Interallelic gene conversion of leukemia-associated single nucleotide variants

Le résumé fourni par la source

• Single nucleotide variants (SNVs) can be altered by interallelic gene conversion (IGC) • SNVs but not wild type alleles are targeted by SNV-specific gRNA protospacers. • IGC of SNVs was accomplished in numerous leukemia cell lines and primary samples. • IGC of SNVs is easy to perform using high-fidelity Cas9 and SNV-specific gRNA. CRISPR-Cas9 is a useful tool for inserting precise genetic alterations through homology-directed repair (HDR), although current methods largely rely on provision of an exogenous repair template. Here, we tested the possibility of interchanging heterozygous single nucleotide variants (SNVs) using mutation-specific guide RNA, and the cell’s own wild-type allele rather than an exogenous template. Using high-fidelity Cas9 to perform allele-specific CRISPR across multiple human leukemia cell lines as well as in primary hematopoietic cells from patients with leukemia, we find high levels of reversion to wild-type in the absence of exogenous template. Moreover, we demonstrate that bulk treatment to revert a truncating mutation in ASXL1 using CRISPR-mediated interallelic gene conversion (IGC) is sufficient to prolong survival in a human cell line-derived xenograft model (median survival 33 days vs 27.5 days; p = 0.0040). These results indicate that IGC is a useful laboratory tool which can be applied to numerous types of leukemia and can meaningfully alter cellular phenotypes at scale. Because our method targets single-base mutations, rather than larger variants targeted by IGC in prior studies, it greatly expands the pool of genetic lesions which could potentially be targeted by IGC. This technique may reduce cost and complexity for experiments modeling phenotypic consequences of SNVs. The principles of SNV-specific IGC demonstrated in this proof-of-concept study could be applied to investigate the phenotypic effects of targeted clonal reduction of leukemogenic SNV mutations.

Ce résumé expose les affirmations des auteurs. BNTIC ne l’interprète pas comme une validation indépendante des résultats.