Analysis of off-target effects in CRISPR-based gene drives in the human malaria mosquito
Analysis of off-target effects in CRISPR-based gene drives in the human malaria mosquito
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DOI：
10.1073/pnas.2004838117
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发表时间：
2021-06-01
影响因子：
11.1
通讯作者：
Simoni, Alekos
中科院分区：
文献类型：
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作者：
Garrood, William T.;Kranjc, Nace;Simoni, Alekos
基于 CRISPR-Cas9 核酸酶的基因驱动器的开发旨在控制人类疟疾媒介冈比亚按蚊。基因驱动基于种系中 Cas9 核酸酶的活性来源，通过同源定向修复（“归巢”）促进转基因的超孟德尔遗传。在该技术进行任何潜在的现场发布之前，了解 CRISPR 诱导的脱靶突变是否会在按蚊中产生是风险评估的一个重要方面。我们比较了四种不同基因驱动菌株中脱靶事件发生的频率和倾向，包括故意混杂的设置，使用 SpCas9 的非种系限制性启动子和在蚊子基因组中具有许多密切相关位点（两个或多个错配）的指导 RNA。在这种情况下，我们观察到脱靶突变的频率不超过 1.42%。我们没有看到任何证据表明 CRISPR 诱导的脱靶突变能够在蚊子种群中积累（或驱动），尽管多代人都暴露于 CRISPR-Cas9 核酸酶构建体。此外，用于 CRISPR 构建体归巢的向导 RNA 的明智设计，再加上 Cas9 表达在种系中的严格时间限制，使得脱靶突变无法检测到。这项研究的结果代表了理解和管理蚊子中 CRISPR-Cas9 特异性的一个重要里程碑，并表明在蚊子基因驱动背景下的 CRISPR 脱靶编辑可以降低到最低水平。
CRISPR-Cas9 nuclease-based gene drives have been developed toward the aim of control of the human malaria vector Anopheles gambiae. Gene drives are based on an active source of Cas9 nuclease in the germline that promotes super-Mendelian inheritance of the transgene by homology-directed repair ("homing"). Understanding whether CRISPR-induced off-target mutations are generated in Anopheles mosquitoes is an important aspect of risk assessment before any potential field release of this technology. We compared the frequencies and the propensity of off-target events to occur in four different gene-drive strains, including a deliberately promiscuous set-up, using a nongermline restricted promoter for SpCas9 and a guide RNA with many closely related sites (two or more mismatches) across the mosquito genome. Under this scenario we observed off-target mutations at frequencies no greater than 1.42%. We witnessed no evidence that CRISPR-induced off-target mutations were able to accumulate (or drive) in a mosquito population, despite multiple generations' exposure to the CRISPR-Cas9 nuclease construct. Furthermore, judicious design of the guide RNA used for homing of the CRISPR construct, combined with tight temporal constriction of Cas9 expression to the germline, rendered off-target mutations undetectable. The findings of this study represent an important milestone for the understanding and managing of CRISPR-Cas9 specificity in mosquitoes, and demonstrates that CRISPR off-target editing in the context of a mosquito gene drive can be reduced to minimal levels.