Scalable CRISPR-Cas9 chemical genetic screens in non-transformed human cells

Kevin Lin; Ya-Chu Chang; Ezequiel Marron Fernandez de Velasco; Kevin Wickman; Chad L Myers; Anja Katrin Bielinsky

doi:10.1016/j.xpro.2022.101675

Scalable CRISPR-Cas9 chemical genetic screens in non-transformed human cells

Kevin Lin, Ya-Chu Chang, Ezequiel Marron Fernandez de Velasco, Kevin Wickman, Chad L Myers, Anja Katrin Bielinsky

Research output: Contribution to journal › Article › peer-review

2 Scopus citations

Abstract

Pooled lentiviral CRISPR-Cas9 screens are utilized for assessing the differential sensitivity or resistance of many single-gene knockouts to a compound. Here, we present a scalable approach for high-throughput compound screening by utilizing a small custom library. We describe steps to perform a proof-of-principle chemical screen in non-transformed hTERT RPE-1 TP53^−/− cells with higher coverage and greater timepoint resolution compared to genome-wide screens. This approach can be adapted for use in various cell lines, compounds, and other focused sgRNA libraries.

Original language	English (US)
Article number	101675
Journal	STAR Protocols
Volume	3
Issue number	4
DOIs	https://doi.org/10.1016/j.xpro.2022.101675
State	Published - Dec 16 2022

Bibliographical note

Funding Information:
We would like to thank the Bielinsky -, Myers -, and Jason Moffat's laboratories for contributing to the development of this protocol. K.L. is supported by the NIH/NCATS TL1 TRACT award (UL1TR002494, TL1R002493) through the University of Minnesota Clinical and Translational Science Institute (UMN CTSI) and by the NIH/NCI Ruth L. Kirschstein National Research F30 award (5F30CA257227-02). This work was also supported by NIH R35 GM141805 to A.K.B. and by a grant from the National Science Foundation (MCB 1818293) to C.L.M. We would also like to acknowledge Aaron Becker (UMN Genomics Center), who consulted on next-generation sequencing options; John Garbe (UMN Genomics Center) as the informatics lead that converted bcl files to fastq files; and Juan Abrahante Lloréns (UMN Informatics Institute) as the genomics analyst who we consulted on converting fastq files to raw read count data. K.L. and Y.C.C. contributed equally to writing and collating this protocol. E.M. and K.W. contributed to the lentiviral library packaging as part of the UMN Viral Vector and Cloning Core (VVCC). A.K.B. and C.L.M. made conceptual contributions to the protocol and oversaw all screening work. The authors declare no competing interests.

Funding Information:
We would like to thank the Bielinsky -, Myers -, and Jason Moffat’s laboratories for contributing to the development of this protocol. K.L. is supported by the NIH / NCATS TL1 TRACT award ( UL1TR002494 , TL1R002493 ) through the University of Minnesota Clinical and Translational Science Institute (UMN CTSI) and by the NIH/ NCI Ruth L. Kirschstein National Research F30 award ( 5F30CA257227-02 ). This work was also supported by NIH R35 GM141805 to A.K.B. and by a grant from the National Science Foundation ( MCB 1818293 ) to C.L.M. We would also like to acknowledge Aaron Becker (UMN Genomics Center), who consulted on next-generation sequencing options; John Garbe (UMN Genomics Center) as the informatics lead that converted bcl files to fastq files; and Juan Abrahante Lloréns (UMN Informatics Institute) as the genomics analyst who we consulted on converting fastq files to raw read count data.

Publisher Copyright:
© 2022 The Author(s)

Keywords

CRISPR
Genomics
High Throughput Screening
Molecular Biology
Sequencing

PubMed: MeSH publication types

Journal Article
Research Support, N.I.H., Extramural
Research Support, U.S. Gov't, Non-P.H.S.

