Rapid Selection of Cas13a/crRNA Targeting HIV-1 and Evaluation of Antiviral Activity
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,N14-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-b1 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.Co-transfection 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-b1 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 N14-3 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.
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