AUTHOR=Lim Hayeon , Choi Soo-Keun 

TITLE=Programmed gRNA Removal System for CRISPR-Cas9-Mediated Multi-Round Genome Editing in Bacillus subtilis

JOURNAL=Frontiers in Microbiology

VOLUME=10

YEAR=2019

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2019.01140

DOI=10.3389/fmicb.2019.01140

ISSN=1664-302X

ABSTRACT=<p>CRISPR/Cas9 has become a simple and powerful genome editing tool for many organisms. However, multi-round genome editing should replace single-guide RNA (sgRNA) every round, which is laborious and time-consuming. Here, we have developed a multi-round genome editing system in which genome editing and the programmed removal of the sgRNA have sequentially occurred in a growth-dependent manner in <italic>Bacillus subtilis</italic>. The system contains two plasmids, one containing a <italic>cas9</italic> gene and the other containing two sgRNAs and a donor DNA for homology directed repair (HDR). The two sgRNAs are chromosome-targeting (sgRNA<sub>ct</sub>) and self-targeting (sgRNA<sub>st</sub>) under the control of a constitutive promoter and sporulation-specific promoter, respectively. In the growth phase, the sgRNA<sub>ct</sub> is transcribed and complexed with the Cas9 to edit the chromosomal target, while the sgRNA<sub>st</sub> is transcribed in the sporulation phase and complexed with the Cas9 to attack its own plasmid. Therefore, the system automatically makes the cell ready for next-round genome editing during cultivation. The system was approved through the sequential deletion of eight extracellular protease genes in the <italic>B. subtilis</italic>, suggesting that it can be used for versatile applications in multi-round genome editing.</p>