摘要
The RNA-guided CRISPR/Cas9 system has been shown to be a powerful tool for genome editing in various organisms. A comprehensive toolbox for multiplex genome editing has been developed for the silkworm,Bombyx mori, a lepidopteran model insect of economic importance. However, as previous methods mainly relied on delivery of transient Cas9/guide RNA（gRNA）, they could not be used in loss-of-function studies of essential genes. Here, we report a simple and versatile tissue-specific genome editing strategy.We perform a proof-of-principle demonstration by establishing and crossing two transgenic B. mori lines,one expressing Cas9 protein in the posterior silk glands（PSGs） and the other constitutively expressing BmlaminA/C（BmLMN） gRNA. All BmLMN alleles in the PSG cells were edited precisely at the target genome region, resulting in diverse mutations. mRNA expression of BmLMN was reduced by up to 75%,and only very low levels of BmLaminA/C protein were detected. Knockout of BmLMN produced obvious defects in gland cell development and cocoon production. In this study, we developed an efficient strategy for spatially controlled genome editing, providing unprecedented opportunities for investigating the function of essential/lethal genes in B. mori, with potential application for other insects.
The RNA-guided CRISPR/Cas9 system has been shown to be a powerful tool for genome editing in various organisms. A comprehensive toolbox for multiplex genome editing has been developed for the silkworm,Bombyx mori, a lepidopteran model insect of economic importance. However, as previous methods mainly relied on delivery of transient Cas9/guide RNA（gRNA）, they could not be used in loss-of-function studies of essential genes. Here, we report a simple and versatile tissue-specific genome editing strategy.We perform a proof-of-principle demonstration by establishing and crossing two transgenic B. mori lines,one expressing Cas9 protein in the posterior silk glands（PSGs） and the other constitutively expressing BmlaminA/C（BmLMN） gRNA. All BmLMN alleles in the PSG cells were edited precisely at the target genome region, resulting in diverse mutations. mRNA expression of BmLMN was reduced by up to 75%,and only very low levels of BmLaminA/C protein were detected. Knockout of BmLMN produced obvious defects in gland cell development and cocoon production. In this study, we developed an efficient strategy for spatially controlled genome editing, providing unprecedented opportunities for investigating the function of essential/lethal genes in B. mori, with potential application for other insects.
基金
supported by the grants from the National Natural Science Foundation of China(No. 31530071)
the Chongqing Postdoctoral Science Foundation (Xm2015024)
