Highly efficient genome editing by homology-directed repair using Cas9 protein in Ceratitis capitata. (October 2018)

• Record Type: Journal Article
• Authors: Aumann, Roswitha A.; Schetelig, Marc F.; Häcker, Irina
• Is Part Of: Insect biochemistry and molecular biology. Volume 101(2018)
• Page Start: 85
• Page End: 93
• Publication Date: 2018-10
• Subjects: ssODN-mediated homology-directed repair -- CRISPR-Cas9 ribonucleoprotein complexes -- Mediterranean fruit fly -- Tephritids -- Sterile insect technique; BFP blue fluorescent protein -- bp base pair(s) -- Cas CRISPR associated -- CRISPR Clustered Regularly Interspaced Short Palindromic Repeats -- DSB double-strand brake -- eGFP enhanced green fluorescent protein -- GMO genetically modified organism -- gRNA guide RNA -- HDR homology directed repair -- IPM integrated pest management -- NHEJ non-homologous end-joining -- PAM protospacer adjacent motif -- SIT sterile insect technique -- ssODN single-stranded oligodeoxynucleotide -- YFP yellow fluorescent protein; Insect biochemistry -- Periodicals; Insects -- Physiology -- Periodicals; Insects -- Molecular aspects -- Periodicals; Biochemistry -- Periodicals; Insectes -- Biochimie -- Périodiques; Insectes -- Composition -- Périodiques; Insectes -- Physiologie -- Périodiques; Insectes -- Aspect moléculaire -- Périodiques; Biochimie -- Périodiques; Biochemistry; Insect biochemistry; Insects -- Molecular aspects; Insects -- Physiology; Periodicals; 572.8157
• DOI: 10.1016/j.ibmb.2018.08.004
• Languages: English
• ISSNs: 0965-1748

Abstract

Abstract: The Mediterranean fruit fly Ceratitis capitata is a highly polyphagous and invasive insect pest, causing enormous economic damage in horticultural systems. A successful and environment-friendly control strategy is the sterile insect technique (SIT) that reduces pest populations through infertile matings with mass-released, sterilized insects. However, the SIT is not readily applicable to each pest species. While transgenic approaches hold great promise to improve critical aspects of the SIT to transfer it to new species, they are suspect to strict or even prohibitive legislation regarding the release of genetically modified (GM) organisms. In contrast, specific mutations created via CRISPR-Cas genome editing are not regulated as GM in the US, and might thus allow creating optimal strains for SIT. Here, we describe highly efficient homology-directed repair genome editing in C. capitata by injecting pre-assembled CRISPR-Cas9 ribonucleoprotein complexes using different guide RNAs and a short single-stranded oligodeoxynucleotide donor to convert an enhanced green fluorescent protein in C. capitata into a blue fluorescent protein. Six out of seven fertile and individually backcrossed G0 individuals generated 57–90% knock-in rate within their total offspring and 70–96% knock-in rate within their phenotypically mutant offspring. Based on the achieved efficiency, this approach could also be used to introduce mutations which do not produce a screenable phenotype and identify positive mutants with a reasonable workload. Furthermore, CRISPR-Cas HDR would allow to recreate mutations formerly identified in classical mutagenesis screens and to transfer them to related species to establish new (SIT-like) pest control systems. Considering the potential that CRISPR-induced alterations in organisms could be classified as non-GM in additional countries, such new strains could potentially be used for pest control applications without the need to struggle with GMO directives. Graphical abstract: Highlights: First-time CRISPR-Cas HDR gene editing in a Tephritid. High HDR efficiency using Cas9 protein and a short, ssDNA repair template, important for mutagenesis without phenotypic marker. Insights into the molecular mechanism of CRISPR-Cas gene editing in Tephritids. Allows the creation of insect strains for environmentally-friendly pest control with the potential to be considered as non-GM.