Targeted Genome Editing On Vitamin E Biosynthesis Related Genes Using CRISPR-Cas System In Barley

CRISPR (clustered regulatory interspersed short palindromic repeat) sequences originate from a kind of prokaryotic adaptive immune system and protect prokaryotes against viruses or conjugative plasmids. Recently CRISPR-Cas9interference machines are utilized to develop novel RNA-guided genome editing tools. The CRISPR-Cas9 system has been demonstrated for genome editing in human, mice, zebrafish, yeast, bacteria, Drosophila, nematode and Arabidopsis, and developed very fast in many optimized aspects since 2013. Barley (Hordeum vulgare) is one of the most important food crops in the world. Vitamin E is important for human health especially α-tocopherol which has the highest Vitamin E activity and is easy to be absorbed by human. The biosynthesis pathway of Vitamin E is very complex, which contains eight kinds of produces (δ、γ、β、α-tocopherol,δ、γ、β、α-tocotrienol). Up to now, few of studies have explored the application of CRIPSR-Cas9system as new genome targeting modification technology in barley. In the study here, we focused on the using of CRIPSR-Cas9system in protoplasts from barley green tissues, compared the expression of Cas9 protein of many different CRIPSR-Cas9system vectors, and successfully performed targeted mutagenesis of homogentisate geranylgeranyl transferase (HGGT) gene. Besides we compared two kinds of mutation test methods (RE-PCR, PCR-RE) and used RE-qPCR to calculate mutation rates. Our main results are as follows: 1. Expression of Cas9 protein in barley protoplastpRGE was selected to construct the CRISPR-Cas9 system vectors which are more compatible in plants. According to western-blot results, the original Cas9 protein is at low expression level in barley. However, Cas9 protein is significantly increased after using UBI10 promoter and codon usage optimization. 2. Expression vectors construction and targeted sites choiceIn our study, HGGT gene which is related to Vitamin E biosynthesis in barley (Hordeum vulgare) was chosen as targeted genes. Briefly, using pRGE32 vector to construct three sgRNA system vectors (pRGE32-HGGT-1, pRGE32-HGGT-2, pRGE32-HGGT-3), a PTG system vector (pRGE32-HGGT-PTG1 (two tandem sgRNAs)) of HGGT gene on three specific sites.3. Mutation detection methodsMutation detection by using traditional T7 endomuclease 1 results poor stability. In our study, we chose sequences containing common restriction enzyme sites as targeted sites. To detect mutation at desired restriction enzyme sites, we used restriction enzyme digestion suppressed PCR (RE-PCR&PCR-RE). PCR-RE is convenient, stable, low-restrictive and easy to recycle mutational fragments.4. Mutation rate, and mutation type statisticsTo identify targeted gene mutation, purified PCR products from restriction enzyme digested template were cloned to pGEM-T easy vector, and resulting random colonies were used for DNA sequencing. Quantitative PCR was also performed to quantify indirectly the amount of mutated genomic DNA in sgRNA system. RE-qPCR showed that the mutation percentage of HGGT-1 site is 4.56%,0.35% for HGGT-2 site, and 1.51% for HGGT-2 site. Sequencing showed that, sgRNA system induced mainly to point mutation (T-C replacement & A-G replacement) and indels (different lengths at 20 bp targeted sites), their frequencies are 89.47% and 10.53% respectively. Thereinto, the mutation percentages of HGGT-1 site is 35%,15% for HGGT-3 site, and 35% for HGGT-TTG1 site. RE-qPCR and sequencing both indicated that the mutation rates of three specific sites in HGGT gene are quite different. The mutation percentages of HGGT-1 site was highest followed by HGGT-3 site, while no mutation was detected at HGGT-2.Taken together, our results demonstrated that CRISPR-Cas%ystem can be applied as a powerful tool for gene targeting and genome editing in barley. Selecting effective target sites at protoplast level can provide reliable experimental basis for Agrobacterium-mediated transformation.