Targeted Mutagenesis Of Three Rice MAPK Genes Using Improved CRISPR/Cas9 Technology

When plant undergoes biotic stresses,the external stress signals can be transmitted to cells and activate the expression of stress responsive genes.Mitogen-activated Protein Kinases(MAPKs)are widely involved in the transduction of stress signals,and enable plants to adapt to diseases and insect pests.Recently,CRISPR/Cas9(Clustered regularly interspaced short palindromic repeat/CRISPR-associated Protein)system becomes the most popular and powerful designer nuclease for genome editing.To date,robust method to express g RNAs for CRISPR genome editing,particular for multiplex genome editing,is still one of the challenges of CRISPR technologies.Here,we presented a new method to express g RNAs and Cas9 in one hybrid gene.We further optimized the structure of hybrid gene of in PTG-Cas9 to simultaneously targeting multiple sites.The results are summaried as follows.(1)The sequence analysis showed that the promoter of rice Ubiquitin(UBI10)gene,which was used in our previous CRISPR vectors(e.g.,p RGEB32),includes a conserved intron in the 5'-UTR(untranslated region).This 5'-UTR intron of UBI10 has a typical splicing donor,branch and acceptor sites as 5'-GU-A-AG-3'.Based on these results,we designed and constructed p RGEB33 and p RGEB34 which express polycistronic t RNA-g RNA from the 5'-UTR intron(in PTG)of Cas9.We named this novel hybrid gene as in PTG-Cas9.(2)We analyzed the mRNA splicing,transcription and translation of in PTG-Cas9 in rice protoplasts.The results showed that the intron with polycistronic t RNA-g RNA(PTG)exhibited normal splicing,but produced slight less mRNA and protein than the normal UBI10p::Cas9 constructs in rice protoplasts.(3)To examine in PTG-Cas9 efficiency,we performed targeted mutation of PDS(phytoene desaturase)and three MAPK(Mitogen-activated protein kinase)genes in rice protoplast.The results showed that in PTG-Cas9 could efficiently target 2-4 sites.(4)In order to further optimize the intron to express g RNAs,we constructed the p RGEB33 T which fuses the in PTG intron at 3'-UTR of Cas9.The protoplast assyas showed that p RGEB33 T has higher editing efficiency than p RGEB33 and p RGEB34 which express in PTG in 5'-UTR.(5)The stable transformation plants of four in PTG-Cas9 constructs were generated by Agrobacterium-mediated transformation.We analyzed the genotype of all targeting site.The results showed that the in PTG-Cas9 could efficiently edit multiple target sites.Further analysis showed that all of these edited sequence were inherited into the T1 generation.(6)We obtained biallelic mutation lines which knock out MPK1,MPK5 and PDS.We also obtained the biallelic double knock-out mutant of MPK1 and MPK5.We used Magnaporthe grisea and root knot nematode to test the resistance of rices that MPK1 and MPK5 genes were knocked out.The results showed that MPK1 and MPK5 knock-out plants exhibited reduced disease resistance than the wild type or the empty vector control.In summary,this work provides a robust and versatile strategy to express many small RNA guides from the intron of Cas9.This method enabled us to construct genetic tools which express Cas9 and the RNA guides from one recombinant gene without trade-off efficiency.Additionally,two rice MAPK genes were targeted and we successfully obtained the knock-out mutants in this study.These MAPKs play essential roles in disease pathway as well as hormone and development signaling pathways.Thus,these genome edited MAPK lines could be helpful to further characterize the interaction between disease resistance and other signaling pathways.