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10.1016/j.xpro.2022.101675

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title = "Scalable CRISPR-Cas9 chemical genetic screens in non-transformed human cells",

abstract = "Pooled lentiviral CRISPR-Cas9 screens are utilized for assessing the differential sensitivity or resistance of many single-gene knockouts to a compound. Here, we present a scalable approach for high-throughput compound screening by utilizing a small custom library. We describe steps to perform a proof-of-principle chemical screen in non-transformed hTERT RPE-1 TP53−/− cells with higher coverage and greater timepoint resolution compared to genome-wide screens. This approach can be adapted for use in various cell lines, compounds, and other focused sgRNA libraries.",

keywords = "CRISPR, Genomics, High Throughput Screening, Molecular Biology, Sequencing",

author = "Kevin Lin and Ya-Chu Chang and {Marron Fernandez de Velasco}, Ezequiel and Kevin Wickman and Myers, {Chad L} and Bielinsky, {Anja Katrin}",

note = "Funding Information: We would like to thank the Bielinsky -, Myers -, and Jason Moffat's laboratories for contributing to the development of this protocol. K.L. is supported by the NIH/NCATS TL1 TRACT award (UL1TR002494, TL1R002493) through the University of Minnesota Clinical and Translational Science Institute (UMN CTSI) and by the NIH/NCI Ruth L. Kirschstein National Research F30 award (5F30CA257227-02). This work was also supported by NIH R35 GM141805 to A.K.B. and by a grant from the National Science Foundation (MCB 1818293) to C.L.M. We would also like to acknowledge Aaron Becker (UMN Genomics Center), who consulted on next-generation sequencing options; John Garbe (UMN Genomics Center) as the informatics lead that converted bcl files to fastq files; and Juan Abrahante Llor{\'e}ns (UMN Informatics Institute) as the genomics analyst who we consulted on converting fastq files to raw read count data. K.L. and Y.C.C. contributed equally to writing and collating this protocol. E.M. and K.W. contributed to the lentiviral library packaging as part of the UMN Viral Vector and Cloning Core (VVCC). A.K.B. and C.L.M. made conceptual contributions to the protocol and oversaw all screening work. The authors declare no competing interests. Funding Information: We would like to thank the Bielinsky -, Myers -, and Jason Moffat{\textquoteright}s laboratories for contributing to the development of this protocol. K.L. is supported by the NIH / NCATS TL1 TRACT award ( UL1TR002494 , TL1R002493 ) through the University of Minnesota Clinical and Translational Science Institute (UMN CTSI) and by the NIH/ NCI Ruth L. Kirschstein National Research F30 award ( 5F30CA257227-02 ). This work was also supported by NIH R35 GM141805 to A.K.B. and by a grant from the National Science Foundation ( MCB 1818293 ) to C.L.M. We would also like to acknowledge Aaron Becker (UMN Genomics Center), who consulted on next-generation sequencing options; John Garbe (UMN Genomics Center) as the informatics lead that converted bcl files to fastq files; and Juan Abrahante Llor{\'e}ns (UMN Informatics Institute) as the genomics analyst who we consulted on converting fastq files to raw read count data. Publisher Copyright: {\textcopyright} 2022 The Author(s)",

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doi = "10.1016/j.xpro.2022.101675",

language = "English (US)",

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T1 - Scalable CRISPR-Cas9 chemical genetic screens in non-transformed human cells

AU - Lin, Kevin

AU - Chang, Ya-Chu

AU - Marron Fernandez de Velasco, Ezequiel

AU - Wickman, Kevin

AU - Myers, Chad L

AU - Bielinsky, Anja Katrin

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PY - 2022/12/16

Y1 - 2022/12/16

N2 - Pooled lentiviral CRISPR-Cas9 screens are utilized for assessing the differential sensitivity or resistance of many single-gene knockouts to a compound. Here, we present a scalable approach for high-throughput compound screening by utilizing a small custom library. We describe steps to perform a proof-of-principle chemical screen in non-transformed hTERT RPE-1 TP53−/− cells with higher coverage and greater timepoint resolution compared to genome-wide screens. This approach can be adapted for use in various cell lines, compounds, and other focused sgRNA libraries.

AB - Pooled lentiviral CRISPR-Cas9 screens are utilized for assessing the differential sensitivity or resistance of many single-gene knockouts to a compound. Here, we present a scalable approach for high-throughput compound screening by utilizing a small custom library. We describe steps to perform a proof-of-principle chemical screen in non-transformed hTERT RPE-1 TP53−/− cells with higher coverage and greater timepoint resolution compared to genome-wide screens. This approach can be adapted for use in various cell lines, compounds, and other focused sgRNA libraries.

KW - CRISPR

KW - Genomics

KW - High Throughput Screening

KW - Molecular Biology

KW - Sequencing

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Scalable CRISPR-Cas9 chemical genetic screens in non-transformed human cells

Abstract

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Keywords

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