Employ CRISPR/Cas System Inducing Target Genome Engineering In Rice

CRISPR(clustered regularly interspaced short palindromic repeats)as a natural defense system is widespread in bacteria and archaea for resist foreign plasmid or bacterial invasion, after ZFNs and TALENs emerged, as the third generation of gene editing technology. This system of Cas9protein guided by double strand RNA (single guide RNA, sgRNA) to site specific identification of the targets and execute cutting, besides Cas protein has a HNH domain and RuvC-like domain cutting double strand DNA one strand, respectively, so as to realized the breaks in targets sites of double strand. With the deepening research on the CRISPR/Cas9system, due to its design simply, highly efficiency and specificity, has been regarded as a kind of broad application prospects of target gene modification technique. Alhough CRISPR/Cas9system has been widely studied, but research on rice has focused on transient expresion or To generation about the target site mutation effect, this paper explored to study on vector construction for express this system cassette and verified the efficiency of targets alteration in rice using this system, otherwise test the progeny of genetic model. It will be of interested to provide reference for the application of CRISPR/Cas9technology widely, genome editing technique could be a main way for crop quality and production improvement in the future, and provide a new method help to cultivate crops more highly quality and multi-resistance.This study designed of Cas9gene and sgRNA in CRISPR/Cas9system, using a binary vector and mediated by Agrobacterium tumefaciens transformation in plants for expression this system, for study fuction of CRISPR/Cas9system in rice. The5target sites were design for expression vector construction, respectively, to explore optimal system,3of them subsequently transformed to rice.The main results are as follows:1. The cassette of Cas9and sgRNA cloned into the binary vector pCAMBIA1301, to form a universial expression vector pOsU3gRNA-2NLSCas9-1301for plant genetic transformation.2.The plant expression vectors Xa5-1-pOsU3gRNA-2NLSCas9-1301?Xa5-2-pOsU3gRNA-2NLSCas9-1301?Badh2-pOsU3gRNA-2NLSCas9-1301?GAI-pOsU3gRNA-2NL SCas9-1301?LTR-pOsU3gRNA-2NLSCas9-1301were constructed to explore the optimal system of target site connected with pOsU3gRNA-2NLSCas9-1301. The result shows that the target sequence in the annealing temperature of55?to60?, and efficiency of cloned into pOsU3gRNA-2NLSCas9-1301at the molar ratio of5:1is the highest.3. Xa5-1-pOsU3gRNA-2NLSCas9-1301?Xa5-2-pOsU3gRNA-2NLSCas9-1301 Badh2-pOsU3gRNA-2NLSCas9-1301binary expression vectors were mediated by agrobacterium transferred into TP309rice callus, respectively. After screen, differentiation, rooting, transgenic plants of total three vectors were acquired. And then the transgenic lines were transplanted in paddy field. The results confirmed that positive transgenic plantlets of Xa5-1-pOsU3gRNA-2NLSCas9-1301were17strains, Xa5-2-pOsU3gRNA-2NLSCas9-1301was of19strains,Badh2-pOsU3gRNA-2NLSCas9-1301was of38strains.4. The mutations of target site were detected in the differentiation stage of transgenic callus. The methods of mutation efficiency of targets were tested by PCR and enzyme digestion. The results confirmed that the target sites of Xa5-1and Xa5-2were displayed of mutation, but no mutations in Badh2target sites.5. Target mutations were detected in To transgenic plants by PCR and enzyme digestion and sequenced. The results indicated that target of Xa5-1with the mutation proportion of41%(7of17), meanwhile the target of Xa5-2with the mutation proportion of63%(12of19), but Badh2were failed to detect any mutagenesis in specific site.6. Target mutations were detected in Ti transgenic plants by PCR and enzyme digestion and sequenced. The results confirmed that target of Xa5-1and Badh2were failed to detect any mutagenesis in specific site, but in the target of Xa5-2there were3strains were occured same mutation with Ti generation with the heritability was25%.7. After inoculating of rice bacterial leaf blight pathogen P2to Xa5-1-pOsU3gRNA-2NLSCas9-1301?Xa5-2-pOsU3gRNA-2NLSCas9-1301for half a month. The results indicated that transgenic lines were compromised resistance against P2.