Deep Sequencing Reveals the Comprehensive CRISPR-Cas9 Editing Spectrum in Bombyx mori

Abstract

Application of the clustered regularly interspaced short palindromic repeats associated 9 (CRISPR-Cas9) technology has revolutionized biology by greatly enhancing the ability to introduce mutations into DNA for research and prospective therapeutic purposes. However, the understanding of Cas9 editing outcomes is still limited. Previously, it was considered that Cas9 introduces stochastic insertions or deletions (indels) at the target site. In the current study, we performed in vivo multiplex editing, deep sequencing, and comprehensive analysis of its editing outcomes in Bombyx mori (B. mori). A total of 31161 editing events from 9 single-guide RNA (sgRNA) sites in 16 individuals were generated and analyzed, and we found that Cas9 introduces mutations with some regularity rather than via stochastic indels. The editing efficiency varies with sgRNA sequences, individuals, and orientation. Small deletions account for the vast majority of mutated sequences, followed by a small fraction of substitutions and insertions. The most likely mutations are deletions between two microhomologous sequences or singlebase deletions at the cleavage site in the absence of microhomologous pairs. Insertions are formed by diverse mechanisms, including direct acquisition of free genomic fragments, duplication of broken ends, replication of adjacent sequences, or random addition of free nucleotides. The above results indicate that the Cas9 editing spectrum is reproducible and predictable. Thus, our findings enable a deeper understanding of Cas9-mediated mutagenesis and better design of genome editing experiments, as well as elucidate the DNA double-strand break repair processes in B. mori.

High-throughput and genome-scale targeted mutagenesis using CRISPR in a nonmodel multicellular organism,Bombyx mori

“…More than 84% of the deletions occurred upstream of the cleavage sites ( Fig. 2 H), which further confirmed the distinct unidirectional feature of DSB repair in B. mori ( Chang et al 2020b ; Ma et al 2021 ). Deletion sizes were generally negatively correlated with their occurrence, with significant occurrence peaks at each 3X base pair ( Fig.…”

“…sgRNAs targeting exons were ranked using criteria described previously ( Chang et al 2020a ). sgRNAs targeting promoters were ranked using an additional criterion; only sgRNAs targeting the sense strand were selected owing to previous findings that the DSBs were repaired in a direction-biased manner ( Chang et al 2020b ; Ma et al 2021 ). A total of 92,917 sgRNAs were chosen (about eight E-sgRNAs and about two P-sgRNAs per gene) and synthesized on a microarray chip ( Supplemental Table S1 ).…”

“…Large-scale deep sequencing of target sites provided the opportunity to investigate editing outcomes in vivo, the patterns of which were previously found to display distinct differences between cultured cells of B. mori and that of other species ( Chang et al 2020b ; Ma et al 2021 ). Of the total 61,720,266 reads, we detected 28,927,046 mutated reads containing 1,387,421 mutation types.…”

High-throughput and genome-scale targeted mutagenesis using CRISPR in a non-model multicellular organism,Bombyx mori

“…sgRNAs targeting exons were ranked using criteria described previously (Chang et al 2020a). sgRNAs targeting promoters were ranked using an additional criterion; only sgRNAs targeting the sense strand were selected due to previous findings that the DSBs were repaired in a direction-biased manner (Chang et al 2020b;Ma et al 2021). A total of 92,917 sgRNAs were chosen (~8 E-sgRNAs and ~2 P-sgRNAs per gene) and synthesized on a microarray chip (Supplemental Table S1).…”

Optimized CRISPR/Cas9 system for gene knockout in chicken DF1 cells

“…In addition to indel frequency, amplicon sequencing also provides accurate estimates of indel size, frequency, and position distribution ( Li et al, 2018 ). These results allow to precisely predict the outcomes of an edit, which enable a deeper understanding of Cas9-mediated mutagenesis and better design of genome editing experiments ( Ma et al, 2021 ). In the present study, the T7E1 and amplicon sequencing assays were compared to evaluate their detections accuracy and sensitivity.…”