furthermore, our combinatorial CRISPR screen helped elucidate the synergistic relationships between putative exhaustion-associated genes described in previous CRISPR / Cas9 single knockout screens, and we show that paired knockdown of these newly identified gene combinations broadly enhances antitumor activity. Specifically, double knockdown of CBLB + FAS best preserved the ability of dysfunctional CAR T cells to control tumor growth upon multiple rechallenges, highlighting potential synergistic mechanisms that require further investigation.

[0236] While our proof-of-concept study focused on a small subset of gene pairs, MEGA is highly scalable and can be used to screen larger subsets of functionally related gene pairs (e.g., metabolic gene sets) or even comprehensive 2D whole genome sets. In such applications, pre-validated spacer sequences reduce false negatives and improve signal-to-noise for quantitative gene interaction (GI) mapping. 55 While spacer-dependent variation is common to all CRISPR systems, especially dCas9-based tools, 29 , our group and others have begun to develop more accurate algorithms for predicting and generating significantly more efficient RfxCas13d spacer sequences. 48、50、73 .

[0237] To date, CRISPR systems have not been stably expressed in primary human T cells for therapeutic applications. 3 A recent groundbreaking Phase 1 clinical trial was the first to use Cas9-edited T cells for infusion into patients. 26 However, this study and other preclinical studies 14relied exclusively on transient Cas9 electroporation to introduce permanent edits into the genome. One group showed that electroporation of recombinant Cas13d could achieve single-gene knockdown in T cells. 74 However, the scope of this method is limited to highly transient perturbations within a specific time frame during production, and it is unclear whether the knockdown efficacy is robust to other endogenous genes. Finally, Cas9-fused effectors either approach or exceed the packaging limits of conventional lentiviruses. 75、76 However, the weak expression levels of these constructs in primary human T cells make them impractical for sustained therapeutic application. 18、77 In contrast to these studies, we report for the first time a powerful genetically encoded CRISPR system that allows for tuning both the magnitude and duration of multiple gene perturbations at any time during the lifespan of a cell.

[0238] In contrast to binary and irreversible gene knockout approaches, precise control of transcript expression may enhance the safety and efficacy of adoptive T cell therapy. Specifically, controlling CAR activation is of great interest, but current cell-intrinsic approaches rely on laborious protein engineering targeted at the receptor itself, and the results can vary dramatically due to inherent differences between receptor structures that affect function. 11 In our study, we show that using MEGA, we can modulate the input / output function of CARs independently of the receptor used by linking the dosage of TMP to the expression of the proximal signaling molecules LCK and ZAP70. Our amplitude limiter functions across a range of input signal strengths, from basal tonic signaling to antigen stimulation by tumor cells.

[0239] Beyond controlling CAR signaling, MEGA allows for precise tuning of gene set expression and pathway activity in T cells, even after manufacturing. This could help find the "sweet spot" between T cell dysfunction and overactivity or autoimmunity in patients after infusion. 78、79 For example, in this study, we confirm previous findings that IRF4 is essential for T cell proliferation, despite its role in driving exhaustion. Although extremely high or low levels of IRF4 can inhibit T cell function, enforcing intermediate levels of expression may be beneficial in the context of antitumor immunity. 7、31、35 Recent studies have also shown that lower TET2 expression correlates with enhanced T cell memory, whereas complete genetic loss of TET2 can generate hyperproliferative T cells with genomic instability. 80 Notably, methods for specifically modulating the metabolism of engineered T cells in patients have been established as an unmet need. 10 , MEGA could therefore open the door to a new set of metabolic interventions that hold promise for cancer immunotherapy.

[0240] Finally, the transient and reversible nature of MEGAs broadens the scope of current gene perturbations to include previously unexplored temporal manipulation of specific genes or gene sets. For example, reversible knockdown of B2M / HLA could be developed as an alternative to traditional "safety switches" by leveraging immune rejection to control T cell persistence in patients. Meanwhile, conditional degradation of specific mRNA transcripts or long non-coding RNA (lncRNA) transcripts during highly dynamic T cell processes (e.g., activation, differentiation, and exhaustion) could provide novel biological and therapeutic insights.

