Application Of An Efficient PTG/Cas9 Gene Editing System In Arabidopsis And Analysis Of Chaperone CPN60 In Structure And Function

CRISPR/Cas9(Clustered Regularly Interspaced Short Palindromic Repeat/CRISPR-associated nuclease 9)gene editing system has been widely used to creating stable homozygous mutants in various organisms.However,the gene editing efficiency of CRISPR/Cas9 in Arabidopsis is much lower than that of other species.PTG(polycistronic tRNA-gRNA)/Cas9 system,a multiple gene editing system in rice,can improve editing efficiency(efficiency can up to 100%),and the simultaneous cutting of multiple target sites is prone to producing PCR-detectable large fragment deletions.In PTG/Cas9 technology,polycistronic tRNA-gRNA structure forms a PTG gene.After transcription,all gRNAs are released from the primary transcripts with the maturation of tRNA by endogenous tRNA-processing system.Multiple released gRNAs guide Cas9 protein to simultaneously cut different target sites of one gene,resulting in fragment deletion mutants.All the components used in this system are conserved in Arabidopsis,therefore,we speculate that the simple and efficient PTG/Cas9 system could also be applied to ArabidopsisTo test our idea,we adopted the polycistronic tRNA-gRNA CRISPR/Cas9(PTG/Cas9)system in Arabidopsis with PTOX as the reporter gene.We selected five specific gRNA target sites in PTOX gene,then constructed a PTOX-PTG/Cas9 system with five gRNAs and introduced it into Arabidopsis.At T1 generation,24.4%of transgenic plants were chimeric with PCR-detectable deletions in PTOX locus,but no homozygous mutant was found,indicating that gene editing occurred predominantly in somatic cells.After a self-cross propagation,60%of T1 chimeric plants were able to produce homozygous,heterozygous,or bi-allelic ptox offsprings.Inheritable homozygous ptox mutants without Cas9 gene can be obtained earliest at T2 generation.We further successfully applied the PTG/Cas9 system to other genes and established a standard and reliable protocol to generate stable inherited deletion mutants in 2?3 generations along with simple PCR screening methods.We conclude that the rice PTG/Cas9 system is an efficient,easy,and rapid tool to edit genes in Arabidopsis.This study showed that rice PTG/Cas9 system also has a highly efficient gene editing function in Arabidopsis thalianaChaperone is a type of protein that can assist the folding and assembly of new translated peptide and misfolded proteins.Arabidopsis chloroplast chaperone CPN60 has six kind of distinct subunits,and processes and assembles target substrates in the form of heptameric complex in vivo.However,the structure and its target substrates of CPN60 complex are not clear.To explore these issues,first,we obtained a striking yellowish stunted mutant of CPN60?1,cpn60?1-trun,harboring a truncated CPN60?1 protein.It was reported that CPN60?1 null mutant showing wild type-like phenotype,which is totally different from that of cpn60?1-trun.By overexpression of the truncated protein CPN60?1-trun in wild type,we found that the CPN60?l-trun protein was the cause of the stunted and yellowish phenotype.Through yeast two-hybrid assay and bimolecular fluorescence complementation assay,we found that the protein CPN60?1-trun was able to insert into CPN60 complex as normal CPN60?1 to form a dysfunctional complex,resulting in unmoral chloroplast structure and reduced photosynthetic efficiency.Moreover,the crossing assay between homologs clarified that that CPN60?1 function with CPN60?2,not CPN60?3 and CPN60?4,in maintaining the leaves color.In addition,the yellowish phenotype could be also complemented by overexpressing CPN60?3 subunits,a member of four CPN60?s.In the end,RNA-seq and iTRAQ assay were performed to investigate the global view of gene expression,and the results revealed that most of genes involved in duplication,transcription,translation,and protein folding were up-regulated in RNA level;the transcriptional level of genes associated with photosynthesis were depend on the gene is located in nucleus(down-regulation)or chloroplast(up-regulation)chromosome,while the protein level of photosynthetic genes were almost all down-regulated;in addition,with the aggregation assay,we predicted that some subunits of the photosynthetic related proteins might be the potential substrates of CPN60 complex.