Work

Insights into the Mechanism of Allele Conversion using a Fluorescent Reporter-Based Compound Heterozygous Allele Conversion Reporter (CHACR) Cell Line Model

Year: 2026

Type: article

Abstract: Compound heterozygous cells have two different disease-causing variants on different alleles of the same gene. Targeting one of the variants in a compound heterozygous cell with Cas9 nuclease or D10A nickase, plus an allele-specific guide RNA that is specific for one of the mutations, has been shown to trigger allele conversion and restore the wild-type sequence to the targeted allele. To gain insights into this process, we designed and created a Compound Heterozygous Allele Conversion Reporter cell line comprising two near-identical alleles with wild-type or mutant versions of three different fluorescent reporter genes. The cell line was designed with mutations that prevent expression of a functional mCherry reporter protein, but create protospacer adjacent motifs for Cas9 recognition which can be selectively targeted with allele-specific gRNAs. When transfected with either Cas9 nuclease, D10A nickase or ABE8e, plus an allele-specific gRNA targeting the allele containing the mCherry fs-2,Y77X variant, >10% of cells recover mCherry fluorescence. Sorting and sequencing these mCherry-fluorescent cells revealed wild-type mCherry sequence indicative of allele conversion of the mCherry fs-2,Y77X variant using the homologous allele as a repair template. Allele conversion efficiency is increased by inhibiting DNA-PKcs but is reduced by overexpression of dnMLH1 or Rad51.

Source: Journal of Biological Chemistry

DOI: 10.1016/j.jbc.2026.113690