CRISPR-based functional genomics screening in human-pluripotent-stem-cell-derived cell types

Li, Kun; Ouyang, Mi; Zhan, Jiangshan; Tian, Ruilin

doi:10.1016/j.xgen.2023.100300

Cited by 11 publications

(14 citation statements)

References 36 publications

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“…While historical methods based on dropout and/or enrichment screening continue to play a major role, recently developed hypothesis-free approaches based on single-cell RNA sequencing (sc-RNA-seq) have taken center stage. Such methods have been particularly successful when coupled with CRISPR/Cas9 technologies (recently reviewed in [197]). In the following sections, we outline how the LoF, GoF, and CoF methods described above can be applied for functional genomic studies in hPSC models, with a particular focus on studies of development and disease mechanisms (Table 2).…”

Section: High-throughput Functional Genomicsmentioning

confidence: 99%

Manipulating and studying gene function in human pluripotent stem cell models

Balmas,

Sozza,

Bottini

et al. 2023

FEBS Letters

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Human pluripotent stem cells (hPSCs) are uniquely suited to study human development and disease and promise to revolutionize regenerative medicine. These applications rely on robust methods to manipulate gene function in hPSC models. This comprehensive review aims to both empower scientists approaching the field and update experienced stem cell biologists. We begin by highlighting challenges with manipulating gene expression in hPSCs and their differentiated derivatives, and relevant solutions (transfection, transduction, transposition, and genomic safe harbor editing). We then outline how to perform robust constitutive or inducible loss‐, gain‐, and change‐of‐function experiments in hPSCs models, both using historical methods (RNA interference, transgenesis, and homologous recombination) and modern programmable nucleases (particularly CRISPR/Cas9 and its derivatives, i.e., CRISPR interference, activation, base editing, and prime editing). We further describe extension of these approaches for arrayed or pooled functional studies, including emerging single‐cell genomic methods, and the related design and analytical bioinformatic tools. Finally, we suggest some directions for future advancements in all of these areas. Mastering the combination of these transformative technologies will empower unprecedented advances in human biology and medicine.

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Section: High-throughput Functional Genomicsmentioning

confidence: 99%

Manipulating and studying gene function in human pluripotent stem cell models

Balmas,

Sozza,

Bottini

et al. 2023

FEBS Letters

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“…High-throughput genetic screening is a powerful approach for identifying cancer therapeutic targets. This process has been greatly facilitated by the recent development of CRISPR/Cas-based genetic manipulation tools, including CRISPR knockout (CRISPRn), CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) [ 33 – 35 ]. CRISPR-based genetic screening has been used in GBM recently to reveal mechanisms of resistance and uncover sensitizing targets for chemotherapy [ 36 – 38 ], radiotherapy [ 39 , 40 ] and immunotherapy [ 41 – 43 ].…”

Section: Introductionmentioning

confidence: 99%

Identification of genetic modifiers enhancing B7-H3-targeting CAR T cell therapy against glioblastoma through large-scale CRISPRi screening

Li,

Sun,

Zhang

et al. 2024

J Exp Clin Cancer Res

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Background Glioblastoma multiforme (GBM) is a highly aggressive brain tumor with a poor prognosis. Current treatment options are limited and often ineffective. CAR T cell therapy has shown success in treating hematologic malignancies, and there is growing interest in its potential application in solid tumors, including GBM. However, current CAR T therapy lacks clinical efficacy against GBM due to tumor-related resistance mechanisms and CAR T cell deficiencies. Therefore, there is a need to improve CAR T cell therapy efficacy in GBM. Methods We conducted large-scale CRISPR interference (CRISPRi) screens in GBM cell line U87 MG cells co-cultured with B7-H3 targeting CAR T cells to identify genetic modifiers that can enhance CAR T cell-mediated tumor killing. Flow cytometry-based tumor killing assay and CAR T cell activation assay were performed to validate screening hits. Bioinformatic analyses on bulk and single-cell RNA sequencing data and the TCGA database were employed to elucidate the mechanism underlying enhanced CAR T efficacy upon knocking down the selected screening hits in U87 MG cells. Results We established B7-H3 as a targetable antigen for CAR T therapy in GBM. Through large-scale CRISPRi screening, we discovered genetic modifiers in GBM cells, including ARPC4, PI4KA, ATP6V1A, UBA1, and NDUFV1, that regulated the efficacy of CAR T cell-mediated tumor killing. Furthermore, we discovered that TNFSF15 was upregulated in both ARPC4 and NDUFV1 knockdown GBM cells and revealed an immunostimulatory role of TNFSF15 in modulating tumor-CAR T interaction to enhance CAR T cell efficacy. Conclusions Our study highlights the power of CRISPR-based genetic screening in investigating tumor-CAR T interaction and identifies potential druggable targets in tumor cells that confer resistance to CAR T cell killing. Furthermore, we devised targeted strategies that synergize with CAR T therapy against GBM. These findings shed light on the development of novel combinatorial strategies for effective immunotherapy of GBM and other solid tumors.

