Efficient somatic and germline genome engineering of Bactrocera dorsalis by the CRISPR/Cas9 system

Abstract

BACKGROUND: Bactrocera dorsalis (Hendel), a very destructive insect pest of many fruits and vegetables, is widespread in many Asian countries. To facilitate control of this pest, it is essential to investigate its genetics and gene function using targeted gene disruption.RESULTS: Here, we describe successful targeted mutagenesis of the white and transformer genes in B. dorsalis through use of the clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) system. Co-injection of the white sgRNA and Cas9 mRNA into B. dorsalis embryos caused eye color change, and the white mutations in the germline were heritable. CRISPR-mediated knockout of the sex determination gene transformer (tra) in B. dorsalis resulted in a male-biased sex ratio and adult flies with abnormal outer and interior reproductive organs. Small indels and substitutions were induced by CRIRPR for both genes.CONCLUSION: Our data demonstrate that somatic and germline genome engineering of the pest B. dorsalis can be performed efficiently using the CRISPR/Cas9 system, opening the door to the use of the CRISPR-mediated method for functional annotations of genes in B. dorsalis and for its population control using, for example, such as gene drive.

Specific Gene Disruption in the Major Livestock PestsCochliomyia hominivoraxandLucilia cuprinaUsing CRISPR/Cas9

“…For example, CRISPR-based gene drive strains could be evaluated in the future for population suppression of these important pests of livestock (Hammond et al 2016; Scott et al 2018; Kyrou et al 2018). Concha and Scott (2009) found that some fully transformed XX L. cuprina flies are fertile, as it was also described by Zhao et al (2019) for the Oriental fruit fly, Bactrocera dorsalis . Thus both XY and fully transformed XX flies could contribute to population suppression.…”

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.…”

Developing genetic tools to control the Oriental Fruit Fly Bactrocera dorsalis [Diptera: Tephritidae]: potential strategies and molecular tools

“…In Bactrocera dorsalis , this technology has been proven effective several times in the study of gene function (Baia et al ., 2019;X. Chen et al ., 2021;Wanget al ., 2020;Zhao et al ., 2019;Zheng et al ., 2019). CRISPR-Cas9 also offers the advantage of a diversity of Cas9 systems depending on the bacterial source (Khalil, 2020) and flexibility of use as DNA in a plasmid (Zheng et al , 2019), as injected mRNA Wang et al ., 2020) or directly as protein (Baia et al , 2019).…”