[0241] In summary, we have developed MEGA, a synthetic biology-driven platform for versatile transcriptome control in primary human T cells using CRISPR / Cas13d. Our work highlights various T cell engineering applications enabled by MEGA and addresses key limitations posed by state-of-the-art CRISPR / Cas9 gene editing technologies (Figure 7). We envision MEGA as an invaluable addition to the synthetic immunology toolkit: engineered MEGA T cells will possess a vast array of novel capabilities for applications ranging from basic biological discovery to improved cell-based immunotherapies for cancer and other diseases.

[0242] References TIFF2025529871000005.tif244160TIFF2025529871000006.tif235160TIFF20255298710 00007.tif232160TIFF2025529871000008.tif232160TIFF2025529871000009.tif151160

[0243] It is understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes in light thereof will be suggested to those skilled in the art and are within the spirit and scope of this application and the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

[0244] Table 1. Exemplary crRNA spacer sequences TIFF2025529871000010.tif173128

[0245] Table 2. Exemplary crRNA spacer sequences TIFF2025529871000011.tif228140TIFF2025529871000012.tif237140TIFF2025529871000013.tif237140TIFF2025529871000014.tif37140

[0246] Table 3: Cloning primers TIFF2025529871000015.tif205142

[0247] Unofficial sequence listing RfxCas13d amino acid sequence (SEQ ID NO: 1) TIFF2025529871000016.tif88159 >RfxCas13d-2XNLS-FLAG nucleic acid sequence (SEQ ID NO: 2) TIFF2025529871000017.tif190159 RfxCas13d-2XNLS-FLAG amino acid sequence (SEQ ID NO: 3) TIFF2025529871000018.tif64160 Bold:RfxCas13d Underline: 2XNLS Italics and underline: FLAG tag DHFR DD domain nucleic acid sequence (SEQ ID NO: 4) TIFF2025529871000019.tif31159 DHFR DD domain amino acid sequence (SEQ ID NO: 5) TIFF2025529871000020.tif12159 >RfxCas13d-2XNLS-DD-FLAG nucleic acid sequence (SEQ ID NO: 6) TIFF2025529871000021.tif222159 RfxCas13d-2XNLS-DD-FLAG amino acid sequence (SEQ ID NO: 7) TIFF2025529871000022.tif69160 Underline: RfxCas13d Bold:2XNLS Italics: DHFR DD domain Bold and underlined: FLAG tag Exemplary 10-plex guide array nucleic acid sequence of RfxCas13d (SEQ ID NO: 8) TIFF2025529871000023.tif36160 Bold: DR30 (30 nucleotide direct repeat) Underlined: DR36 (36 nucleotide direct repeat) Direct repeat sequences: TIFF2025529871000024.tif13128 Linker sequence: TIFF2025529871000025.tif4128

Claims

1. (i) a class 2 VI-D CRISPR effector with RNA-guided RNA endonuclease activity; and (ii) A guide array comprising multiple CRISPR-associated RNA (crRNA) molecules, each of which independently comprises a direct repeat sequence and a spacer sequence that binds to a target RNA, and each of which binds to different target mRNAs or different regions of the same target mRNA. Genetically modified T cells, including

2. 2. The genetically modified T cell of claim 1, comprising a nucleic acid encoding (i), a nucleic acid encoding (ii), or a nucleic acid encoding both (i) and (ii), wherein the nucleic acid encoding (i) or (ii), or both (i) and (ii), is stably integrated into the genome of the T cell.

3. The genetically modified T cell of claim 1 or 2, wherein the class 2 VI-D CRISPR effector having RNA-guided RNA endonuclease activity constitutes a fusion protein comprising a destabilization domain (DD).

4. The genetically modified T cell of any one of claims 1 to 3, wherein the class 2 VI-D CRISPR effector having RNA-guided RNA endonuclease activity is a Cas13 nuclease selected from the group consisting of Cas13a, Cas13b, 13bt1, Cas13bt2, Cas13c, Cas13d, RfxCas13d, Cas13e, and functional variants thereof.

5. The genetically modified T cell of any one of claims 1 to 4, wherein the guide array is a multicistronic array comprising multiple crRNA molecules.

