Establishment Of A New Type Of CRISPR/Cas9 Detection System In Vivo And Identification Of The Activity Of The System

ObjectiveCRISPR/Cas9 is a new type of gene editing techniques, the operation is simple and can be cut to specific sites of genome. However, the major concern of the off-target still needs further research. This study was performed to establish a one step and sequence independent method for molecular clone. Applying the method to construct the recombinant plasmids p Tac-Cas9 in E.coli and p MV261-Cas9, p MV261(+)-Clp C2 and a fluorescence report plasmid p MV261(+)-m Cherry in M.smegmatis. On this basis, this study intends to establish a detection system basing on homologous recombination and m Cherry fluorescence protein reporter gene with homologous arm, then applying it to detect the cutting efficiency and off-target of target RNA sequence for Cas9 protein and different guide design. This study laid a foundation for further study on CRISPR/Cas9off-target effects, guide RNA design and biological applications.Methods1. The target gene and plasmid vector were amplified by PCR, the primers contain methylation site. PCR amplification fragments were purified, then the purpose fragments were mixed with PCR plasmid vector taking the same number of moles. The recombinant plasmids p Tac-Cas9,p MV261-Cas9, p MV261-Clp C2 and p MV261-m Cherry were fast constructed by the method using the restriction enzyme Fsp EⅠ which was only recognize the methylation base Cm C which was added in the PCR products of mixing T4 DNA ligase, Fsp EⅠ enzyme, the activator, Fsp EⅠenzyme buffer, blending and ATP. The recombinant plasmid p Tac-Cas9 was transformed into E.coli DH5α and the recombinant plasmids p MV261-Cas9,p MV261-Clp C2 and p MV261-m Cherry was transformed into M.smegmatis respectively. The expression products were analyzed by Western-blot.2. The guide RNA target sequence was inserted into the ORF of the fluorescent reporter gene m Cherry, and both sides of the target sequence upstream and downstream of the different lengths of homologous coding regions were reserved. The fluorescent reporter gene m Cherry was cut by the Cas9 protein, then the divided m Cherry gene were be repair by homologous recombination in vivo and the red colonies were observed by naked eyes. On this basis, the different lengths of guide RNA complementary target sequence were designed and the mismatch base between target sequence and guide RNA was added for cutting experiment.The red colonies were counted from the resistant plate and the results were analyzed by SPSS software.Results1. Using a one step method for molecular clone based on Fsp EⅠenzyme and T4 ligase, the expression plasmid p Tac-Cas9 in E.coli DH5αwas constructed successfully. The recombinant plasmid p Tac-Cas9 was identified by sequencing. The Cas9 protein was successfully expressed in E.coli DH5α by Western-blot. In addition, the expression plasmids p MV261-Cas9, p MV261-Clp C2 and p MV261-m Cherry in mycobacterium was constructed respectively by the method and the r Clp C2 and rm Cherry was successfully expressed in M.smegmatis respectively, but the recombinant plasmid p MV261-Cas9 was not transformed into M.smegmatis, which was needed further research.2. The guide RNA target sequence was inserted into the ORF of the fluorescent reporter gene m Cherry, and both sides of the target sequence upstream and downstream of the different lengths of homologous coding regions were reserved. This study showed red colon was not observed if the plasmid with no homologous regions was cut by Cas9 protein and red colonies were observed and counted if the plasmids with homologous regions contain 42 bp, 78 bp and 136 bp were cut by Cas9 protein and be repair by homologous recombination, similar recombination efficiency was discovered. The cutting efficiency was compared among the matching lengths, 19 bp the highest cutting efficiency, 20 bp the higher one than other lengths. The off-target effects were detected if the mismatch base of guide RNA compared with the target sequence was located in the penultimate or six(5 ′- 3′) which was still cut by Cas9 protein.Conclusions1. A one step and sequence independent method for molecular clone was Successfully established. The expression plasmid p Tac-Cas9 in E.coli DH5α,the mycobacterium plasmids p MV261-Cas9, p MV261-Clp C2 and p MV261-m Cherry was constructed successfully respectively. The Cas9 protein was successfully expressed in E.coli DH5α and the r Clp C2 was successfully expressed in M.smegmatis.2. A detection system in vivo basing on homologous recombination and m Cherry fluorescence protein reporter gene with homologous arm was successfully established. The cutting efficiency and off-target of target RNA sequences for Cas9 protein and different guide design were detected by the detection system.