Kulkarni Aditi, Yu Wanqin, Moon Alex S, Pandey Ashmita, Hanley Kathryn A, Xu Jiannong
Department of Biology, New Mexico State University, PO Box 30001 MSC 3AF, Las Cruces NM, 88003, USA.
J Genomics. 2020 Mar 1;8:30-36. doi: 10.7150/jgen.43928. eCollection 2020.
In the CRISPR-Cas systems, Cas13a is an RNA-guided RNA nuclease specifically targeting single strand RNA. We developed a Cas13a mediated CRISPR interference tool to target mRNA for gene silencing in mosquitoes. A expressing plasmid was delivered to mosquitoes by intrathoracic injection, and transcripts were detectable at least 10 days post-delivery. The target specific crRNA was synthesized using T7 RNA polymerase. The Cas13a plasmid and target crRNA can be delivered by intrathoracic injection together, or the Cas13a construct can be provided first, and then target crRNA can be given later when appropriate. The machinery was tested in two mosquito species. In , gene was silenced by Cas13a/crRNA, which was accompanied by a significant reduction in egg production. In , the α- and δ-subunits of genes were silenced by Cas13a/crRNA, which resulted in mortality and fragile midguts, reproducing a phenotype reported previously. Co-silencing genes simultaneously is achievable when a cocktail of target crRNAs is given. No detectable collateral cleavages of non-target transcripts were observed in the study. In addition to dsRNA or siRNA mediated RNA interference, the programmable CRISPR interference method offers an alternative to knock down genes in mosquitoes.
在CRISPR-Cas系统中，Cas13a是一种RNA引导的RNA核酸酶，专门靶向单链RNA。我们开发了一种Cas13a介导的CRISPR干扰工具，用于在蚊子中靶向mRNA以实现基因沉默。通过胸腔注射将表达质粒递送至蚊子体内，并且在递送后至少10天可检测到转录本。使用T7 RNA聚合酶合成靶特异性crRNA。Cas13a质粒和靶crRNA可以一起通过胸腔注射递送，或者可以先提供Cas13a构建体，然后在适当的时候再给予靶crRNA。该机制在两种蚊子中进行了测试。在其中一种蚊子中，Cas13a/crRNA使某个基因沉默，这伴随着产卵量的显著减少。在另一种蚊子中，Cas13a/crRNA使某个基因的α和δ亚基沉默，这导致了死亡和脆弱的中肠，重现了先前报道的一种表型。当给予靶crRNA混合物时，可以同时共沉默多个基因。在该研究中未观察到对非靶转录本的可检测的旁切。除了dsRNA或siRNA介导的RNA干扰外，可编程的CRISPR干扰方法为在蚊子中敲低基因提供了一种替代方法。
