Highly efficient genome editing by homology-directed repair using Cas9 protein in Ceratitis capitata

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 G 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.

Female-to-male sex conversion in Ceratitis capitata by CRISPR/Cas9 HDR-induced point mutations in the sex determination gene transformer-2

“…In conclusion, we demonstrated the successful creation of the D. melanogaster tra2 ts2 point mutation in C. capitata via markerless CRISPR/Cas9-HDR gene editing and the importance of the respective amino acid for the correct function of TRA2 in the female sex-determination. The previously shown high HDR efficiency in medfly using a ssODN repair template to convert the marker gene eGFP (enhanced green fluorescent protein) into BFP (blue), could be confirmed in this study, where we achieved 100% knock-in efficiency (2 out of 2 fertile G 0 ) compared to 86% (6 out of 7 fertile G 0 ) in the previous study 39 . Also, the high penetrance of mutant offspring within the G 1 with 75–83% in this study is similar compared to 90% in the previous one.…”

“…Hence, only the tra ts SNPs, but no exogenous DNA was inserted, to help facilitate a potential field release of Cctra2 ts strains. This was possible due to the high mutagenesis rate achieved with our previously published CRISPR/Cas9-HDR protocol 39 , which we now successfully applied for the first time without using a visible phenotype.…”

“…Hence, we used our previously established protocol for markerless CRISPR/Cas9-HDR in medfly yielding high-efficiency mutagenesis 39 to integrate the D. melanogaster tra2 ts1 and tra2 ts2 mutations into the Cctra2 homolog. Omitting the use of a fluorescent marker gene should facilitate the use of non-transgenic strains in SIT programs, as CRISPR/Cas9-induced single nucleotide polymorphisms (SNP) are even considered non-GMO in certain countries 40 .…”

A generic white pupae sex selection phenotype for insect pest control

“…Guide RNA was synthesized by in vitro transcription of linear double-stranded DNA template as previously described 67 to previous studies 67,68 . The mix was freshly prepared on ice followed by an incubation step for 10 min at 37°C to allow pre-assembly of gRNA-Cas9 ribonucleoprotein complexes and stored on ice prior to injections.…”

“…Microinjections were conducted in wild type EgII C. capitata embryos. Eggs were collected over a 30-40 min time period and prepared for injection as previously described 67 . Injections were performed using siliconized quartz glass needles (Q100-70-7.5; LOT171381; Science Products, Germany), drawn out on a Sutter P-2000 laserbased micropipette puller.…”