Genome-scale CRISPR screens are efficient in non-homologous end-joining deficient cells

Joana Ferreira Da Silva,Alexander Hanzl,Joanna I Loizou,Patrick Essletzbichler,Giulio Superti-Furga,Sejla Salic,Georg Winter,Christoph Bock,Paul Datlinger,Marc Wiedner

doi:10.1038/s41598-019-52078-9

Joana Ferreira Da Silva, Alexander Hanzl + Show 8 more

Open Access

https://doi.org/10.1038/s41598-019-52078-9

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Abstract

The mutagenic repair of Cas9 generated breaks is thought to predominantly rely on non-homologous end-joining (NHEJ), leading to insertions and deletions within DNA that culminate in gene knock-out (KO). In this study, by taking focused as well as genome-wide approaches, we show that this pathway is dispensable for the repair of such lesions. Genetic ablation of NHEJ is fully compensated for by alternative end joining (alt-EJ), in a POLQ-dependent manner, resulting in a distinct repair signature with larger deletions that may be exploited for large-scale genome editing. Moreover, we show that cells deficient for both NHEJ and alt-EJ were still able to repair CRISPR-mediated DNA double-strand breaks, highlighting how little is yet known about the mechanisms of CRISPR-based genome editing.

Highlights

The mutagenic repair of Cas[9] generated breaks is thought to predominantly rely on non-homologous end-joining (NHEJ), leading to insertions and deletions within DNA that culminate in gene knock-out (KO)
In order to address the NHEJ dependency of mutagenic repair of Cas9-breaks, a NHEJ-deficient cell line was generated in the human HAP1 cell line, by knocking-out DNA Ligase IV (LIG4), an essential factor for the ligation of the two DNA ends[13] (Supplementary Fig. 1A)
In line with the function of NHEJ, ∆LIG4 cells were hypersensitive to the DNA double-strand break (DSB)-inducing agents neocarcinostatin (NCS), doxorubicin and etoposide[14], but not to the alkylating agent methyl methanesulfonate (MMS), providing a specific phenotypic confirmation of NHEJ abrogation in this cell line (Supplementary Fig. 1B)

Summary

Introduction

The mutagenic repair of Cas[9] generated breaks is thought to predominantly rely on non-homologous end-joining (NHEJ), leading to insertions and deletions within DNA that culminate in gene knock-out (KO). DSBs are mostly repaired by the error-prone non-homologous end-joining (NHEJ) pathway that induces insertions and deletions (indels), disrupting gene function. Supported by studies based on pharmacologic inhibition, it is widely accepted that NHEJ is the major DNA repair pathway that deals with Cas[9] lesions[10,11,12]. Confounders such as incomplete inhibition, off-target effects and dominant-negative patterns can skew the results of such studies, prompting us to develop genetic tools to investigate the mutagenic repair of Cas[9] generated DNA breaks, using isogenic cell line models fully deficient in NHEJ. We observed a differential indel signature with larger deletions in the absence of NHEJ, as well as residual editing in cells deficient for both NHEJ and alt-EJ, suggesting the existence of an alternative mechanism for the repair of Cas9-generated breaks

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Conclusion