Protocol for in vivo CRISPR screening using selective CRISPR antigen removal lentiviral vectors

Lane-Reticker, Sarah Kate; Kessler, Emily; Muscato, Audrey J.; Kim, Sarah Y.; Doench, John G.; Yates, Kathleen B.; Manguso, Robert T.; Dubrot, Juan

doi:10.1016/j.xpro.2023.102082

Cited by 4 publications

(3 citation statements)

References 10 publications

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“…We also included 5 non-targeting control guides and a set of 3 depletion and 2 enrichment control genes, including Cd47 and Pten , respectively, based on previous studies (32). We then transduced the non-rejected SCC 35 cell line with the sgRNA library and Cas9 using selective CRISPR antigen removal (SCAR) lentiviral vectors in order to induce per- cell knock-outs in vitro, while avoiding immune-mediated rejection of CRISPR/Cas9 components in vivo ( Figure 4B ) (33,34). The edited SCC35 cell lines were then split into 3 replicates at day 0 and sequenced to verify high sgRNA recovery rates, which ranged from 93.5%-95.6% of the original guide library ( Supplementary Figure 4 A).…”

Section: Resultsmentioning

confidence: 99%

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Temporal genomic analysis of melanoma rejection identifies regulators of tumor immune evasion

Shvefel,

Pai,

Cao

et al. 2023

Preprint

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Decreased intra-tumor heterogeneity (ITH) correlates with increased patient survival and immunotherapy response. However, even highly homogenous tumors may display variability in their aggressiveness, and how immunologic-factors impinge on their aggressiveness remains understudied. Here we studied the mechanisms responsible for the immune-escape of murine tumors with low ITH. We compared the temporal growth of homogeneous, genetically-similar single-cell clones that are rejected vs. those that are not- rejected after transplantationin-vivousing single-cell RNA sequencing and immunophenotyping. Non- rejected clones showed high infiltration of tumor-associated-macrophages (TAMs), lower T-cell infiltration, and increased T-cell exhaustion compared to rejected clones. Comparative analysis of rejection- associated gene expression programs, combined within-vivoCRISPR knockout screens of candidate mediators, identifiedMif(macrophage migration inhibitory factor) as a regulator of immune rejection.Mifknockout led to smaller tumors and reversed non-rejection-associated immune composition, particularly, leading to the reduction of immunosuppressive macrophage infiltration. Finally, we validated these results in melanoma patient data.Statement of significanceHere, we uncover the association ofMifexpression with tumor growth and aggressiveness, specifically in low ITH tumors. These findings could facilitate the development of new strategies to treat patients with homogeneous, high-MIFexpressing tumors that are unresponsive to immune checkpoint therapy.

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Section: Resultsmentioning

confidence: 99%

“…Edited SCC lines were generated following the protocol as described by Lane-Reticker et al, 2023 (34). In brief, plated SCCs were infected with pSCAR-Cas9-Blast-GFP lentivirus along with 4 ug/ml of polybrene.…”

Section: Generation Of Crispr-edited Cell Linesmentioning

confidence: 99%

Temporal genomic analysis of melanoma rejection identifies regulators of tumor immune evasion

Shvefel,

Pai,

Cao

et al. 2023

Preprint

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“…Rigorous analytical methods mitigate the false discovery rates generated by CRISPR screens via a Bayesian classifier of gene essentiality [ 102 ]. Sequence quality control can also be carried out under the guide of GPP Pooled Screen Analysis ( https://portals.broadinstitute.org/gpp/broad/ ), and statistical enrichment and gene depletion were calculated by hit calling algorithm STARS ( http://www.broadinstitute.org/rnai/public/software/index ) based on normalized fold changes [ 103 ]. High-content downstream gRNA library sequence validation in tumor immunology were summarized in the recent review [ 29 ].…”

Section: The Application Of Crispr Screening System In Cancermentioning

confidence: 99%

CRISPR-Cas and CRISPR-based screening system for precise gene editing and targeted cancer therapy

Qin,

Deng,

Wen

et al. 2024

J Transl Med

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Target cancer therapy has been developed for clinical cancer treatment based on the discovery of CRISPR (clustered regularly interspaced short palindromic repeat) -Cas system. This forefront and cutting-edge scientific technique improves the cancer research into molecular level and is currently widely utilized in genetic investigation and clinical precision cancer therapy. In this review, we summarized the genetic modification by CRISPR/Cas and CRISPR screening system, discussed key components for successful CRISPR screening, including Cas enzymes, guide RNA (gRNA) libraries, target cells or organs. Furthermore, we focused on the application for CAR-T cell therapy, drug target, drug screening, or drug selection in both ex vivo and in vivo with CRISPR screening system. In addition, we elucidated the advantages and potential obstacles of CRISPR system in precision clinical medicine and described the prospects for future genetic therapy.In summary, we provide a comprehensive and practical perspective on the development of CRISPR/Cas and CRISPR screening system for the treatment of cancer defects, aiming to further improve the precision and accuracy for clinical treatment and individualized gene therapy.

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In vivo CRISPR screens identify key modifiers of CAR T cell function in myeloma

Korell,

Knudsen,

Kienka

et al. 2024

Preprint

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Chimeric antigen receptor (CAR) T cells are highly effective in hematologic malignancies. However, loss of CAR T cells can contribute to relapse in a significant number of patients. These limitations could potentially be overcome by targeted gene editing to increase CAR T cell persistence. Here, we performed in vivo loss-of-function CRISPR screens in BCMA-targeting CAR T cells to investigate genes that influence CAR T cell persistence, function and efficacy in a human multiple myeloma model. We tracked the expansion and persistence of CRISPR-library edited T cells in vitro and then at early and late timepoints in vivo to track the performance of gene modified CAR T cells from manufacturing to survival in tumors. The screens revealed several context-specific regulators of CAR T cell expansion and persistence. Ablation of RASA2 and SOCS1 enhanced T cell expansion in vitro, while loss of PTPN2, ZC3H12A, and RC3H1 conferred early selective growth advantages to CAR T cells in vivo. Strikingly, we identified cyclin-dependent kinase inhibitor 1B (CDKN1B), a cell cycle regulator, as the most important factor limiting CAR T cell fitness at late timepoints in vivo. CDKN1B ablation increased BCMA CAR T cell proliferation and effector function in response to antigen, significantly enhancing tumor clearance and overall survival. Thus, our findings reveal differing effects of gene-perturbation on CAR T cells over time and in different selective environments, highlight CDKN1B as a promising target to generate highly effective CAR T cells for multiple myeloma, and underscore the importance of in vivo screening as a tool for identifying genes to enhance CAR T cell function and efficacy.

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Protocol for in vivo CRISPR screening using selective CRISPR antigen removal lentiviral vectors

Cited by 4 publications

References 10 publications

Temporal genomic analysis of melanoma rejection identifies regulators of tumor immune evasion

Temporal genomic analysis of melanoma rejection identifies regulators of tumor immune evasion

CRISPR-Cas and CRISPR-based screening system for precise gene editing and targeted cancer therapy

In vivo CRISPR screens identify key modifiers of CAR T cell function in myeloma

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