6. The genetically modified T cell of any one of claims 1 to 5, wherein the guide array comprises 2 to 10 crRNA molecules.

7. The genetically modified T cell of any one of claims 1 to 5, wherein the guide array comprises more than 10 crRNA molecules.

8. The genetically modified T cell of any one of claims 1 to 7, wherein the guide array comprises a crRNA molecule that binds to an mRNA encoding a protein associated with T cell exhaustion.

9. 9. The genetically modified T cell of claim 8, wherein the protein associated with T cell exhaustion is selected from the group consisting of TOX, TOX2, NR4A1, NR4A2, NR4A3, TET2, IRF4, JUNB, BATF3, DHX37, FLI1, ZC3H12A, SOCS1, TCEB2, PDCD1 (PD-1), HAVCR2 (TIM3), LAG3, CTLA4, TIGIT, FAS, TRAC, CBLB, RASA2, PTPN2, CD5, ENTPD1 (CD39), CD46, B2M, ZAP70, LCK, AKT1, AKT2, HK1, HK2, NT5E (CD73), ADORA2A, ADORA2B, LDHA, LDHB, CD147, MCT1, MCT4, GAPDH, and combinations thereof.

10. 10. The genetically modified T cell of any one of claims 1 to 9, further comprising a chimeric antigen receptor (CAR).

11. The genetically modified T cell of claim 10, wherein the CAR binds to an antigen expressed by a tumor.

12. CAR is Her-2, B7-H3, GPC2, GD2, CD19, CD20, CD22, MAGE, BAGE, CAGE, GAGE, HAGE, LAGE, PAGE, PRAME, NY-ESO-1 , NY-SEO-1, Tyrosinase, Melan-A / MART, gpl00, TRP-1, TRP-2, CD30, EGFR, EGFRvIII, FAP, CD33, CD123, PD-L1, IGF1 12. The genetically modified T cell of claim 10 or 11, which binds to an antigen selected from the group consisting of R, CD4, CSPG4, B7-H4, NKG2D, CS1, CD138, EpCAM, EBNA3C, GPA7, CD244, CA-125, ETA, CEA, CD52, MUC5AC, c-Met, FAB, WT-1, PSMA, AFP, BCMA, mesothelin, GPC3, MUC1, and CTAG1B.

13. The genetically modified T cell of any one of claims 10-12, wherein intracellular signaling by the CAR upregulates a T cell exhaustion marker in a control T cell that does not comprise (i) and (ii) of claim 1.

14. The genetically modified T cell of claim 13, wherein the exhaustion marker is selected from the group consisting of LAG3, PDCD1 (PD-1), HAVCR2 (TIM3), CTLA4, ENTPD1, ​​and combinations thereof.

15. The genetically modified T cell of any one of claims 10-14, wherein the guide array comprises crRNA molecules that bind to mRNA encoding LAG3, PDCD1 (PD-1), HAVCR2 (TIM3), CTLA4, ENTPD1, ​​or a combination thereof.

16. The genetically modified T cell of any one of claims 10-15, wherein the guide array comprises one or more crRNA molecules that bind to the mRNA expressed by the CAR.

17. 17. The genetically modified T cell of any one of claims 1 to 16, wherein the T cell is selected from the group consisting of a human T cell and a primary human T cell.

18. (i) a class 2 VI-D CRISPR effector with RNA-guided RNA endonuclease activity; and (ii) A guide array comprising multiple CRISPR-associated RNA (crRNA) molecules, each of which independently comprises a direct repeat sequence and a spacer sequence that binds to a target RNA, and each of which binds to different target mRNAs or different regions of the same target mRNA. A nucleic acid encoding

19. 19. The nucleic acid of claim 18, wherein the class 2 VI-D CRISPR effector having RNA-guided RNA endonuclease activity is a Cas13 nuclease selected from the group consisting of Cas13a, Cas13b, 13bt1, Cas13bt2, Cas13c, Cas13d, RfxCas13d, Cas13e, and functional variants thereof.

20. 20. The nucleic acid of claim 18 or 19, wherein the guide array is a multicistronic array comprising multiple crRNA molecules.

