靶向HIV-1的Cas13a/crRNA的快速筛选和抗病毒活力测定

Rapid Selection of Cas13a/crRNA Targeting HIV-1 and Evaluation of Antiviral Activity

目前基于规律成簇的间隔短回文重复(Clustered regularly interspaced short palindromic repeats,CRISPR)/CRISPR相关蛋白(Cas)系统的基因治疗在遗传病、肿瘤及感染性疾病等领域均取得重大突破,其中Cas13a属于VI型CRISPR系统,Cas13a蛋白在HIV感染细胞内crRNA的引导下定位目标RNA,切割核酸,同时激活Cas13a蛋白的附属效应(Collateral effect)可高效切割细胞内非特异性单链RNA,从而诱导感染细胞凋亡,进一步破坏病毒库,是最有可能功能性治愈HIV的策略。本研究旨在筛选靶向HIV-1的高灵敏度crRNA,进一步探索组合不同crRNA对HIV-1的抑制效果,并在细胞转染水平证明Cas13a/crRNA系统的附属切割活性及其诱导细胞凋亡。在LANL数据库中比对HIV-1各亚型毒株序列,获得各功能蛋白高度保守区。同时,结合HIV数据库中RNA的剪接供体及受体位点信息;然后利用MPRDOCK软件进行crRNA与LwCas13a的分子对接并计算结合复合物的自由能大小选定一组crRNA池;再以荧光标记RNA探针与LwCas13a蛋白组成可视化检测体系,以FAM荧光信号强弱确定了最有效的crRNA。构建LwCas13a和crRNA的共表达质粒,通过共转染HIV全长感染性克隆质粒和含单个或多个crRNA组合的LwCas13a的表达质粒到HEK293T细胞中,根据转染细胞及其上清再感染细胞中萤光素酶和病毒结构蛋白RNA表达水平来判定CRISPR-Cas13a系统对HIV基因表达的抑制效率。初步筛选出优选的27条crRNA并结合切割荧光强弱从中筛选出8条高灵敏度crRNA。细胞共转染实验表明,靶向病毒结构基因的单个crRNA,crGag2,crPol1,crA53能降低HIV-1子代病毒RNA表达降低了43.2%~58.3%,同样,再次感染实验也证明子代病毒活性滴度降低了38%~74.6%。进一步的结果证明,4个crRNA(crD15,crA53,crGag2,crPol1)联用比单个crRNA表达降低了86.4%,比两个crRNAs降低了64.9%,同时在共转染HEK293T细胞中观察到Cas13a蛋白的附属切割活性及其诱导细胞凋亡。我们建立的CRISPR/Cas13a系统能够显著抑制HIV病毒复制和子代病毒产生并诱导细胞凋亡,为HIV的治疗提供了一种新的工具。

Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein(CRISPR/Cas)systems have recently become widely used in research for generating potential therapeutic tools as well as for muscular dystrophy,infectious diseases(e.g.,malaria),and complex diseases(e.g.,cancer).Cas13a belongs to the class-2 type-VI CRISPR-Cas system,which exhibits two distinct ribonuclease activities.Cas13a possesses sequence-specific RNA-cleavage activity in human immunodeficiency virus(HIV)-infected cells under the guidance of the CRISPR-RNA(crRNA)complementary sequence to the interested site.After specific cleavage,Cas13a activates collateral cleavage activity towards adjacent non-specific single-stranded RNA sequences,which can induce the apoptosis of the infected virus and decrease the production of the progeny virus.Therefore,with purging of the viral reservoir,it is the most probable strategy for the functional cure of HIV infection.We screened high-sensitivity crRNA targeting HIV-1 and determined anti-HIV activity by a combination of multiple crRNAs.Meanwhile,the collateral cleavage activity of Cas13a/crRNA and apoptosis were measured in HEK293T transiently transfected plasmids encoding Cas13a,Nl4-nanoLuc,and targeting crRNAs.HIV-1 sequences from different types were aligned in the LANL database.Regions with high identity were selected for designing crRNA.Then,a set of crRNA candidates was chosen by molecular docking of crRNA and LwCas13a using MPRDOCK software.crRNA/Cas13a with the lowest binding free energy was selected for further analyses.Next,a visual detection system was established by combination of a RNA probe labeled with 5'-FAM and 3'-BHQ1,with LwCas13a protein.The FAM fluorescence signal was used to determine which crRNAs were the most effective.Single crRNA was inserted into the expression vector of LwCas13a and the resultant plasmids verified by sequencing.Their constructs were co-transfected into HEK293T cells with HIV-1 full-length infectious clone plasmids.This supernatant was collected and used to infect new Tzm-bl cells.The viral RNA expression and luciferase activity of transfected and infected cells were determined for evaluation of antiviral activity of the CRISPR-Cas13a/crRNA system.Based on the intensity of the fluorescence signal,eight highly sensitive crRNAs were identified from a pool of 27 favored crRNAs.Cotransfection of HEK293T cells indicated that individual crRNAs targeting viral function-related genes(crGag2,crPol,and crA53)could decrease HIV-1 RNA from 43.2%–58.3%.Meanwhile,analyses of infectious viral titers in the supernatant of Tzm-bl cells revealed that the viral infectivity from CRISPR-Cas13a/crRNA-treated cell decreased 38%–74.6%compared with that in an untreated panel.Combination of the four crRNAs(crD15,crA53,crGag2,crPol1)could reduce HIV RNA expression by 86.4%and 64.9%compared with that using single crRNA and two crRNAs during co-transfection of HEK293T cells.Meanwhile,the collateral cleavage activity of Cas13a protein and apoptosis were observed in transfected HEK293T cells with plasmids encoding Nl4-3 and CRISPR-Cas13a/crRNA.Our results demonstrate that the CRISPR-Cas13a system is a novel and effective technology to inhibit HIV replication.This programmable method could be developed further into a novel therapeutic strategy for HIV and other RNA viruses.