2.3.2 Vector for CRISPR
The searching pattern of CRISPR target sites is set as 5’-GGX18NGG-3’, 5’-GX19NGG-3’ or 5’-X20NGG-3’, where N and X is any base, NGG is the PAM sequence. In addition, CRISPR target sequences should align to the whole genome scale to choose the less off-target ones, improving the cleavage efficiency and specificity of CRISPR-Cas9 system [35]. Therefore, we designed the CRISPR target sequence of the LAC1 gene as 5’- GGGCATGGTTTGCGGCAGCT-GGG-3’ with the use of sgRNAcas9 target designing software[35]. Besides, we added 5’ BspQI-compatible overhangs to the target sequence (LAC1-CRI-F) and its complementary sequence (LAC1-CRI-R) in order to ligate with the BspQI-digested PNK003 vector. The LAC1-CRI-F annealed with LAC1-CRI-R in a PCR thermocycler at 95℃ for 5 min, cooling down at room temperature for 1 h. Then, the hybridized oligonucleotides consisting of target sequences and 5’ overhangs were diluted to 1:50 with ddH20 and ligated into BspQI-digested PNK003 vectors (pre-digested at 50℃ for 20-30 min) at 4 ℃ for 4 h. The PNK003 vectors inserted with annealed LAC1 target oligonucleotides (PNK-LAC1, shown in Fig. 2A) were transformed into E. coli DH5α competent cells incubated on ampicillin-containing agar plates at 37 ℃ for 16 h. Finally, three random colonies were picked out for the sequencing of the gRNA expression cassette by T3 universal primer (its sequencing was displayed in supplementary Fig. 4 and sequence 4).