Key result
A therapeutically relevant BCL11A-targeting gRNA generated ~9.6% off-target indels in a strictly allele-specific manner at a site created by a non-reference allele common in African-ancestry populations.
Why the study?
Standard computational and biochemical methods for predicting CRISPR off-target genotoxicity focus on reference genomes and do not consider the impact of human genetic diversity.
Human genetic diversity, particularly non-reference alleles, can create clinically significant off-target sites for CRISPR gene editing that are missed by standard reference genome-based tools.
Answers are grounded in this paper and cite their sources.
Non-reference alleles may create undetected off-target sites during CRISPR editing of HSPCs; leaves open need for variant-aware screening before clinical translation.
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-thalassemia 1 . 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.
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Cancellieri et al. (2021) studied Sickle cell disease and β-thalassemia (context) (n=7). SpCas9 with BCL11A-targeting gRNA #1617 vs. Reference allele (rs114518452-G) was evaluated on Off-target indel frequency at rs114518452. A therapeutically relevant BCL11A-targeting gRNA generated ~9.6% off-target indels in a strictly allele-specific manner at a site created by a non-reference allele common in African-ancestry populations.
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