Timed inhibition of CDC7 increases CRISPR-Cas9 mediated templated repair

Beeke Wienert,Matthew A M Carter,Chris D Richardson,Alexis Guenther,Jiyung Shin,David N Nguyen,Alex Marson,Stacia K Wyman,Francis Wright,Jacob E Corn,Katelynn R Kazane,Bruce R Conklin,Sharon J Feng,Melissa N Locke,Georgia L Gregory

doi:10.1038/s41467-020-15845-1

Abstract

Repair of double strand DNA breaks (DSBs) can result in gene disruption or gene modification via homology directed repair (HDR) from donor DNA. Altering cellular responses to DSBs may rebalance editing outcomes towards HDR and away from other repair outcomes. Here, we utilize a pooled CRISPR screen to define host cell involvement in HDR between a Cas9 DSB and a plasmid double stranded donor DNA (dsDonor). We find that the Fanconi Anemia (FA) pathway is required for dsDonor HDR and that other genes act to repress HDR. Small molecule inhibition of one of these repressors, CDC7, by XL413 and other inhibitors increases the efficiency of HDR by up to 3.5 fold in many contexts, including primary T cells. XL413 stimulates HDR during a reversible slowing of S-phase that is unexplored for Cas9-induced HDR. We anticipate that XL413 and other such rationally developed inhibitors will be useful tools for gene modification.

Highlights

Repair of double strand DNA breaks (DSBs) can result in gene disruption or gene modification via homology directed repair (HDR) from donor DNA
To investigate the various HDR mechanisms in greater detail, we previously developed a reporter[8] that allowed us to interrogate the genetic requirements of Cas9mediated HDR using single-stranded donor DNA and discovered that single strand template repair (SSTR) requires the Fanconi Anemia (FA) DNA repair pathway[9]
Our prior work defined the Fanconi Anemia (FA) pathway as necessary for Cas9-mediated SSTR9, and we report that almost the entire FA repair pathway is required for Cas9-induced homologous recombination (HR). 31 of 40 FA and FA-related genes were required for HR, suggesting that this is an activity of the overall FA pathway (Supplementary Fig. 1b) and indicating its importance for all forms of Cas9-mediated HDR13

Summary

Introduction

Repair of double strand DNA breaks (DSBs) can result in gene disruption or gene modification via homology directed repair (HDR) from donor DNA. One strategy for efficient genome editing in eukaryotic cells introduces a ribonucleotide protein (RNP) complex comprised of the type II endonuclease Cas[9] and a guide RNA (gRNA), which create a double strand DNA break (DSB) at a targeted location in the genome[1,2] This DSB is repaired by cellular DNA repair pathways to produce two outcomes: error-prone sequence disruption by insertion or deletion (indels) at the DSB, or precise sequence modification via homology directed repair (HDR) that copies donor DNA sequences into the DSB. We found that SSTR does not depend on the classic RAD51 pathway, unlike homologous recombination (HR) repair from a double stranded DNA donor (dsDonor) These distinct requirements for HDR from ssDonor and dsDonor implied that different donors produce molecularly identical gene modifications via different mechanistic routes. We investigate factors whose knockdown increases Cas9-induced HDR, discovering that timed administration of a small molecule inhibitor of one of these factors, cycle 7-related protein kinase (CDC7), increases HR and SSTR by up to 3.5-fold in multiple contexts including primary human T cells

Methods

Results

Conclusion