Activity Evaluation Of Promoters Identified By RNA Polymerase ? And Mutagenesis Of The Waxy1 Gene Using CRISPR-Cas9 System In Maize

Genome editing tools can be used into inducing intended sit-specific mutagenesis,such as insertion,deletion and substitution,in the recipient genome.Recently,the clustered regularly interspaced short palindromic repeats(CRISPR)associated Cas9 nuculease had been developed as the major gene editing technology due to its' high mutation frequency,simpleness use and flexibility in design.It could provide an ideal approach to use into genetic improvement in higher plant including maize.The RNA pol? promoter is one of the key components of the CRISPR-Cas9 technology.However,the RNA Pol? promoters had not been well characterized in maize.It is the major limiting for using CRISPR-Cas9 technology in maize.To meet this end,we establish a transient expression system to evaluate the activity of all RNA pol? promoters in CRISPR-Cas9 system.Based on the identified RNA pol? promoter,we developed an efficient CRISPR-Cas9 gene editing technology for targeting ZmWaxy1 gene to create the knockout mutant in maize.The key results of this study were as follows.(1)A stable and efficient transient expression system in maize had been established.By using leaves from Zheng 58,B73,Zhongdan 99,Black Mexican Sweet(BMS),high proportion of healthy mesophyll protoplast could be prepared.Stained with FDA,the results showed that Zheng 58,B73,Zhongdan 99,Black Mexican Sweet(BMS)and other varieties can extract high viable mesophyll protoplasts.A construct of DsRed expression cassette had been transformed maize protoplasts meditated by polyethylene glycol(PEG).Our results indicated that 45% PEG would be optional concentration.A protocols with an transformation efficiency up to 80% had been optimized in our study.(2)An efficient in vitro CRISPR-Cas9 gene editing reporting system,Traffic Light Reporter(TLR),had been established.The TLR reporting system is composed of frameshift mutations eGFP and DsRed gene.We added a CRISPR-Cas9 recognition sequence site in the front of TLR.In the protoplast,when the TLR system is edited by the CRISPR-Cas9,the reporter gene will be recovered.Different colors,such as green,red and yellow will be displayed in the protoplast.The flow cytometry is used to count the fluorescing protoplasts.(3)Candidate RNA Pol? promoters had been identified.The maize U6 snRNA gene and sequences were found by searching in maize genome sequence archieves.The sequence about 400-500 bp upstream of the gene was chosed as the potential U6 snRNA promoters and they were isolated by PCR.A total of six potential maize U6 snRNA promoters had been evaluated and identified their activities by using our former TLR reporting system.A high mutation efficiency U6 snRNA promoter had been identified and used into our subsequent CRISPR-Cas9 system.(4)The exon 7 of ZmWaxy1 gene was selected as the target site and the CRISPR-Cas9 gene editing vector had been counstructed.(5)The ZmWaxy1 gene editing of targeted knockout had been accomplished in maize.The CRISPR-Cas9 editing construct had been stably transformed in maize mediated by Agrobacterium.We isolate thirty six transgenic plants and sequence the transgenic T0.We found thirteen homozygous mutants,twelve biallelic mutant,none heterozygous,and one wild type in thirty six transgenic T0,and the mutation rate was 97.22 %.T0 generation was hybridized with wild-type maize and we obtained the T1 generation.The T0,T1 endosperm starch granules and pollen is stained by iodine and observed by microscopic and the result show that the phenotypic and genotype can be stability inherited to the progeny.In summary,We establish an efficient CRISPR-Cas9 efficient editing techniques system in maize.