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Revolutionizing DNA repair research and cancer therapy with CRISPR–Cas screens

Nature Reviews Molecular Cell Biology volume 24, pages 477–494 (2023)Cite this article

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Abstract

All organisms possess molecular mechanisms that govern DNA repair and associated DNA damage response (DDR) processes. Owing to their relevance to human disease, most notably cancer, these mechanisms have been studied extensively, yet new DNA repair and/or DDR factors and functional interactions between them are still being uncovered. The emergence of CRISPR technologies and CRISPR-based genetic screens has enabled genome-scale analyses of gene–gene and gene–drug interactions, thereby providing new insights into cellular processes in distinct DDR-deficiency genetic backgrounds and conditions. In this Review, we discuss the mechanistic basis of CRISPR–Cas genetic screening approaches and describe how they have contributed to our understanding of DNA repair and DDR pathways. We discuss how DNA repair pathways are regulated, and identify and characterize crosstalk between them. We also highlight the impacts of CRISPR-based studies in identifying novel strategies for cancer therapy, and in understanding, overcoming and even exploiting cancer-drug resistance, for example in the contexts of PARP inhibition, homologous recombination deficiencies and/or replication stress. Lastly, we present the DDR CRISPR screen (DDRcs) portal, in which we have collected and reanalysed data from CRISPR screen studies and provide a tool for systematically exploring them.

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Fig. 1: Overview of CRISPR–Cas strategies and tools.
Fig. 2: High-throughput CRISPR–Cas9 screening approaches.
Fig. 3: Overview of novel DNA repair factors and associations discovered using CRISPR screens.

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Acknowledgements

The authors thank R. Belotserkovskaya and G. D’Alessandro for critical reading of the manuscript, K. Dry for editorial assistance and other members of the S.P.J. laboratory for advice and discussions. The authors apologize to authors whose work they did not cite owing to space constraints. S.W.A. is supported by the Mark Foundation for Cancer Research and was a recipient of an Outstanding Postdoctoral Women Fellowship from the Israeli Council for Higher Education. A.S.-B. and J.C.T. are supported by European Research Council (ERC) Synergy grant no 855741 (DDREAMM). J.C.T. and V.G. were supported by Wellcome Investigator Award (206388/Z/17/Z). The S.P.J. laboratory is supported by Cancer Research UK and the ERC.

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  1. These authors contributed equally: Samah W. Awwad, Almudena Serrano-Benitez.

Authors and Affiliations

  1. Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge, UK

    Samah W. Awwad, Almudena Serrano-Benitez, John C. Thomas & Stephen P. Jackson

  2. The Gurdon Institute and Department of Biochemistry, University of Cambridge, Cambridge, UK

    Samah W. Awwad, Almudena Serrano-Benitez, John C. Thomas, Vipul Gupta & Stephen P. Jackson

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S.P.J., S.W.A. and A.S.-B. researched data for the article, substantially contributed to discussion of the content, wrote the article and reviewed the manuscript before submission. J.C.T. and V.G. researched data for the article and substantially contributed to discussion of the content.

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Related links

BioGRID ORCS: https://orcs.thebiogrid.org/

ClinVar: https://www.ncbi.nlm.nih.gov/clinvar/

DDR CRISPR screen (DDRcs) portal: https://stevejacksonlab.org/ddrcs

DepMap Portal: https://depmap.org/

Glossary

6-Thioguanine

A nucleotide (guanine) analogue that, when incorporated into DNA, is toxic to mismatch repair (MMR)-proficient cells, which are unable to effectively repair 6-thioguanine-induced lesions.

BRCA1-A complex

A protein complex (BRCC45–ABRAXAS–MERIT40–RAP80–BRCC36) that has been reported to modify double-strand break (DSB) resection dynamics and limit homologous recombination (HR) repair, somehow counteracting ataxia telangiectasia mutated (ATM) function.

Class-switch recombination

(CSR). A DNA recombination process that occurs in B cells to switch between the production of immunoglobulin isotypes.

Clustered regularly interspaced short palindromic repeat

(CRISPR). A form of immunity against viruses in prokaryotes, comprising genomic loci of short repeats interspersed with DNA sequences of viral origin called ‘spacers’, and the CRISPR-associated (Cas) family of nucleases.

Dropout hits

Genes that, when depleted or lost, affect a specific condition measured in a screen.

Dual-guide

A construct including two single guide RNA (sgRNA) sequences targeting two regions of the same gene.

Homologous recombination

(HR). A conserved type of DNA repair process that relies on use of an extensively homologous template from a sister chromatid, another homologous sequence elsewhere in the genome or an experimentally delivered DNA molecule.

Non-homologous end joining

(NHEJ). The primary pathway in higher eukaryotes that repairs double-strand breaks (DSBs) by directly tethering the break ends without use of a homologous template, and after potential modification of the ends, ligating them in a way that often introduces mutations.

Non-productive HR intermediates

Aberrant intermediates observed in response to unsuccessful homologous recombination (HR) (for example, in POLQ/53BP1 double-knockout cells), which are often associated with larger than usual RecA-like protein (RAD51) foci.

Polθ-mediated end joining

(TMEJ). A double-strand break (DSB) repair mechanism that entails exposure of microhomology sequences internal to the DSB ends before ligation, leading to deletion of the sequence flanking the DSB; sometimes associated with chromosomal rearrangements.

Sanitizer of cellular nucleotide pools

An enzyme involved in preventing the incorporation of aberrant nucleotides into genomic DNA.

Saturation genome editing

Clustered regularly interspaced short palindromic repeat (CRISPR)–Cas9 genome editing aimed at introducing all possible single-nucleotide variants into a targeted genomic region.

Single guide RNAs

(sgRNAs). Artificial fusions of the clustered regularly interspaced short palindromic repeat (CRISPR) RNA, which recognizes the target sequence in DNA, and the scaffold trans-activating CRISPR RNA, which includes secondary structures crucial for its loading onto Cas9.

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Awwad, S.W., Serrano-Benitez, A., Thomas, J.C. et al. Revolutionizing DNA repair research and cancer therapy with CRISPR–Cas screens. Nat Rev Mol Cell Biol 24, 477–494 (2023). https://doi.org/10.1038/s41580-022-00571-x

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