21. 21. The nucleic acid of any one of claims 18 to 20, wherein the guide array comprises 2 to 10 or more crRNA molecules.

22. 22. The nucleic acid of any one of claims 18-21, wherein said guide array comprises a crRNA molecule that binds to an mRNA encoding a protein associated with T cell exhaustion.

23. 23. The nucleic acid of claim 22, wherein the protein associated with T cell exhaustion is selected from the group consisting of TOX, TOX2, NR4A1, NR4A2, NR4A3, TET2, IRF4, JUNB, BATF3, DHX37, FLI1, ZC3H12A, SOCS1, TCEB2, PDCD1 (PD-1), HAVCR2 (TIM3), LAG3, CTLA4, TIGIT, FAS, TRAC, CBLB, RASA2, PTPN2, CD5, ENTPD1 (CD39), CD46, B2M, ZAP70, LCK, AKT1, AKT2, HK1, HK2, NT5E (CD73), ADORA2A, ADORA2B, LDHA, LDHB, CD147, MCT1, MCT4, GAPDH, and combinations thereof.

24. (i) an expression cassette comprising a nucleic acid sequence encoding a class 2 VI-D CRISPR effector having RNA-guided RNA endonuclease activity; and (ii) An expression cassette comprising a nucleic acid sequence encoding a guide array comprising multiple crRNA molecules, wherein the crRNA molecules comprise a direct repeat sequence and a spacer sequence that binds to a target mRNA, and the crRNA molecules bind to different target mRNAs or different regions of the same target mRNA. A system for multiplexed transcriptome control, comprising:

25. 25. The system of claim 24, wherein the class 2 VI-D CRISPR effector having RNA-guided RNA endonuclease activity is a Cas13 nuclease selected from the group consisting of Cas13a, Cas13b, 13bt1, Cas13bt2, Cas13c, Cas13d, RfxCas13d, Cas13e, and functional variants thereof.

26. 26. The system of claim 24 or 25, wherein the guide array is a multicistronic array comprising a plurality of crRNA molecules.

27. 27. The system of any one of claims 24-26, wherein the guide array comprises 2 to 10 or more crRNA molecules.

28. 28. The system of any one of claims 24-27, wherein the guide array comprises crRNA molecules that bind to mRNAs encoding proteins associated with T cell exhaustion.

29. 29. The system of claim 28, wherein the protein associated with T cell exhaustion is selected from the group consisting of TOX, TOX2, NR4A1, NR4A2, NR4A3, TET2, IRF4, JUNB, BATF3, DHX37, FLI1, ZC3H12A, SOCS1, TCEB2, PDCD1 (PD-1), HAVCR2 (TIM3), LAG3, CTLA4, TIGIT, FAS, TRAC, CBLB, RASA2, PTPN2, CD5, ENTPD1 (CD39), CD46, B2M, ZAP70, LCK, AKT1, AKT2, HK1, HK2, NT5E (CD73), ADORA2A, ADORA2B, LDHA, LDHB, CD147, MCT1, MCT4, GAPDH, and combinations thereof.

30. (iii) the system of any one of claims 24 to 29, further comprising T cells or primary T cells.

31. The system of claim 30, wherein the T cells or primary T cells further comprise a chimeric antigen receptor (CAR).

32. 32. The system of claim 31, wherein the CAR binds to an antigen expressed by the tumor.

33. CAR is Her-2, B7-H3, GPC2, GD2, CD19, CD20, CD22, MAGE, BAGE, CAGE, GAGE, HAGE, LAGE, PAGE, PRAME, NY-ESO- 1, NY-SEO-1, Tyrosinase, Melan-A / MART, gpl00, TRP-1, TRP-2, CD30, EGFR, EGFRvIII, FAP, CD33, CD123, PD-L1, IG The system of claim 31 or 32, which binds to an antigen selected from the group consisting of F1R, CD4, CSPG4, B7-H4, NKG2D, CS1, CD138, EpCAM, EBNA3C, GPA7, CD244, CA-125, ETA, CEA, CD52, MUC5AC, c-Met, FAB, WT-1, PSMA, AFP, BCMA, mesothelin, GPC3, MUC1, and CTAG1B.

