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

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 nonmodel multicellular organism, Bombyx mori We developed a piggyBac-delivered binary genome editing strategy, which can simultaneously meet the requirements of mixed microinjection, efficient multipurpose 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 pollutant 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 functional B. mori genomics, as well as a powerful resource for identifying the potential of key candidate genes for improving various economic traits. This study also shows the effectiveness, practicality, and convenience of large-scale mutant libraries in other nonmodel organisms.

Effects of poly (ADP-ribose) polymerase 1 (PARP1) on silk proteins in the silkworm, Bombyx mori.

Animal silk is economically important, while silk secretion is a complex and subtle mechanism regulated by many genes. We identified the poly (ADP-ribose) polymerase (PARP1) gene of the silkworm and successfully cloned its coding sequence (CDS) sequence. Using clustered regularly interspaced short palindromic repeat (CRISPR/Cas9) technology, we screened single guide RNA (sgRNA) with high knockout efficiency by cellular experiments and obtained PARP1 mutants by knocking out the PARP1 gene of the silkworm at the individual level. We found that the mutants mainly exhibited phenotypes such as smaller cocoon size and reduced cocoon shell rate than the wild type. We also detected the expression of silk protein genes in the mutant by quantitative real-time PCR (qPCR) and found that the expression of some silk protein genes was slightly down-regulated. Meanwhile, together with the results of transcriptomic analysis, we hypothesized that PARP1 may affect the synthesis of silk proteins, resulting in their failure to function properly. Our study may provide an important reference for future in-depth refinement of the molecular mechanism of silk protein expression in silk-producing animals, as well as a potential idea for future development of molecular breeding lines of silkworms to improve silk production.

Ectopic expression of BmeryCA in Bombyx mori increases silk yield and mechanical properties by altering the pH of posterior silk gland.

The silk glands are the specialized tissue where silk protein synthesis, secretion, and conformational transitions take place, with pH playing a critical role in both silk protein synthesis and fiber formation. In the present study, we have identified erythrocyte carbonic anhydrase (BmeryCA) belonging to the α-CA class in the silk gland, which is a Zn2+ dependent metalloenzyme capable of efficiently and reversibly catalyzing the hydrated reaction of CO2 to HCO3-, thus participating in the regulation of acid-base balance. Multiple sequence alignments revealed that the active site of BmeryCA was highly conserved. Tissue expression profiling showed that BmeryCA had relatively high expression levels in hemolymph and epidermis but is barely expressed in the posterior silk gland (PSG). By specifically overexpressing BmeryCA in the PSG, we generated transgenic silkworms. Ion-selective microelectrode (ISM) measurements demonstrated that specifically overexpression of BmeryCA in the PSG led to a shift in pH from weakly alkaline to slightly neutral conditions. Moreover, the resultant PSG-specific BmeryCA overexpression mutant strain displayed a significant increase in both silk yield and silk fiber mechanical properties. Our research provided new insights into enhancing silk yield and improving the mechanical properties of silk fibers.