![]() Dual injections with two appropriate gRNAs have also been used to delete large fragments of DNA, spanning thousands of bp. These modifications are often indels, ranging from one to tens of base pairs (bp) that can generate null alleles, e.g., carrying premature stop codons in the coding region. During the subsequent DNA repair by non-homologous end-joining, random mutations frequently occur upstream of the PAM site. Briefly, the gRNA and Cas9 localize to the genomic site of interest, where Cas9 causes a double-strand break in the DNA upstream to a protospacer adjacent motif (PAM). The mechanism of CRISPR/Cas9 requires a guide RNA (gRNA) and Cas9 endonuclease. In recent years, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 has become the dominant method of targeted mutagenesis due to its reduced costs, simplified workflow, and significantly increased efficiency. mori, several techniques, including Zinc Finger Nucleases and Transcription Activator-Like Effector Nucleases (TALENS), have been developed to generate silkworm strains, carrying specific mutations in endogenous genes, or expressing exogenous factors. Since Tamura and colleagues introduced the transposon-mediated germline transformation in B. mori strains has been fundamental for both basic research and applied purposes. Therefore, the production of modified or mutant B. In addition to the textile industry, silk fiber is also used in the biomedical and cosmetic sectors, and several studies have suggested silkworm larvae are suitable bioreactors, producing valuable recombinant proteins or modified silk useful for applied research. The domesticated silkworm Bombyx mori has been extensively studied as a model organism, allowing the characterization of numerous biological processes, and for its economic value in silk production. This protocol could be further applied to screen CRISPR/Cas9 mutations in haploid insects. The use of these methodologies in a sequential combination allows the identification of CRISPR/Cas9-induced mutations in genes mapping on both autosomes and sex chromosomes, and the selection of appropriate individuals to found stable mutant B. This approach relies on the use of different molecular methods, the heteroduplex assay, cloning followed by Sanger sequencing, and the amplification refractory mutation system PCR. mori lines, stably inheriting single CRISPR/Cas9-induced mutations. Here we illustrate a complete and efficient workflow to screen, characterize rapidly and follow mutations through generations, allowing the generation of B. In recent years, CRISPR/Cas9 technology is being rapidly adopted as the most efficient molecular tool for generating silkworm lines carrying mutations in target genes. mori strains significantly enhances basic and applied silkworm research. Silkworms also have applications in biomedical and cosmetic industries, and the production of mutant B. The domestic silkworm Bombyx mori is extensively studied as a model organism for lepidopteran genetics and has an economic value in silk production.
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