CRISPR/Cas9-induced gene conversion between ATAD3 paralogs

Paralogs and pseudogenes are abundant within the human genome, and can mediate non-allelic homologous recombination (NAHR) or gene conversion events. The ATAD3 locus contains three paralogs situated in tandem, and is therefore prone to NAHR-mediated deletions and duplications associated with severe neurological phenotypes. To study this locus further, we aimed to generate biallelic loss-of-function variants in ATAD3A by CRISPR/Cas9 genome editing. Unexpectedly, two of the generated clones underwent gene conversion, as evidenced by replacement of the targeted sequence of ATAD3A by a donor sequence from its paralog ATAD3B. We highlight the complexity of CRISPR/Cas9 design, end-product formation, and recombination repair mechanisms for CRISPR/Cas9 delivery as a nucleic acid molecular therapy when targeting genes that have paralogs or pseudogenes, and advocate meticulous evaluation of resultant clones in model organisms. In addition, we suggest that endogenous gene conversion may be used to repair missense variants in genes with paralogs or pseudogenes.