Local recruitment of DNA repair proteins enhances CRISPR-ssODN-HDR editing

Abstract

CRISPR-Cas technologies enable precise editing of genomic sequences. One major way to introduce precise editing is through homology directed repair (HDR) of DNA double strand breaks (DSB) templated by exogenously supplied single-stranded oligodeoxyribonucleotides (ssODN). Competing pathways determine the outcome of edits. Non-homologous end-joining pathways produce destructive insertions/deletions (indels) at target sites and are dominant over the precise homology directed repair pathways. In this study, we aim to favor HDR and use two strategies to recruit DNA repair proteins (DRPs) to Cas9 cut site, the Casilio-DRP approach that recruits RNA-binding protein-tethered DRPs to target site via aptamers appended to guide RNA; and the 53BP1-DRP approach that recruits DRPs to DSBs via DSB-sensing activity of 53BP1. We conducted two screens using these approaches and identified DRPs such as FANCF and BRCA1 that when recruited to Cas9 cut site, enhance ssODN-templated HDR and increase the proportion of precise edits. This study provides not only new constructs for enhanced CRISPR-ssODN-HDR but also a collection of DRP fusions for studying DNA repair processes.