Single nucleotide editing without DNA cleavage using CRISPR/Cas9‐deaminase in the sea urchin embryo

Background: A single base pair mutation in the genome can result in many congenital disorders in humans. The recent gene editing approach using CRISPR/Cas9 has rapidly become a powerful tool to replicate or repair such mutations in the genome. These approaches rely on cleaving DNA, while presenting unexpected risks. Results: In this study, we demonstrate a modified CRISPR/Cas9 system fused to cytosine deaminase (Cas9‐DA), which induces a single nucleotide conversion in the genome. Cas9‐DA was introduced into sea urchin eggs with sgRNAs targeted for SpAlx1, SpDsh , or SpPks , each of which is critical for skeletogenesis, embryonic axis formation, or pigment formation, respectively. We found that both Cas9 and Cas9‐DA edit the genome, and cause predicted phenotypic changes at a similar efficiency. Cas9, however, resulted in significant deletions in the genome centered on the gRNA target sequence, whereas Cas9‐DA resulted in single or double nucleotide editing of C to T conversions within the gRNA target sequence. Conclusions: These results suggest that the Cas9‐DA approach may be useful for manipulating gene activity with decreased risks of genomic aberrations. Developmental Dynamics 246:1036–1046, 2017 . © 2017 Wiley Periodicals, Inc.