Analysis of Single Nucleotide Variants in CRISPR-Cas9 Edited Zebrafish Exomes Shows No Evidence of Off-Target Inflation

Abstract

Therapeutic applications of CRISPR-Cas9 gene editing have spurred innovation in Cas9 enzyme engineering and single guide RNA (sgRNA) design algorithms to minimize potential off-target events. While recent work in rodents outlines favorable conditions for specific editing and uses a trio design (mother, father, offspring) to control for the contribution of natural genome variation, the potential for CRISPR-Cas9 to induce de novo mutations in vivo remains a topic of interest. In zebrafish, we performed whole exome sequencing (WES) on two generations of offspring derived from the same founding pair: 54 exomes from control and CRISPR-Cas9 edited embryos in the first generation (F0), and 16 exomes from the progeny of inbred F0 pairs in the second generation (F1). We did not observe an increase in the number of transmissible variants in edited individuals in F1, nor in F0 edited mosaic individuals, arguing that in vivo editing does not precipitate an inflation of deleterious point mutations.