Establishing CRISPR/Cas13a immune system conferring RNA virus resistance in both dicot and monocot plants

Besides its powerful capability for genome editing, the clustered, regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) (CRISPR/Cas) systems, has been exploited to combat virus infection in eukaryotic organisms (Zaidi et al., 2016). By harnessing CRISPR/Cas system, its compelling inhibiting activities against DNA viruses (Ali et al., 2015; Baltes et al., 2015; Ji et al., 2015) or RNA viruses (Aman et al., 2018; Zhang et al., 2018) were reported in many cases. Moreover, due to the facts that the eukaryotic viruses themselves do not equip the ability to counter this prokaryotic immune defense, by utilization of such strategy, we could establish effective control and eradiation strategy against the eukaryotic virus. For dicot plant, the systems were well-established and reported. However, for monocot plants, encompassing many important grain crops, whose yield was significantly influenced by serious viral diseases, no effective and valid method has been reported by using CRISPR/Cas system to build safeguard for them against viruses. RNA viruses cause serious losses in crops and significant damage to agricultural production, and there are two types of CRISPR/Cas effectors, Cas9 from Francisella novicida (FnCas9) and Cas13a from Leptotrichia shahii (LshCas13a) or Leptotrichia wadei (LwaCas13a), have been introduced to target RNA in vivo (Abudayyeh et al., 2016; Sampson et al., 2013). Previously we have successfully established FnCas9 immune system conferring RNA virus in tobacco and Arabidopsis (Zhang et al., 2018), in this study, we reprogrammed and expressed the LshCas13a system in plants, and employed several RNA viruses to test the antivirus effect of the CRISPR/Cas13a system from L. shahii. Our study demonstrates this system can target and degrade viral RNA genomes, and confer resistance to RNA viral diseases in monocot grain plants. To establish invading RNA virus resistance in plants, we constructed pCambia1300-derived vectors pCR11 and pCR12, which were used to express the CRISPR/Cas13a machinery from L. shahii driven by suitable promoters for dicot or monocot plants, respectively (Figure 1a). Tobacco mosaic virus, which is a classic virus infecting dicot plants, was employed to evaluate the defense efficiency of the CRISPR/Cas13a system. Five crRNAs targeting TMV genome were synthesized and inserted into pCR11 to create corresponding pCR11-crRNA vectors. A recombinant Tobacco mosaic virus agro-infectious clone which is expressing GFP (TMV-GFP) and the pCR11-crRNA vector were simultaneously injected into 25-day-old Nicotiana benthamiana leaves. At 1 week post-inoculation, bright green fluorescence was observed in control plants, which were inoculated by TMV-GFP only, or TMV-GFP plus pCR11 (Figure 1c). In pCR11-TA, pCR11-TB, pCR11-TC, pCR11-TD and pCR11-TE inoculated tobacco plants, the green fluorescence was obviously weaker compared with control (Figure 1c), which reflects that the TMV infection was significantly attenuated by the CRISPR/Cas13a system. Quantification of the TMV titre by RT-qPCR further confirmed that the TMV-GFP levels in CRISPR targeted plants were significantly decreased (Figure 1d). To exclude the possibility that the crRNA bind to the viral genome and inhibit the infection without the help of LshCas13a, a GUS gene was substituted for the LshCas13a to produce pCR11_Gus (Figure 1b). The pCR11_Gus-TA, which is targeting the TMA TA site, lost the ability to suppress the TMV-GFP infection as pCR11-TA (Figure 1c and d). Then, alanine point mutations in the two higher eukaryotes and prokaryotes nucleotide-binding (HEPN) RNases domains of LshCas13a (R597A, H602A, R1278A and H1283A; Abudayyeh et al., 2016), were generated to test whether the endonucleolytic activity was involved in the repression of virus infection (Figure 1b). The pCR11_dCas-TA abrogated the repression of TMV-GFP infection compared with pCR11-TA (Figure 1c and d). These results indicate that the cleavage sites of LshCas13a were essential for inhibiting virus infection in our system. Plant