PT - JOURNAL ARTICLE AU - Samuele Cancellieri AU - Jing Zeng AU - Linda Yingqi Lin AU - Manuel Tognon AU - My Anh Nguyen AU - Jiecong Lin AU - Nicola Bombieri AU - Stacy A. Maitland AU - Marioara-Felicia Ciuculescu AU - Varun Katta AU - Shengdar Q. Tsai AU - Myriam Armant AU - Scot A. Wolfe AU - Rosalba Giugno AU - Daniel E. Bauer AU - Luca Pinello TI - Human genetic diversity alters therapeutic gene editing off-target outcomes AID - 10.1101/2021.05.20.445054 DP - 2022 Jan 01 TA - bioRxiv PG - 2021.05.20.445054 4099 - http://biorxiv.org/content/early/2022/07/04/2021.05.20.445054.short 4100 - http://biorxiv.org/content/early/2022/07/04/2021.05.20.445054.full AB - CRISPR gene editing holds great promise to modify somatic genomes to ameliorate disease. In silico prediction of homologous sites coupled with biochemical evaluation of possible genomic off-targets may predict genotoxicity risk of individual gene editing reagents. However, standard computational and biochemical methods focus on reference genomes and do not consider the impact of genetic diversity on off-target potential. Here we developed a web application called CRISPRme that explicitly and efficiently integrates human genetic variant datasets with orthogonal genomic annotations to nominate and prioritize off-target sites at scale. The method considers both single-nucleotide variants (SNVs) and indels, accounts for bona fide haplotypes, accepts spacer:protospacer mismatches and bulges, and is suitable for personal genome analyses. We tested the tool with a guide RNA (gRNA) targeting the BCL11A erythroid enhancer that has shown therapeutic promise in clinical trials for sickle cell disease (SCD) and β-thalassemia1. We find that the top candidate off-target site is produced by a non-reference allele common in African-ancestry populations (rs114518452, minor allele frequency (MAF)=4.5%) that introduces a protospacer adjacent motif (PAM) for SpCas9. We validate that SpCas9 generates indels (∼9.6% frequency) and chr2 pericentric inversions in a strictly allele-specific manner in edited CD34+ hematopoietic stem/progenitor cells (HSPCs), although a high-fidelity Cas9 variant mitigates this off-target. The CRISPRme tool highlights alternative allele-specific off-target editing as a prevalent risk of gRNAs considered for therapeutic gene editing. Our report illustrates how population and private genetic variants should be considered as modifiers of genome editing outcomes. We suggest that variant-aware off-target assessment should be considered in therapeutic genome editing efforts going forward and provide a powerful approach for comprehensive off-target nomination.Competing Interest StatementL.P. has financial interests in Edilytics, Inc., Excelsior Genomics, and SeQure Dx, Inc. L.P.'s interests were reviewed and are managed by Massachusetts General Hospital and Partners HealthCare in accordance with their conflict of interest policies.