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CRISPR Cas9 介导的性别决定途径基因敲除。

CRISPR Cas9 mediated knockout of sex determination pathway genes in .

机构信息

Department of Zoology, Government College University Faisalabad, Faisalabad, Pakistan.

Centre of Department of Biochemistry/US-Pakistan Center for Advance Studies in Agriculture and Food Security (USPCAS-AFS), University of Agriculture Faisalabad, Faisalabad, Pakistan.

出版信息

Bull Entomol Res. 2023 Apr;113(2):243-252. doi: 10.1017/S0007485322000505. Epub 2022 Oct 19.

Abstract

The vector role of for viral diseases including dengue and dengue hemorrhagic fever makes it imperative for its proper control. Despite various adopted control strategies, genetic control measures have been recently focused against this vector. CRISPR Cas9 system is a recent and most efficient gene editing tool to target the sex determination pathway genes in . In the present study, CRISPR Cas9 system was used to knockout () and () genes in embryos. The injection mixes with Cas9 protein (333 ng ul) and gRNAs (each at 100 ng ul) were injected into eggs. Injected eggs were allowed to hatch at 26 ± 1°C, 60 ± 10% RH. The survival and mortality rate was recorded in knockout and . The results revealed that knockout produced low survival and high mortality. A significant percentage of eggs (38.33%) did not hatch as compared to control groups ( value 0.00). Highest larval mortality (11.66%) was found in the knockout of female isoform, whereas, the emergence of only male adults also showed that the knockout of (female isoform) does not produce male lethality. The survival (3.33%) of knockout for eggs to the normal adults suggested further study to investigate as an efficient upstream of to target for sex transformation in mosquitoes.

摘要

该病毒的载体作用包括登革热和登革出血热,因此必须对其进行适当的控制。尽管采取了各种控制策略,但最近的研究重点是针对这种载体的遗传控制措施。CRISPR Cas9 系统是一种最新的、最有效的基因编辑工具,可以靶向 的性别决定途径基因。在本研究中,CRISPR Cas9 系统被用于敲除 和 基因。将 Cas9 蛋白(333ng/ul)和 gRNA(各 100ng/ul)混合的注射混合物注入卵中。将注射后的卵在 26±1°C、60±10%RH 的条件下孵化。记录敲除 和 中的存活率和死亡率。结果表明,敲除会导致低存活率和高死亡率。与对照组相比,未孵化的卵的比例显著增加( 值 0.00)。在 雌性同工型的敲除中,幼虫死亡率最高(11.66%),而仅出现雄性成虫表明, (雌性同工型)的敲除不会导致雄性致死。敲除 卵的存活率(3.33%)可发育至正常成虫,这表明进一步研究 可作为 的有效上游基因,用于在 蚊中进行性别转化。

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