viruses not only harm dicot plants, but also monocot plants, such as rice, which suffered serious yield losses by many viruses. For example, Southern rice black-streaked dwarf virus (SRBSDV) causes a striking disease on rice in several East Asian countries (Zhou et al., 2013). Here we synthesized three crRNAs targeting the double strand RNA genome of SRBSDV and inserted into pCR12. The resulting vectors, pCR12-SA, pCR12-SB, pCR12-SC and along with the control vector pCR12 were transformed into rice plants mediated by agrobacterium. T1 transgenic lines for each construct, along with control wild-type rice plants, were selected and infected with SRBSDV by its viruliferous vector feeding. Forty days later, typical symptoms were observed in the control plants, including significant dwarfing and failure to head (Figure 1e). In the transgenic lines, most plants showed mild symptoms, and the pCR12-SB lines in particular had no obvious symptoms (Figure 1e). Quantification of virus accumulation by RT-qPCR showed that SRBSDV infection was indeed inhibited in these transgenic plants (Figure 1f). Rice Stripe Mosaic Virus (RSMV) is a novel cytorhabdovirus and became a new threat to rice production in south China (Yang et al., 2017). We also generate transgenic rice plants harbouring the CRISPR/Cas13a system targeting the single strand RNA genome of RSMV. Virus attacking experiment showed that the control plants had typical symptoms, including slight dwarfing, with leaves showing yellow stripes and excessive tillering, while the transgenic plants with crRNA targeting RSMV (pCR12-RA, pCR12-RB and pCR12-RC) had very mild symptoms (Figure 1g) and less viral RNA accumulation(Figure 1h). To test the inheritability of the resistance, we harvest the T3 homozygous lines to attack by SRBSDV or RSMV. Inspiringly, all the T3 transgenic plants we tested showed stable resistance to SRBSDV (Figure 1i) or RSMV (Figure 1j). Our results showed that overexpressing of crRNA–LshCas13a specifically targeting the viral genome was an effective way to generate stable RNA virus resistance in monocot plants. In the past decades, RNA interference-mediated resistance has been used to confer immunity against viruses in plants. However, through long-term co-evolution, eukaryotic viruses have developed methods of antagonize RNAi, which limited the applications in agriculture. In recent years, the CRISPR/Cas9 machinery has been exploited to combat eukaryotic viruses in dicot plants (Ali et al., 2015; Aman et al., 2018; Baltes et al., 2015; Ji et al., 2015; Zhang et al., 2018). To our knowledge, this is the first report of a method targeting the viral RNA to control viral diseases in monocot plants. In addition, we have used two distinct type of CRISPR/Cas system, Cas9 from F. novicida (Zhang et al., 2018) and Cas13a from L. shahii (this study), both of them showed high efficiency in generating RNA virus-resistant plants. The difference is that the former depends on FnCas9 binding viral RNA, while the latter requires LshCas13a having RNases activity to cleave viral RNA. These findings provide us with more options for developing antiviral strategies, and combination of multiple strategies may provide reference to generate viral-immune crops in the future. Our findings demonstrate that the L. shahii CRISPR/Cas13a system we established in this study could enable the plant acquire potent defense against viral infection in both dicot and monocot plants, which imply that the method has the potential to develop into a universal applicable system in various kinds of crop species. This work was supported by grants from the National Natural Science Foundation of China (31871928 and 31601608), and Science and Technology Planning Project of Guangdong Province (2017A020208058). T.Z. and G.Z. have filed a patent application in China (priority filing with serial number 201811493466.4). T.Z. and G.Z. designed the experiments; T.Z., Y.Z., J.Y., X.C., C.X., B.C., H.A., Y.J. and F.Z. performed the experiments; T.Z., Y.Z., H.A. and G.Z. analysed the results; T.Z. and G.Z. wrote the manuscript. All authors read and approved the final manuscript.