Widespread impact of natural genetic variations in CRISPR/Cas9 outcomes

Abstract

CRISPR/Cas9 is a genome editing tool widely used in biological research and clinical therapeutics. Naturally occurring human genomic variations, through altering the sequence context of CRISPR/Cas9 target regions, can significantly affect its DNA repair outcomes and ultimately lead to different editing efficiencies. However, these effects have not been systematically studied or documented, even as CRISPR/Cas9 is broadly applied to primary cells and patient samples that harbor such genetic diversity. Herein, we present CROTONdb (https://croton.princeton.edu/), a publicly available database that enables fast and comprehensive investigations of single nucleotide variant (SNV) effects on CRISPR/Cas9 outcomes across the human genome. CROTONdb provides predictions for all possible CRISPR/Cas9 target sites in the coding region, spanning over 5.38 million gRNA targets, 32.32 million predicted editing outcomes, and 90.82 million estimated variant effects. Critically, variant effects presented in CROTONdb accurately cover both common and rare/understudied variants. We illustrate the utility of CROTONdb through two case studies, showcasing its impact on both preclinical discoveries and recent clinical trials. Our case studies demonstrate pitfalls of failing to account for genetic variations in Cas9 target selection, and how they can be effectively examined and avoided using our tool. We anticipate CROTONdb having broad clinical utilities in gene and cellular therapies.