Comparative analysis of genome editing systems, Cas9 and BE3, in silkworms

Abstract The clustered regularly interspaced short palindromic repeats (CRISPR) system and the Cas9-derived proteins have been applied to genome editing in various organisms. Traditional Cas9 is typically used to knockout genes or specific DNA fragments based on the generation of double-stranded breaks, whereas nCas9 and dCas9 are fused with effectors to perform base pair transitions or epigenetic modification and regulation. However, this system has off-target effects and can cause genomic structure variations. Here, we comparatively analyzed Cas9 and BE3, an initial base editor based on the nCas9 fusion protein, in silkworms. Our results showed that base editing was superior to Cas9 in silkworm cultured cells. BE3 introduced accurate termination codons, whereas Cas9 did not. Moreover, Cas9 induced chromosome translocation, chromosome fragment repetition, and chromosome fragment deletion, with the deletion frequency reaching up to 4.29%. BE3 was not able to induce these changes in our study. Furthermore, Cas9-derived proteins blocked ribosome advance and mRNA transcription for 9 days, with a 9.40% repression effect by combining with double-stranded DNA when single guide RNAs were targeted in the coding region in silkworms. Overall, our findings established a strategy for choosing suitable editing tools for various applications in different organisms.