CRISPR/dCas9-TET1cd-targeted DNA Demethylation Editing Of The Spontaneous Epiallele NMR19-4 In Arabidopsis

In organisms,DNA methylation can regulate gene expression through epialles.Epigenetic variations can be induced by natural and artificial approaches.In naturally occurring epigenetic variation,the spontaneous epialles are relatively rare in species and little is known about their functions and regulations.In our previous study,we identified a spontaneous epialleles NMR19-4(naturally occurring methylation region)in Arabidopsis regulating leaf senescence which is a LINE1 transposon located in the PPH(Pheophytin Pheophorbide Hydrolase)promoter region.NMR19-4 show obvious DNA methylation variation in different ecotypes and the expression of PPH gene was highly negatively correlated with the methylation level of NMR19-4.However,the above studies can only be limited to correlation analysis,and it is difficult to carry out causal verification.The emergence of CRISPR/dCas9 tools provides effective support for solving this problem.Therefore,we want to ask whether artificially mediated NMR19-4 demethylation will cause changes in PPH gene expression? After hybridization does the hybrid progeny conforms to the Mendelian inheritance? These questions attract us to continue to conduct further research.In this study,demethylated lines were obtained by using demethylating epigenetic editing system CRISPR/dCas9-TET1 cd on spontaneous epialleles-NMR19-4 in Arabidopsis.Through molecular biology,physiological experimental methods and bioinformatics analysis,the epigenetic modified individuals have been deeply explored which provides a new approach and theoretical basis for the study of spontaneous epialleles.The main conclusions of this experiment are as follows:(1)CRISPR/dCas9-TET1cd-mediated NMR19-4 demethylation can be inherited.NMR19-4m(methylated)Arabidopsis ecotypes were transformed with the CRISPR/dCas9-TET1 cd demethylation vector,and the methylation level of NMR19-4 in Krot0-65 dc line decreased most significantly.Demethylated NMR19-4 in Krot 0-65 dc selfed progeny can stably inherit through single seed descent and significantly up-regulate PPH gene expression.Since PPH gene can degrade chlorophyll and promote leaf senescence,after dark induction,it was found that individual leaf senescence of T4 epigenetic editing generation increased significantly,and chlorophyll content decreased significantly,showing that CRISPR/dCas9-TET1 cd targeted epigenetic editing of NMR19-4,resulting in decreased methylation of the target region,increased PPH gene expression,accelerated leaf senescence and effective editing.(2)The chromatin states of the region at NMR19-4 may influence the efficiency of epigenetic editing.To detect the involvement of chromatin state in epigenetic editing,we try to analyzed the chromatin states using ATAC-seq.The results showed that ATAC-seq peaks had the highest enrichment degree in Chromosome 1 and the lowest enrichment degree in Chromosome 4.The enrichment length of Peaks of ATAC-seq is mainly concentrated in the range of 250-500 bp.Peaks of ATAC-seq are mainly enriched in the gene and promoter regions and have different preferences in different ecotypes.The relatively high openness of the NMR19-4 locus of Krot 0 and its flanking genes may promote the editing efficiency of CRISPR/dCas9-TET1 cd,indicating the degree of chromatin opening associated with the editing efficiency of CRISPR/dCas9-TET1 cd.(3)CRISPR/dCas9-TET1 cd did not cause drastic changes in genome-wide methylation levels.The bisulfite sequencing of NMR19-4 locus showed that CG,CHG and CHH decreased significantly in T4 epigenetic editing demethylated individuals with all exceeding 90%compared to Krot 0 WT,while whole-genome methylation did not show drastic changes except for NMR19-4 position.Transcriptomic analysis found that the overall transcript level of the whole genome was not drastically affected by CRISPR/dCas9-TET1 cd,but the PPH gene expression was significantly increased in T4 epigenetic editing individuals,indicating the rare off-target in the whole genome.(4)Demethylated NMR19-4 conforms to Mendelian inheritance.To examine the inheritance of epigenetic editing,we crossed two NMR19-4m ecotypes,Per1 and C24,with the T4 epigenetic editing individuals and obtained the F2 individuals.After genotyping and Chop-PCR,we found that in the progeny of F2,the NMR19-4 locus of all NMR19-4u(unmethylated)individuals was inherited from the T4 epigenetic editing parent,in contrast the NMR19-4 locus of all NMR19-4m individuals was inherited from the parent of NMR19-4m,and no methylation interaction occurred.The chi-square test of NMR19-4m individuals and NMR19-4u individuals in F2 progeny was 3:1 ratio,just consistent with Mendelian inheritance.The chlorophyll level after dark induction and PPH gene expression analysis of F2 generation individuals showed that NMR19-4u individuals had faster chlorophyll degradation rate and higher PPH gene expression,on the contrary,the chlorophyll degradation rate of NMR19-4m individuals was slow and the PPH gene expression was low,which was highly consistent with the phenotype of the parents.These results showed that epigenetic editing of spontaneous epialleles follow the Mendelian inheritance,which is universal in different Arabidopsis accesions.Our results confirmed that the CRISPR/dCas9-TET1 cd system can effectively target and decrease the DNA methylation of the spontaneous epiallele-NMR19-4,increase the expression level of the PPH gene significantly,and promote the leaf senescence.Methylation changes in NMR19-4 can be stably inherited in progenies,and the CRISPR/dCas9-TET1 cd system has little effect on genome-wide methylation changes,and no methylation interaction occurs after hybridization with the NMR19-4 hypermethylated ecotype,which is consistent with Mendelian inheritance.Thus,our results provide a powerful tool for our study of spontaneous epigenetic variation mechanisms in the future and crop epigenetic breeding.