ReMOT control delivery of CRISPR-Cas9 RNP complex to induce germline mutagenesis in the disease vector mosquitoes Culex pipiens pallens
ReMOT control delivery of CRISPR-Cas9 RNP complex to induce germline mutagenesis in the disease vector mosquitoes Culex pipiens pallens
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ReMOT 控制 CRISPR-Cas9 RNP 复合物的传递以诱导疾病媒介蚊子淡色库蚊种系突变
作者：
li X;Xu Y;Zhang H;Yin H;Zhou D;Sun Y;Ma L;Shen B;Zhu C
尖音库蚊复合体的广泛分布使得很难预防蚊媒疾病在人类中的传播。基于CRISPR/Cas9的基因编辑是一种强大的方法，有可能解决日益严重的蚊媒疾病问题。本研究使用ReMOT控制技术在淡色库蚊中产生转基因蚊子。通过注射60只成年雌性蚊子建立了显微注射系统-需要14 µL注射混合物，且≤1 µL内体释放试剂（氯喹或皂苷）不会发生沉淀。当在血餐后24小时（PBM 24小时）注射时，P2 C-EGFP-Cas9（EGFP：增强型绿色荧光蛋白）核糖核蛋白复合物递送到卵巢中的效率为100%。我们使用这种方法进行KMO敲除;我们发现当将P2C-Cas9 RNP复合物注射到成年Cx的血淋巴中时，卵巢中的基因编辑也可能发生。通过ReMOT Control对pipiens pallens进行检测。在氯喹组中，在筛选的2251个G 0后代中，有9个个体表现出白色和马赛克眼表型。在皂苷组中，在筛选的2462个G 0后代中，观察到8个突变个体。测序结果显示有13个bp缺失，进一步证实了基因编辑发生的事实。总之，ReMOT控制在Cx. pipiens pallens不仅为该方法提供了基本参数（注射参数和注射时间），而且还促进了蚊子生物学的研究和更好地控制该疾病媒介。
The wide distribution of Culex pipiens complex mosquitoes makes it difficult to prevent the transmission of mosquito-borne diseases in humans. CRISPR/Cas9-based gene editing is a powerful method with the potential to solve the growing problem of mosquito-borne diseases. The present study uses the ReMOT Control technique in Culex pipiens pallens to produce genetically-modified mosquitoes. A microinjection system was established by injecting sixty adult female mosquitoes—14 µL injection mixture was required, and no precipitation occurred with ≤1 µL of endosomal release reagents (chloroquine or saponin). The efficiency of delivery of the P2C-EGFP-Cas9 (EGFP: Enhanced Green Fluorescent Protein) ribonucleoprotein complex into the ovary was 100% when injected at 24 hours post blood meal (24 h PBM). We used this method for KMO knockout; we found that gene editing in the ovary could also occur when P2C-Cas9 RNP complex was injected into the hemolymph of adult Cx. pipiens pallens by ReMOT Control. In the chloroquine group, of the 2251 G0 progeny screened, nine individuals showed with white and mosaic eye phenotypes. In the saponin group, of the 2462 G0 progeny screened, eight mutant individuals were observed. Sequencing results showed 13 bp deletions, further confirming the fact that gene editing occurred. In conclusion, the successful application of ReMOT Control in Cx. pipiens pallens not only provides the basic parameters (injection parameters and injection time) for this method but also facilitates the study of mosquito biology and better control of this disease vector.