CRISPR/Cas9 mediated multiplex genome editing and heritable mutagenesis of BmKu70 in Bombyx mori

Abstract

CRISPR/Cas9, a bacterial adaptive immune system derived genome-editing technique, has become to be one of the most compelling topics in biotechnology. Bombyx mori is an economically important insect and a model organism for studying lepidopteran and arthropod biology. Here we reported highly efficient and multiplex genome editing in B. mori cell line and heritable site-directed mutagenesis of Bmku70, which is required for NHEJ pathway and also related to antigen diversity, telomere length maintenance and subtelomeric gene silencing, using CRISPR/Cas9 system. We established a simple and practicable method and obtained several Bmku70 knockout B. mori lines, and showed that the frequency of HR was increased in embryos of the Bmku70 knockout B. mori. The mutant lines obtained in this study could be a candidate genetic resource for efficient knock-in and fundamental research of DNA repair in B. mori. We also provided a strategy and procedure to perform heritable genome editing of target genes with no significant phenotype effect.

Highly efficient Cas9-mediated gene drive for population modification of the malaria vector mosquitoAnopheles stephensi

“…Additionally, dsRNA was injected targeting the putative ortholog of Ku70, a protein essential for nonhomologous end joining (45,46). Knockdown of this protein may increase the possibility of repair by homologous recombination (47,48). The putative ortholog for Ku70 in An.…”

A Practical Guide to CRISPR/Cas9 Genome Editing in Lepidoptera

“…The HDR pathway can replace an endogenous genome segment with a homologous donor sequence and can thus be used for knockin of foreign DNA into a selected genomic locus. To our knowledge, there are only two published examples of this approach in Lepidoptera, both of which (Ma et al 2014;Zhu et al 2015). To test the feasibility of this approach in butterflies, we sought to insert an in-frame EGFP coding sequence into to the V. cardui dopa decarboxylase (Ddc) locus using a donor plasmid containing the EGFP coding sequences and homologous arms matching endogenous sequences flanking the Cas9 cut sites ( Fig.…”

“…The earliest published reports of Cas9-mediated genome editing in Lepidoptera, from 2013 and 2014, all describe work done in B. mori (Wang et al 2013;Ma et al 2014;Wei et al 2014) -an experimental system that benefits from a large research community that had already developed efficient methods for injection, rearing, and genotyping. To our knowledge, Wang et al (2013) represent the first published report of Cas9-mediated genome editing in Lepidoptera and set three important precedents.…”

“…Third, they showed that deletions could occur in the germ line at a high enough frequency to generate stable lines. After Wang et al (2013), one of the next most important technical advancements came from Ma et al (2014), who showed that knockins could be achieved using a donor plasmid to insert a DsRed expression cassette using~1 kb homology arms. Following this, Zhu et al showed successful epitope tagging of BmTUDOR-SN gene by CRISPR/Cas9-mediated knockin in Bombyx cells (Zhu et al 2015).…”

“…Compared to NHEJ-mediated high efficiency knockouts (69% in the case of V. cardui Ddc deletion knockouts (Zhang and Reed 2016)), the rate of HDR-mediated targeted integration is low, at~3% in our most recent trials. It has been shown that knocking out factors in the NHEJ pathway can enhance the HDR pathway and increase gene targeting efficiency in Bombyx (Ma et al 2014;Zhu et al 2015). Some Cas9-mediated homology-independent knockin approaches have shown higher efficiency rates in zebrafish (Auer et al 2014) and human cell lines (He et al 2016), suggesting NHEJ repair may provide an alternate strategy to improve incorporation of donor DNA in Lepidoptera.…”