34. The system of any one of claims 31-33, wherein expression and intracellular signaling by the CAR upregulates a T cell exhaustion marker in control T cells that do not comprise (i) and (ii) of claim 24.

35. The system of claim 34, wherein the exhaustion marker is selected from LAG3, PDCD1 (PD-1), HAVCR2 (TIM3), CTLA4, ENTPD1, ​​or a combination thereof.

36. The system of any one of claims 31-35, wherein the guide array comprises crRNA molecules that bind to mRNA encoding LAG3, PDCD1 (PD-1), HAVCR2 (TIM3), CTLA4, ENTPD1, ​​or a combination thereof.

37. 37. The system of any one of claims 31-36, wherein said guide array comprises one or more crRNA molecules that bind to the mRNA expressed by the CAR.

38. The system of any one of claims 30 to 37, wherein the T cells are selected from the group consisting of human T cells and primary human T cells.

39. (i) transducing T cells with an expression vector comprising a nucleic acid sequence encoding a class 2 VI-D CRISPR effector with RNA-guided RNA endonuclease activity; and (ii) transducing the T cells with an expression vector comprising a nucleic acid sequence encoding a guide array comprising a plurality of CRISPR-associated RNA (crRNA) molecules, wherein the crRNA molecules independently comprise a direct repeat sequence and a spacer sequence that binds to a target mRNA, and the crRNA molecules bind to different target mRNAs or different regions of the same target mRNA. wherein the modified T cells are produced by a method for producing the modified T cells, the method comprising:

40. 40. The method of claim 39, further comprising (iii) transducing the T cells with an expression vector comprising a nucleic acid sequence encoding a CAR.

41. 41. The method of claim 39 or 40, wherein step (i) is performed before step (ii); step (iii) is performed before step (ii); or steps (i) and (iii) are performed before step (ii).

42. 42. The method of any one of claims 39-41, wherein (i), (ii), and / or (iii) are stably integrated into the genome of said T cell.

43. 43. The method of any one of claims 39 to 42, wherein the class 2 VI-D CRISPR effector having RNA-guided RNA endonuclease activity is a Cas13 nuclease selected from the group consisting of Cas13a, Cas13b, 13bt1, Cas13bt2, Cas13c, Cas13d, RfxCas13d, Cas13e, and functional variants thereof.

44. 44. The method of any one of claims 39 to 43, wherein the guide array is a multicistronic array comprising a plurality of crRNA molecules.

45. 45. The method of any one of claims 39-44, wherein said guide array comprises 2 to 10 or more crRNA molecules.

46. The method of any one of claims 39-45, wherein said guide array comprises crRNA molecules that bind to mRNAs encoding proteins associated with T cell exhaustion.

47. 47. The method of claim 46, wherein the protein associated with T cell exhaustion is selected from the group consisting of TOX, TOX2, NR4A1, NR4A2, NR4A3, TET2, IRF4, JUNB, BATF3, DHX37, FLI1, ZC3H12A, SOCS1, TCEB2, PDCD1 (PD-1), HAVCR2 (TIM3), LAG3, CTLA4, TIGIT, FAS, TRAC, CBLB, RASA2, PTPN2, CD5, ENTPD1 (CD39), CD46, B2M, ZAP70, LCK, AKT1, AKT2, HK1, HK2, NT5E (CD73), ADORA2A, ADORA2B, LDHA, LDHB, CD147, MCT1, MCT4, GAPDH, and combinations thereof.

48. The method of any one of claims 40 to 47, wherein the CAR binds to an antigen expressed by the tumor.

49. CAR is Her-2, B7-H3, GPC2, GD2, CD19, CD20, CD22, MAGE, BAGE, CAGE, GAGE, HAGE, LAGE, PAGE, PRAME, NY-ESO-1 , NY-SEO-1, Tyrosinase, Melan-A / MART, gpl00, TRP-1, TRP-2, CD30, EGFR, EGFRvIII, FAP, CD33, CD123, PD-L1, IGF1 The method of any one of claims 40-48, wherein the antibody binds to an antigen selected from the group consisting of R, CD4, CSPG4, B7-H4, NKG2D, CS1, CD138, EpCAM, EBNA3C, GPA7, CD244, CA-125, ETA, CEA, CD52, MUC5AC, c-Met, FAB, WT-1, PSMA, AFP, BCMA, mesothelin, GPC3, MUC1, and CTAG1B.