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“…High-throughput genetic screening is a powerful approach for identifying cancer therapeutic targets. This process has been greatly facilitated by the recent development of CRISPR/Cas-based genetic manipulation tools, including CRISPR knockout (CRISPRn), CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) [31][32][33]. CRISPR-based genetic screening has been used in GBM recently to reveal mechanisms of resistance and uncover sensitizing targets for chemotherapy [34][35][36], radiotherapy [37,38] and immunotherapy [39][40][41].…”

Section: Introductionmentioning

confidence: 99%

Identification of genetic modifiers enhancing B7-H3-targeting CAR T cell therapy against glioblastoma through large-scale CRISPRi screening

Li,

Sun,

Zhang

et al. 2024

Preprint

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Background Glioblastoma multiforme (GBM) is a highly aggressive brain tumor with a poor prognosis. Current treatment options are limited and often ineffective. CAR T cell therapy has shown success in treating hematologic malignancies, and there is growing interest in its potential application in solid tumors, including GBM. However, current CAR T therapy lacks clinical efficacy against GBM due to tumor-related resistance mechanisms and CAR T cell deficiencies. Therefore, there is a need to improve CAR T cell therapy efficacy in GBM. Methods We conducted large-scale CRISPR interference (CRISPRi) screens in GBM cell line U87 cells co-cultured with B7-H3 targeting CAR T cells to identify genetic modifiers that can enhance CAR T cell-mediated tumor killing. Flow cytometry-based tumor killing assay and CAR T cell activation assay were performed to validate screening hits. Bioinformatic analyses on bulk and single-cell RNA sequencing data and the TCGA database were employed to elucidate the mechanism underlying enhanced CAR T efficacy upon knocking down the selected screening hits in U87 cells. Results We established B7-H3 as a targetable antigen for CAR T therapy in GBM. Through large-scale CRISPRi screening, we discovered genetic modifiers in GBM cells, including ARPC4, PI4KA, ATP6V1A, UBA1, and NDUFV1, that regulated the efficacy of CAR T cell-mediated tumor killing. Furthermore, we discovered that TNFSF15 was upregulated in both ARPC4 and NDUFV1 knockdown GBM cells and revealed an immunostimulatory role of TNFSF15 in modulating tumor-CAR T interaction to enhance CAR T cell efficacy. Conclusions Our study highlights the power of CRISPR-based genetic screening in investigating tumor-CAR T interaction and identifies potential druggable targets in tumor cells that confer resistance to CAR T cell killing. Furthermore, we devised targeted strategies that synergize with CAR T therapy against GBM. These findings shed light on the development of novel combinatorial strategies for effective immunotherapy of GBM and other solid tumors.

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CRISPR-based functional genomics screening in human-pluripotent-stem-cell-derived cell types

Cited by 11 publications

References 36 publications

Manipulating and studying gene function in human pluripotent stem cell models

Manipulating and studying gene function in human pluripotent stem cell models

Identification of genetic modifiers enhancing B7-H3-targeting CAR T cell therapy against glioblastoma through large-scale CRISPRi screening

Identification of genetic modifiers enhancing B7-H3-targeting CAR T cell therapy against glioblastoma through large-scale CRISPRi screening

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