Highly efficient genome editing by homology-directed repair using Cas9 protein in Ceratitis capitata
Highly efficient genome editing by homology-directed repair using Cas9 protein in Ceratitis capitata
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DOI：
10.1016/j.ibmb.2018.08.004
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发表时间：
2018-10-01
影响因子：
3.8
通讯作者：
Haecker, Irina
中科院分区：
文献类型：
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作者：
Aumann, Roswitha A.;Schetelig, Marc F.;Haecker, Irina
地中海实蝇Ceratitis capitate是一种高度杂食性和入侵性的害虫，在园艺系统中造成巨大的经济损失。一个成功的和环境友好的控制策略是不育昆虫技术（SIT），通过不育交配与大量释放，绝育昆虫减少害虫种群。然而，SIT并不容易适用于每种害虫。虽然转基因方法在改善SIT的关键方面以将其转移到新物种方面有很大的希望，但它们被怀疑会对关于释放转基因生物的严格甚至禁止性立法产生影响。相比之下，通过CRISPR-Cas基因组编辑产生的特定突变在美国不像GM那样受到监管，因此可能允许创建SIT的最佳菌株。在这里，我们描述了高效的同源定向修复基因组编辑在C。通过注射使用不同指导RNA和短单链寡脱氧核苷酸供体的预组装CRISPR-Cas9核糖核蛋白复合物来转化C.变成蓝色荧光蛋白。七个可育的和单独回交的Go个体中的六个在其总后代中产生57-90%的敲入率，并且在其表型突变后代中产生70-96%的敲入率。基于所实现的效率，该方法还可以用于引入不产生可筛选表型的突变，并以合理的工作量鉴定阳性突变体。此外，CRISPR-Cas HDR将允许重建以前在经典诱变筛选中鉴定的突变，并将它们转移到相关物种中以建立新的（SIT样）害虫控制系统。考虑到CRISPR诱导的生物体改变在其他国家可能被归类为非转基因，这些新菌株可能用于害虫控制应用，而无需与转基因指令作斗争。
The Mediterranean fruit fly Ceratitis capitate 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. capitate 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. capitate into a blue fluorescent protein. Six out of seven fertile and individually backcrossed Go 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.