50. 50. The method of any one of claims 40-49, wherein expression and intracellular signaling by the CAR upregulates T cell exhaustion markers in control T cells that do not contain the expression vector of steps (i) and (ii).

51. 51. The method of claim 50, wherein the exhaustion marker is selected from LAG3, PDCD1 (PD-1), or HAVCR2 (TIM3), or a combination thereof.

52. 52. The method of any one of claims 40-51, wherein said guide array comprises crRNA molecules that bind to the mRNA expressed by the CAR.

53. 53. The method of any one of claims 39 to 52, wherein the T cells are selected from the group consisting of human T cells and primary human T cells.

54. Class 2 VI-D CRISPR effectors with RNA-guided RNA endonuclease activity covalently linked to a destabilization domain (DD) polypeptide A fusion protein comprising:

55. 55. The fusion protein of claim 54, wherein the class 2 VI-D CRISPR effector having RNA-guided RNA endonuclease activity is a Cas13 nuclease selected from the group consisting of Cas13a, Cas13b, 13bt1, Cas13bt2, Cas13c, Cas13d, RfxCas13d, Cas13e, and functional variants thereof.

56. 56. The fusion protein of claim 54 or 55, wherein the DD comprises an E. coli dihydrofolate reductase DD linked to the C-terminus of a class 2 VI-D CRISPR effector having RNA-guided RNA endonuclease activity.

57. A nucleic acid encoding the fusion protein of any one of claims 54 to 56.

58. (a) (i) an expression vector comprising a nucleic acid sequence encoding the fusion protein of any one of claims 54 to 56; and (ii) an expression vector comprising a nucleic acid sequence encoding a guide array comprising a crRNA molecule, the crRNA molecule comprising a direct repeat sequence and a spacer sequence that binds to a target mRNA expressed by the target gene; transducing T cells by (b) contacting the T cells with a compound that binds to and stabilizes the DD, wherein expression of the target gene is decreased in the presence of the compound compared to expression of the target gene in the absence of the compound. thereby controlling gene expression in T cells. A method for controlling gene expression in T cells.

59. 59. The method of claim 58, wherein the fusion protein is degraded in the T cell in the absence of the compound.

60. 60. The method of claim 58 or 59, wherein expression of the target gene is increased following removal of the compound, thereby reversibly regulating expression of the target gene.

61. 61. The method of any one of claims 58-60, wherein said guide array comprises multiple crRNA molecules that bind to different target mRNAs or different regions of the same target mRNA.

62. 62. The method of any one of claims 58 to 61, wherein expression of the target gene is regulated by said compound in a dose-dependent manner.

63. 63. The method of any one of claims 58-62, wherein the compound is trimethoprim (TMP).

64. In a T cell or population of T cells, (i) a class 2 VI-D CRISPR effector with RNA-guided RNA endonuclease activity; and (ii) A library of guide arrays, wherein each guide array comprises one or more CRISPR-associated RNA (crRNA) molecules, each crRNA molecule comprising a direct repeat sequence and a spacer sequence that binds to a target RNA, and wherein the one or more crRNA molecules bind to different target mRNAs or different regions of the same target mRNA. expressing the culturing the T cells to produce a clonal population of expanded T cells; and determining whether the expanded clonal population of T cells is enriched or depleted for guide arrays.

1. A method for screening to identify regulators of T cell activity, comprising: wherein when the guide array is enriched, the target mRNA encodes a negative regulator of T cell activity, or when the guide array is depleted, the target mRNA encodes a positive regulator of T cell activity. The method.

65. 65. The method of claim 64, wherein the T cell activity is T cell proliferation, increased cytokine secretion, or increased tumor cell killing.

66. The method of claim 64 or 65, wherein the T cells comprising the enriched guide array have an effector memory phenotype and the T cells comprising the depleted guide array have a central memory phenotype or a stem cell memory phenotype.

