Establishing an Immune System Conferring DNA and RNA Virus Resistance in Plants Using CRISPR/Cas12a Multiplex Gene Editing

Two types of CRISPR/Cas systems (Cas9 and Cas13) have been used to combat eukaryotic viruses successfully. In this study, we established resistance to the DNA virus BSCTV and RNA virus TMV in <i>Nicotiana benthamiana</i> using the CRISPR-Cas12a multiplex gene editing system. We employed two effector proteins LbCas12a and FnCas12a coupled with six guide RNAs targeting virus genome and a novel mRNA-gRNA nucleic acid complex to transport gRNA efficiently. Compared with the BSCTV accumulation in the wild-type <i>N. benthamiana</i>, it was reduced by more than 90% by most transgenic events derived at 7 days post-inoculation. Additionally, the shoot-tip leaves were normal in the transgenic plants, whereas they appeared severely curled and stunted in wild-type <i>N. benthamiana</i> at 15 days post-infection. Target sites evaluation revealed that the editing system can directly destroy the structure of BSCTV viral genomes via large fragment deletions. We quantified TMV virus accumulation in the transgenic <i>N. benthamiana</i> lines by monitoring dynamic changes in GFP fluorescence and quantitative analysis by qPCR showed that the CRISPR-Cas12a system can introduce TMV virus resistance to <i>N. benthamiana</i> by preventing its systemic spread. Our study provides an innovative strategy-an mRNA-gRNA nucleic acid complex-which has proven to be highly effective in the gene-editing system and offers an efficient antiviral approach for generating virus-resistant plants.