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.
Population
Edited CD34+ hematopoietic stem/progenitor cells and human genetic variant datasets
Comparison
SpCas9 vs high-fidelity Cas9 variant across variant-aware target sites
Design
In silico tool development and in vitro validation study
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.
Authors
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Non-reference alleles may create undetected off-target sites during CRISPR editing of HSPCs; leaves open need for variant-aware screening before clinical translation.
Absolute Event Rate: 9.6% vs 0%
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.
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.