Research On Inhibit Replication Of Zika Virus Using CRISPR-Cas13

Zika Virus(ZIKV)is a mosquito-borne positive,single-strand RNA virus,which belong to the genus Flavivirus(family Flaviviridae).ZIKV is similar to other flaviviruses,such as yellow fever virus(YFV),dengue virus(DENV),Japanese encephalitis virus(JEV)and West Nile virus(WNV),causing mild to severe life-threatening infections in humans.After the first outbreak of ZIKV in 2007,it has become an important human pathological gene for severe neurological complications.it causes adult Guillain-Barre syndrome(GBS)and various fetal abnormalities,such as microcephaly,which have attracted widespread attention.At present,there are no targeted vaccines and drugs around the world,and the outbreak and epidemic of ZIKV have become a global public health problem.To explore new ways to suppress the ZIKV,we used a tool CRISPR-Cas13 system targeting the cleavage of RNA virus to inhibit ZIKV replication.CRISPR-Cas13 system(formerly known as C2c2)is the class 2 type VI of the CRISPR-Cas system.This system is similar to the CRISPR-Cas9 system,but unlike Cas9,which targets DNA,it is an RNA targeting and editing system based on the bacterial immune system.CRISPR-Cas13 is the specific targeting RNA system found in the CRISPR-Cas family.Itcontains a single effector protein Cas13 and CRISPR RNA(cr RNA)assembled to form a cr RNA-guided RNA targeting complex.Since the CRISPR-Cas13 discovery,the system has been used for RNA editing,RNA knockout,RNA detection,RNA tracking,and imaging.The CRISPR-Cas13 system,such as Cas13a?Cas13b,etc.its RNA knockout technology has been applied to many fields,such as plant RNA virus-Tu MV,Potato virus Y(PVY),etc.Single-stranded RNA virus knockout of mammalian cells-influenza A virus(IAV),vesicular stomatitis virus(VSV),and lymphocytic meningitis virus(LCMV),etc.All these have proved that CRISPR-Cas13 is an effective tool for destroying RNA viruses.The CRISPR-Cas13 system will play an important role in studying the inhibition of RNA viruses.In the research,using CRISPR-Cas13 to target the function of cutting single-stranded RNA,we evaluated the inhibitory effect of CRISPR-Cas13 on mammalian cells infected with ZIKV.By establishing a CRISPR-Cas13:g RNA dual-plasmid fluorescent labeling system and designing and selectingcorresponding target sites on the ZIKV genome,CRISPR-Cas13 targeted to cut the ZIKV single-stranded RNA genome.Then use fluorescence microscope technology to observe the reduction of fluorescently labeled ZIKV,and further use flow cytometry to analyze the experimental results to determine the ability of the CRISPR-Cas13 system to target the ZIKV.The experiment played a potential role of CRISPR-Cas13 on RNA virus suppression and provided a new technical tool for the prevention and treatment of ZIKV.First,we designed and constructed mammalian cells CRISPR-Cas13: g RNA dual plasmid system model,including the construction of CRISPR-Cas13 and related g RNA plasmids.We selected CRISPR-Psp Cas13 b from Prevotella sp.P5-125,and added the NES(nuclear exportsignal)to the CRISPR-Psp Cas13 b protein and fused a green fluorescent protein GFP(Psp Cas13b-GFP-NES).In addition,we also constructed g RNA plasmids corresponding to Psp Cas13 b targeting ZIKV.We selected 9target sites on the ZIKV genome(gpr M,g E,g NS1-1,g NS1-2,g NS2 B,g NS3,g NS4 B,g NS5 and gm C).Then,we transfected the constructed Psp Cas13-GFP-NES: g RNA-(ZIKV)dual plasmid system into mammalian cells 293 T DC-SIGN,and then infected the Zika virus fluorescent reporter system(ZIKV-m Cherry).The experimental results show that the Psp Cas13-GFP-NES: g RNA-(ZIKV)dual plasmid system can inhibit the replication of ZIKV in mammalian cells.In a word,our research demonstrates the effective suppression of ZIKV by CRISPR-Psp Cas13 b.Based on CRISPR-Cas13: g RNA's ability to degrade ss RNA in cells,we developed a CRISPR-Cas13:g RNA dual plasmid system to disrupt ZIKV replication.This evidences suggest that the CRISPR-Cas13 system has broad potential for targeting ss RNA viruses in mammals and will be an effective tool for intervention against RNA viruses.