| With the post-genomic era coming on, it is in urgent need to analyze the interaction of the genes and regulatory networks. However, in the growth and development process of plants, examples of redundant genes abound in nature. Then, only by knocking out multiplex genes or sites together can we get the material for current research of functional genomics and genetic breeding. As the gene editing technology develops, it has achieved successfully to knock out one or two genes/sites at the same time on plant level. However, if we want to create stable genetic multiple genes mutation, according to traditional breeding and genetic engineering, it is time-consuming to accumulate mutations and to bring into homozygous stage. In addition, as the growth number of mutation genes, the big workload of genotyping would be unbearable. Moreover, multiplex genome editing is very meaningful for polyploidy plants, such as wheat, oats, tobacco and cotton.Although multiplex genome editing is meaningful and a few years ago knocking out one gene in plant has been made by ZFN, TALEN and other genome editing technologies, in the botany fields multiplex genome editing just halted for the limitations of the technologies. After TALEN has been used successfully in our lab, when modifying the TALEN for multiplex genes editing descended into deadlock, the CRISPR-Cas9 system appeared. Because CRISPR/Cas9 system has independent Cas9 protein and sg RNA, and the sg RNA determines editing direction at the posttranscriptional level. In addition, if there are sufficient Cas9 proteins, the g RNAs do not interfere with each other. All of above make it possible to edit multiplex genes/sites. What’s more, it will significantly reduce the time to make multiplex genes mutations. Based on these, the multiplex genes editing is the most attractive part different from others.With the advantages and experiences of TALEN platform and CRISPR/Cas9 system, we successfully created the DNA modifications within genome loci of interest for single gene targeted editing. In this paper, with the CRISPR/Cas9 system of single g RNA, we got Arabidopsis idm3(Increased DNA Methylation 3) homologous T2 mutation. We reported that IDM3 was an anti-silencing factor, which prevents gene repression and DNA hypermethylation. Moreover, by this vector system, two g RNAs were used to delete the long fragment in order to modify the genome region of interest.Based on the results above, we designed a development of CRISPR/Cas9 systems to perform multiplex gene editing in Arabidopsis, which allowed construction of sixindividual g RNAs in a single vector by three kinds of promoters. PYR/PYL/RCAR family includes 14 members in plants, which are ABA receptors in ABA signal transduction. The functional redundancy of receptor proteins hinders the functional study of such big family.In order to find out the g RNA target sites that match the rules and cover multiplex sites, we analyzed the family members with method of exhaustion one by one. Based on the genes conserved regions, we designed six g RNAs to target members of Arabidopsis PYL gene family: one g RNAs was designed in the conserved region of PYL10, 11, 12 and 13, respectively;one g RNA was targeted in the conserved region of PYL 7 and 8; and four g RNAs were targeted the regions within four PYLs loci, i.e. PYL3, 5, 6 and 9. Because of the different between the transgenic methods of plants and animals, we cannot inject m RNA or active proteins into plant directly which worked very well in some animals. Even using particle gun, the injected type and does of vector/protein are uncontrollable. To study the biological function of the receptor proteins, U6, U3 b and 7SL promoter driven single g RNAs were connected to each other by Golden Gate Assembly method. Then the cassette of six g RNAs with Cas9 was sub-cloned into an expression vector, which was integrated into Arabidopsis genome by Argrobacteriamediated transformation. We grow the plants of transformation under the constant humidity from beginning. When we got the population of transgenic lines, to examine if each of six g RNAs would launch Cas9 in the target sites respectively, and if the single g RNA in the conserved region would launch Cas9 to edit two or four PYLs simultaneously, in T1 plants we used the primer pairs flanked the modified DNA region and amplified the fragments from different PYLs and cloned the PCR products into Tvector. Sequencing method was used to verify the modification in desired loci of PYLs. And except for PYL8 and PYL9 in which no editing events were detected in the samples examined, various small fragment indels were detected within the PYL3, 5, 6, 7,10, 11, 12 and 13 loci.The result of T1 illustrated that the multiplex genes editing system of six g RNAs driven by U6, U3 b, 7SL promoters worked in Arabidopsis. Although all of the examined T1 seedlings were chimera, homozygotes for targeted gene modifications would segregate at the next generation. Finally, from the ABA treatment plates(100μM), we screened out some lines showed the phenotypes of insensitive to ABA as the early reports predicted. And the confirmed sequencing results showed that most of them were homozygous or heterozygous multiplex mutation. What’s more, we found some multiplex mutation from 112458 background were sensitive to moisture in the air as well. These results indicated that through the CRISPR/Cas9 system including sixg RNAs the multiplex gene editing indeed occurred simultaneously in Arabidopsis plants and the PYL multiplex mutation stocks were useful for future functional diverse analyses of the gene family. We made it operable and practical to edit multiplex genes/sites and screen out the mutations. Although we have not got the whole families mutation yet, the stock of kinds of mutations is very useful for redundancy genes functional study. And we solved the issues: 1. Express the Cas9 protein on a defined level to satisfy the need of more g RNAs; 2. Co-express multiple g RNAs and the g RNA don’t interfere with each other; 3. Transform the Cas9 and g RNAs into plants together; 4. Argrobacteria-mediated transformation of 112458 background; 5. High throughput detection and storage.Although CRISPR/Cas9 system has been used successfully, it is just a kind of model plant. Then we also proved that CRISPR/Cas9 system worked very well in tomato, an important vegetable crop. Two tomato genes were targeted: Sl IAA9 and one unreported gene Sl MET1. Sl IAA9 is an Aux/IAA gene, and is involved in tomato leaf morphology. Sequencing results showed that the CRISPR/Cas9-induced mutations in targeted endogenous genes were readily detectable in T0 tomato plants, and we obtained expected phenotypes of targeted mutagenesis of Sl IAA9 to convert compound leaves to simple leaves in tomato in the T0 generation.Form the results above, CRISPR/Cas9-based genomic editing simultaneously could create the multiplex mutations, and the mutations are heritable, which are precious resources for gene function study. Moreover, it is fair to expect that this CRISPR/Cas9-based genomic editing technology will be widely adopted for routine gene functional research even functional genomics research in Arabidopsis and other plants in the near future. |
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