Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR) – CRISPR-associated protein 9 (Cas9) has revolutionized the field of genome engineering. However, low efficiency of targeted gene integration represents one of the major obstacles in therapeutic genome editing.

Previous reports have established that small molecules can enhance gene insertions in various cell types. However, most studies using compounds to increase knock-in efficiency have been limited to a small number of tested substances and to HDR pathway for integration. We first tested 19,516 well annotated compounds to enhance the efficiency of genome editing in a high-throughput screening. Successful hits were then validated using a novel assay, to evaluate different knock-in strategies (like ObLiGaRe, HITI, PITCh, SSDR) in various genomic loci and cell types. The results contributed to our understanding of the interplay between DNA Damage Repair (DDR) and CRISPR-mediated knock-ins. These findings provide useful tools to improve cell line generation and pave the way for gene therapy where high knock-in efficiencies are needed.