67. The method of any one of claims 64-66, wherein each guide array comprises a crRNA molecule that binds to an mRNA encoding a protein associated with T cell exhaustion.

68. 68. The method of claim 67, wherein the protein associated with T cell exhaustion is selected from the group consisting of TOX, TOX2, NR4A1, NR4A2, NR4A3, TET2, IRF4, JUNB, BATF3, DHX37, FLI1, ZC3H12A, SOCS1, TCEB2, PDCD1 (PD-1), HAVCR2 (TIM3), LAG3, CTLA4, TIGIT, FAS, TRAC, CBLB, RASA2, PTPN2, CD5, ENTPD1 (CD39), CD46, B2M, ZAP70, LCK, AKT1, AKT2, HK1, HK2, NT5E (CD73), ADORA2A, ADORA2B, LDHA, LDHB, CD147, MCT1, MCT4, GAPDH, and combinations thereof.

69. The method of any one of claims 64-68, wherein said library of guide arrays comprises one or more individual guide arrays, including a multicistronic array comprising a plurality of crRNA molecules.

70. 70. The method of any one of claims 64-69, wherein said individual guide array comprises 2 to 10 crRNA molecules.

71. 71. The method of any one of claims 64-70, wherein said individual guide array comprises a pair of crRNA molecules.

72. 72. The method of any one of claims 64 to 71, wherein the class 2 VI-D CRISPR effector having RNA-guided RNA endonuclease activity is a Cas13 nuclease selected from the group consisting of Cas13a, Cas13b, 13bt1, Cas13bt2, Cas13c, Cas13d, RfxCas13d, Cas13e, and functional variants thereof.

73. The method of any one of claims 64-72, wherein the T cell further expresses a chimeric antigen receptor (CAR).

74. The method of claim 73, wherein the CAR binds to an antigen expressed by the tumor.

75. CARs express Her-2, B7-H3, GPC2, GD2, CD19, CD20, CD22, MAGE, BAGE, CAGE, GAGE, HAGE, LAGE, PAGE, PRAME, NY-ESO-1, NY-SEO-1, tyrosinase, Melan-A / MART, gpl00, TRP-1, TRP-2, CD30, EGFR, EGFRvIII, FAP, CD33, CD123, PD-L1, I The method of claim 73 or 74, wherein the antibody binds to an antigen selected from the group consisting of GF1R, CD4, CSPG4, B7-H4, NKG2D, CS1, CD138, EpCAM, EBNA3C, GPA7, CD244, CA-125, ETA, CEA, CD52, MUC5AC, c-Met, FAB, WT-1, PSMA, AFP, BCMA, mesothelin, GPC3, MUC1, and CTAG1B.

76. 76. The method of any one of claims 64-75, wherein determining whether the guide array is enriched or depleted in a clonal population of expanded T cells comprises sequencing guide RNAs present in said T cells.

77. (i) an expression vector comprising a nucleic acid sequence encoding a class 2 VI-D CRISPR effector with RNA-guided RNA endonuclease activity; and (ii) An expression vector comprising a nucleic acid sequence encoding a guide array comprising one or more crRNA molecules, wherein the crRNA molecules independently comprise a direct repeat sequence and a spacer sequence that binds to a target mRNA, and the crRNA molecules bind to different target mRNAs or different regions of the same target mRNA.

1. A method for increasing T cell proliferation, comprising transducing T cells with wherein proliferation of the T cells is increased relative to control T cells expressing a non-targeting control guide array comprising one or more crRNA molecules that do not bind to the target mRNA of (ii). The method.

78. 78. The method of claim 77, wherein said guide array comprises crRNA molecules that bind to mRNAs encoding proteins associated with T cell exhaustion.

79. 79. The method of claim 78, wherein the protein associated with T cell exhaustion is selected from the group consisting of TOX, TOX2, NR4A1, NR4A2, NR4A3, TET2, IRF4, JUNB, BATF3, DHX37, FLI1, ZC3H12A, SOCS1, TCEB2, PDCD1, HAVCR2, LAG3, CTLA4, TIGIT, FAS, TRAC, CBLB, RASA2, PTPN2, CD5, ENTPD1 (CD39), CD46, B2M, ZAP70, LCK, AKT1, AKT2, HK1, HK2, NT5E (CD73), ADORA2A, ADORA2B, LDHA, LDHB, CD147, MCT1, MCT4, GAPDH, and combinations thereof.

