2.3. sgRNA design

For dCas9 system, it is necessary to design a RNA oligonucleotide containing 20 base pairs, which can target any genome site and is an important part of CRISPR Cas9 system. Many factors need to be considered when designing gRNA sequences. In order to find a suitable gRNA sequence, the online gRNA design tool (https://chopchop.cbu.uib.no/) was used to design the gRNA of the target gene zwf. The NGG sequence in which the first bit of protospacer adjacent motif (PAM) in gRNA library is A, T, C, and G was selected as PAM sequence. Using plasmid pFST-porb as template and primers sg1F/sgR, sg2F/sgR, sg3F/sgR, sg4F/sgR, PCR amplification was carried out to obtain four sgRNA containing 20N sequence. PCR products were recovered by 2% agarose gel, and the same volume of isopropanol was added to mix in the sample. The recovered product and plasmid pXMJ19-dCas9 1 were digested with EcoR I and XbaI for 2h, respectively, and then agarose gel was recovered. The T4 DNA Ligase was used to mix the fragments with the vector in proportion and incubate at 22 ​°C for 10 ​min, and 10 ​μl of the ligation products were transformed to E. coli JM109.