Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated 9‐mediated mutagenesis of the multiple edematous wings gene induces muscle weakness and flightlessness in Bactrocera dorsalis (Diptera: Tephritidae)

Abstract

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated 9 (Cas9) system is a versatile, efficient and heritable gene editing tool that can be useful for genome engineering. Bactrocera dorsalis (Hendel) is a major pest of agriculture that causes great economic losses. We used the B. dorsalis multiple edematous wings (Bdmew) gene as the target gene to explore the effectiveness of CRISPR/Cas9 for B. dorsalis genome manipulation. We studied the physiological functions of the Bdmew gene, particularly those related to muscle development. Site‐specific genome editing was feasible using direct microinjection of specific guide RNA and the Cas9‐plasmid into B. dorsalis embryos. Mutation frequencies ranged from 12.1 to 30.2% in the injected generation. Mosaic G0, with the mew mutation, was heritable to the next generation. The G1 displayed a series of defective phenotypes including muscle weakness, flightlessness, failure to eclose, wing folds and unbalanced movement. These results demonstrated that CRISPR/Cas9 can act as a highly specific, efficient, heritable tool for genome manipulation in B. dorsalis and this has significance for gene function research and genetic control of pests. The Bdmew gene possesses key functions in muscle development of B. dorsalis. Bdmew mutations cause a series of serious defects by interfering with muscle development and may provide a means for controlling B. dorsalis via a gene‐based method such as gene drive.

CRISPR‐mediated mutagenesis of the odorant receptor co‐receptor (Orco) gene disrupts olfaction‐mediated behaviors in Bactrocera dorsalis

“…Meanwhile, the mutant efficiency in the surviving G 0 flies was 23% (Table 1). This was similar to previous research on B. dorsalis (Zheng et al, 2019) but lower than some previously reported insect mutants induced by the CRISPR/Cas9 system (Yu et al, 2013;Awata et al, 2015). However, the mutant efficiency depends on many factors, including the selection of gRNA target sites, the concentration of gRNA and the Cas9 protein, microinjection of the eggs, and the methods used for screening mutants (Li et al, 2016).…”

CRISPR/Cas9‐mediated knockout of the eye pigmentation gene white leads to alterations in colour of head spots in the oriental fruit fly, Bactrocera dorsalis

“…We synthesized two sgRNAs, named BdWhite‐g1 and BdWhite‐g2 , which are located in exons 1 and 3 respectively, and we injected a mixture of Cas9 protein and sgRNA, transcribed in vitro , into fresh eggs that resulted in highly efficient editing of the BdWhite locus. We observed a 100% editing frequency in the surviving flies of the BdWhite‐g2 ‐injected generation; this 100% germline transmission rate was higher than reported for other insects (Wang et al ., ; Xue et al ., ; Chen et al ., ), including B. dorsalis (Zhao et al ., ; Zheng et al ., ; Sim et al ., ). The high mutation efficiency may be due to the high concentration of sgRNA (500 ng/μl) used in this study, and rapid injection of the embryos following oviposition (within 1 h) and the injection of cas9 protein into embryos, rather than cas9 mRNA or plasmid (Zhao et al ., ; Zheng et al ., ).…”

“…The CRISPR/Cas9 system is a simple and efficient genome‐editing technique that shows significant potential for genetic editing and exploring gene function in nonmodel animals (Choo et al ., ; Wang et al ., ; Xue et al ., ; Chen et al ., ). The system has been used to edit genomes of several Tephritidae, including B. dorsalis (Meccariello et al ., ; Zhao et al ., ; Zheng et al ., ; Li and Handler, ; Sim et al ., ), and here we successfully applied it to study the function of the white gene in B. dorsalis . We synthesized two sgRNAs, named BdWhite‐g1 and BdWhite‐g2 , which are located in exons 1 and 3 respectively, and we injected a mixture of Cas9 protein and sgRNA, transcribed in vitro , into fresh eggs that resulted in highly efficient editing of the BdWhite locus.…”

Precise single base substitution in the shibire gene by CRISPR/Cas9-mediated homology directed repair in Bactrocera tryoni

“…Targeted mutagenesis using the CRISPR/Cas9 genome editing technology has now been successfully achieved in a number of agricultural pest species, where nonspecific indel mutations were generated through the NHEJ pathway [10,[23][24][25][26][27][28]. Whilst NHEJ-induced indel mutations have been useful for enabling the study of gene function, a greater focus has now been placed on using the HDR repair pathway particularly to introduce precise mutations to generate specific mutants without producing transgenic strains that contain DNA from other species.…”