3.2 Successful construction of LAC1 knock-out strain and its phenotypic stability
For gene knock-out, we used the PNK003 vector gifted by Ping Zhang and Xudong Zhu, Beijing Key Laboratory of Genetic Engineering Drug and Biotechnology, College of Life Sciences, Beijing Normal University. PNK003 contains two reverse BspQI restriction sites between the U6 promoter of H99 (serotype A) and the gRNA component, allowing the hybridized target DNA with 5’ overhangs to be conveniently introduced into the BspQI-cut plasmids. Apart from the simplified construction, the elimination of Cas9 and gDNA cassettes after gene editing is another highlight of the vector. In this study, we proved that the PNK003 could conduct the function of genic knockout in B-3501A (serotype D) (Fig. 2). We picked the white strains out of the DOPA agar containing hygromycin B (Fig. 2A) and passaged them by plate streaking on DOPA agar every 5 days to screen out the completely knock-out strains. Finally, we found that the albino mutant strains of group PNK-LAC1 were stable to maintain and pass on the albino phenotype (Fig. 2C, at the 5thpassage). Meanwhile, the qRT-PCR result of the albino strains was no amplification within 40 PCR cycles (Fig. 2D), indicating that PNK003 was also effective for gene editing in serotype D.