| Autor: |
Cancellieri, Samuele, Zeng, Jing, Lin, Linda Yingqi, Tognon, Manuel, Nguyen, My Anh, Lin, Jiecong, Bombieri, Nicola, Maitland, Stacy A., Ciuculescu, Marioara-Felicia, Katta, Varun, Tsai, Shengdar Q., Armant, Myriam, Wolfe, Scot A., Giugno, Rosalba, Bauer, Daniel E., Pinello, Luca |
| Zdroj: |
Nature Genetics; 20220101, Issue: Preprints p1-10, 10p |
| Abstrakt: |
CRISPR gene editing holds great promise to modify DNA sequences in somatic cells to treat disease. However, standard computational and biochemical methods to predict off-target potential focus on reference genomes. We developed an efficient tool called CRISPRme that considers single-nucleotide polymorphism (SNP) and indel genetic variants to nominate and prioritize off-target sites. We tested the software with a BCL11Aenhancer targeting guide RNA (gRNA) showing promise in clinical trials for sickle cell disease and β-thalassemia and found that the top candidate off-target is produced by an allele common in African-ancestry populations (MAF 4.5%) that introduces a protospacer adjacent motif (PAM) sequence. We validated that SpCas9 generates strictly allele-specific indels and pericentric inversions in CD34+hematopoietic stem and progenitor cells (HSPCs), although high-fidelity Cas9 mitigates this off-target. This report illustrates how genetic variants should be considered as modifiers of gene editing outcomes. We expect that variant-aware off-target assessment will become integral to therapeutic genome editing evaluation and provide a powerful approach for comprehensive off-target nomination. |
| Databáze: |
Supplemental Index |
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