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.