Strand asymmetry influences mismatch resolution during single-strand annealing

Abstract

Biases of DNA repair can shape the nucleotide landscape of genomes at evolutionary timescales. However, such biases have not yet been measured in chromatin for lack of technologies. Here we develop a genome-wide assay whereby the same DNA lesion is repaired in different chromatin contexts. We insert thousands of barcoded transposons carrying a reporter of DNA mismatch repair in the genome of mouse embryonic stem cells. Upon inducing a double-strand break between tandem repeats, a mismatch is generated when the single strand annealing repair pathway is used. Surprisingly, the mismatch repair machinery favors the same strand 60-80% of the time. The location of the lesion in the genome and the type of mismatch have little influence on the repair bias in this context. Using machine learning, we further show that both the repair bias and the efficiency of the repair are independent of known chromatin features. These results suggest that some intrinsic property of the lesion can have a large influence on the outcome of DNA repair, irrespective of the surrounding chromatin context.Recent insight into this question came from cancer genome sequencing (Pleasance et al . 2010;Hoadley et al . 2014) . In particular, this made it possible to show that the mismatch repair system in healthy cells is more accurate at some loci than others. For instance, mismatches in late-replicating regions are repaired less efficiently (Supek and Lehner 2015) , a feature that seems to be shared among eukaryotes (Weber, Pink, and Hurst 2012) . It is presently unknown whether the chromatin context can bias the mismatch repair toward one nucleotide or another, mostly because it is difficult to tease apart the contributions of DNA damage and DNA repair to mutation patterns.

Impact of chromatin context on Cas9-induced DNA double-strand break repair pathway balance

“…This work complements a recent study that used a multiplexed reporter for DNA mismatch repair, which did not reveal significant effects of chromatin context on the repair outcome ( Pokusaeva et al., 2019 ). Another multiplexed integrated reporter study also found evidence that genomic location can affect Cas9 editing efficiency ( Gisler et al., 2019 ), but these results were more difficult to interpret because the reporter sequence itself was not transcriptionally inert.…”

Impact of chromatin context on Cas9-induced DNA double-strand break repair pathway balance

“…This work complements a recent study that employed a multiplexed reporter for DNA mismatch repair, which did not find significant effects of chromatin context on the repair outcome (Pokusaeva et al, 2019). Another multiplexed integrated reporter study also found evidence that genomic location can affect Cas9 editing efficiency (Gisler et al, 2019), but these results were more difficult to interpret because the reporter sequence itself was not transcriptionally inert.…”

Protocol: A Multiplexed Reporter Assay to Study Effects of Chromatin Context on DNA Double-Strand Break Repair

“…It will be interesting to apply DSB-TRIP with other CRISPR endonucleases such as Cas12a (Zetsche et al, 2015) or rare-cutter restriction enzymes such as I-SceI (Niu et al, 2008) or AsiSI (Iacovoni et al, 2010) (Figure 2A, bottom). I-SceI was successfully applied to study Single Strand Annealing in a TRIP assay (Pokusaeva et al, 2021). Another option are transcription activator-like effector nucleases (TALENs) (Boch et al, 2009;Gaj et al, 2013;Moscou and Bogdanove, 2009), especially since it was found that these nucleases are more efficient in heterochromatin, compared to Cas9 (Jain et al, 2021).…”

“…Several studies have demonstrated that the local chromatin state is one of the factors that influences which pathway is preferentially used to repair a DSB. These studies used methods ranging from using single imprinted endogenous loci (Kallimasioti-Pazi et al, 2018), single transgenic loci (Lemaitre et al, 2014), hundreds of endogenous loci (Iacovoni et al, 2010;Massip et al, 2010;Aymard et al, 2014) to thousands of integrated reporters as presented here (Gisler et al, 2019;Pokusaeva et al, 2021;Schep et al, 2021). All these studies rely on endonucleases creating a DSB at a defined locus in the genome, targeting either a definite sequence (e.g., restriction enzymes) (Iacovoni et al, 2010) or a user-defined one (Kallimasioti-Pazi et al, 2018;van Overbeek et al, 2016;Chakrabarti et al, 2019) [e.g., with CRISPR/Cas9 (Jinek et al, 2012;Cong et al, 2013;Jinek et al, 2013;Mali et al, 2013)].…”