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Large-scale genetic mutant libraries are powerful approaches to interrogating genotype-phenotype correlations and identifying genes responsible for certain environmental stimuli, both of which are the central goal of life science study. We produced the first large-scale CRISPR/Cas9-induced library in a non-model multicellular organism,Bombyx mori. We developed apiggyBac-delivered binary genome editing strategy, which can simultaneously meet the requirements of mixed microinjection, efficient multi-purpose genetic operation, and preservation of growth-defect lines. We constructed a single-guide RNA (sgRNA) plasmid library containing 92,917 sgRNAs targeting promoters and exons of 14,645 protein-coding genes, established 1726 transgenic sgRNA lines following microinjection of 66,650 embryos, and generated 300 mutant lines with diverse phenotypic changes. Phenomic characterization of mutant lines identified a large set of genes responsible for visual phenotypic or economically valuable trait changes. Next, we performed pooled context-specific positive screens for tolerance to environmental pollute cadmium exposure, and identified KWMTBOMO12902 as a strong candidate gene for breeding applications in sericulture industry. Collectively, our results provide a novel and versatile approach for functionalB. morigenomics, and a powerful resource for identifying key candidate genes potential for improving various economic traits. This study also demonstrates the effectiveness, practicality, and convenience of large-scale mutant libraries in other non-model organisms.

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.