In recent years, CRISPR-Cas9 has emerged as a site-specific genome editing tool that has become fundamental to basic research and holds the potential to transform modern medicine. In particular, the ability of CRISPR-Cas9 to generate precise knock-in mutations at target loci presents a novel therapeutic strategy for genetic disorders. To insert exogenous DNA into the genome, CRISPR-Cas9 induces a double-strand break and relies on template-driven homology-directed repair (HDR) to resolve the lesion. However, due to the prevalence of the competing and more error-prone nonhomologous end-joining (NHEJ) repair pathway, the efficiency of CRISPR-Cas9 gene editing is severely limited and prohibits its use in many applications. Small molecules that inhibit NHEJ and/or promote HDR have been shown to enhance CRISPR-Cas9 editing efficiencies and could help overcome this limitation. 53BP1 is a chromatin methyl-lysine reader protein that initiates NHEJ by preventing DNA end-resection needed for HDR. In previous work, we have developed fragment-like inhibitors of 53BP1 with modest affinity and cellular activity. Here we show that our small molecule inhibitors of 53BP1 increase GFP expression following CRISPR-Cas9 transfection by nearly 2-fold in mouse embryonic fibroblasts. When used in combination with other small molecules targeting alternate proteins implicated in DNA damage repair, we have achieved additional boosts in GFP expression. Importantly, our small molecules are highly cell penetrant, non-toxic reagents that can easily be incorporated into any CRISPR-Cas9 gene editing experiment. With co-crystal structures of 53BP1 and our small molecule inhibitors in hand, we aim to develop more potent antagonists through iterative medicinal chemistry and structure-guided design to further increase their efficacy as CRISPR-Cas9 efficiency enhancing reagents. Based on our preliminary data, we believe that the development of a high-quality chemical probe of 53BP1 would provide a universal reagent to enable more efficient gene editing using CRISPR-Cas9, thereby benefitting basic researchers and gene-directed therapeutics as well.

Transforming CRISPR-Cas9 genome editing efficiency with 53BP1 chemical probes

ACS Fall 2021

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