80. 80. The method of any one of claims 77-79, wherein said guide array comprises a multicistronic array comprising a plurality of crRNA molecules.

81. 81. The method of any one of claims 77-80, wherein said guide array comprises 2 to 10 or more crRNA molecules.

82. 82. The method of any one of claims 77 to 81, wherein the class 2 VI-D CRISPR effector having RNA-guided RNA endonuclease activity is a Cas13 nuclease selected from the group consisting of Cas13a, Cas13b, 13bt1, Cas13bt2, Cas13c, Cas13d, RfxCas13d, Cas13e, and functional variants thereof.

83. The method of any one of claims 77-82, further comprising (iii) transducing the T cell with an expression vector comprising a nucleic acid sequence encoding the CAR.

84. The method of claim 83, wherein the CAR binds to an antigen expressed by the tumor.

85. CARs express Her-2, B7-H3, GPC2, GD2, CD19, CD20, CD22, MAGE, BAGE, CAGE, GAGE, HAGE, LAGE, PAGE, PRAME, NY-ESO-1, NY-SEO-1, tyrosinase, Melan-A / MART, gpl00, TRP-1, TRP-2, CD30, EGFR, EGFRvIII, FAP, CD33, CD123, PD-L1, I The method of claim 83 or 84, wherein the antibody binds to an antigen selected from the group consisting of GF1R, CD4, CSPG4, B7-H4, NKG2D, CS1, CD138, EpCAM, EBNA3C, GPA7, CD244, CA-125, ETA, CEA, CD52, MUC5AC, c-Met, FAB, WT-1, PSMA, AFP, BCMA, mesothelin, GPC3, MUC1, and CTAG1B.

86. 20. A method for treating a tumor in a subject, comprising administering to the subject the genetically modified T cells of any one of claims 1 to 17, wherein the modified T cells kill tumor cells in the subject, thereby treating the tumor.

87. 20. A method for enhancing anti-tumor activity of T cells, comprising contacting a tumor cell with the genetically modified T cell of any one of claims 1-17, wherein contacting the tumor cell with the genetically modified T cell increases expression of anti-tumor cytokines or kills the tumor cell, thereby enhancing anti-tumor activity compared to control T cells that do not comprise (i) or (ii), or both (i) and (ii).

88. 88. The method of claim 87, which is an in vitro method.

89. 88. The method of claim 87, which is an in vivo method.

90. A guide array comprising multiple CRISPR-associated RNA (crRNA) molecules, wherein the crRNA molecules comprise a direct repeat sequence and a spacer sequence that binds to a target RNA, and the crRNA molecules bind to different target mRNAs or different regions of the same target mRNA.

91. 91. The guide array of claim 90, wherein said guide array is a multicistronic array comprising a plurality of crRNA molecules.

92. 92. The guide array of claim 90 or 91, comprising 2 to 10 crRNA molecules.

93. 92. The guide array of claim 90 or 91, comprising more than 10 crRNA molecules.

94. 94. The guide array of any one of claims 90-93, comprising a crRNA molecule that binds to an mRNA encoding a protein associated with T cell exhaustion.

95. 95. The guide array of claim 94, wherein the protein associated with T cell exhaustion is selected from the group consisting of TOX, TOX2, NR4A1, NR4A2, NR4A3, TET2, IRF4, JUNB, BATF3, DHX37, FLI1, ZC3H12A, SOCS1, TCEB2, PDCD1 (PD-1), HAVCR2 (TIM3), LAG3, CTLA4, TIGIT, FAS, TRAC, CBLB, RASA2, PTPN2, CD5, ENTPD1 (CD39), CD46, B2M, ZAP70, LCK, AKT1, AKT2, HK1, HK2, NT5E (CD73), ADORA2A, ADORA2B, LDHA, LDHB, CD147, MCT1, MCT4, GAPDH, and combinations thereof.

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