Interallelic gene conversion of leukemia-associated single nucleotide variants

Area of Science:

• * Molecular Biology
• * Genetics
• * Cancer Research

Background:

• * CRISPR-Cas9 facilitates genetic alterations via homology-directed repair (HDR), typically requiring external DNA templates.
• * Current HDR methods for precise genetic editing in leukemia are limited by the need for exogenous repair templates.

Purpose of the Study:

• * To investigate the potential of using allele-specific CRISPR and the cell's endogenous wild-type allele to correct heterozygous single nucleotide variants (SNVs).
• * To evaluate CRISPR-mediated interallelic gene conversion (IGC) as a therapeutic strategy for leukemia by reverting specific mutations.

Main Methods:

• * Employed high-fidelity Cas9 for allele-specific CRISPR-mediated interallelic gene conversion (IGC) in human leukemia cell lines and primary patient hematopoietic cells.
• * Tested the efficacy of reverting a truncating ASXL1 mutation using CRISPR-IGC in a human cell line-derived xenograft model.

Main Results:

• * Achieved high levels of reversion to wild-type alleles without the need for exogenous repair templates.
• * Demonstrated that CRISPR-mediated IGC to revert ASXL1 mutations significantly prolonged survival in a leukemia xenograft model (median survival increase from 27.5 to 33 days).

Conclusions:

• * CRISPR-mediated IGC is a viable laboratory tool for correcting SNVs in various leukemia types, offering a template-free approach.
• * This method expands the range of targetable genetic lesions for IGC, potentially simplifying and reducing the cost of experiments modeling SNV consequences.
• * The SNV-specific IGC technique provides a novel strategy for investigating the phenotypic effects of targeted clonal reduction of leukemogenic mutations.