Design and Construction of Portable CRISPR-Cpf1-Mediated Genome Editing in Bacillus subtilis 168 Oriented Toward Multiple Utilities

Abstract

Bacillus subtilis is an important Gram-positive bacterium for industrial biotechnology, which has been widely used to produce diverse high-value added chemicals and industrially and pharmaceutically relevant proteins. Robust and versatile toolkits for genome editing in B. subtilis are highly demanding to design higher version chassis. Although the Streptococcus pyogenes ( Sp ) CRISPR-Cas9 has been extensively adapted for genome engineering of multiple bacteria, it has many defects, such as higher molecular weight which leads to higher carrier load, low deletion efficiency and complexity of sgRNA construction for multiplex genome editing. Here, we designed a CRISPR-Cpf1-based toolkit employing a type V Cas protein, Cpf1 from Francisella novicida. Using this platform, we precisely deleted single gene and gene cluster in B. subtilis with high editing efficiency, such as sacA , ganA, ligD & ligV , and bac operon. Especially, an extremely large gene cluster of 38 kb in B. subtilis genome was accurately deleted from the genome without introducing any unexpected mutations. Meanwhile, the synthetic platform was further upgraded to a version for multiplex genome editing, upon which two genes sacA and aprE were precisely and efficiently deleted using only one plasmid harboring two targeting sequences. In addition, we successfully inserted foreign genes into the genome of the chassis using the CRISPR-Cpf1 platform. Our work highlighted the availability of CRISPR-Cpf1 to gene manipulation in B. subtilis , including the flexible deletion of a single gene and multiple genes or a gene cluster, and gene knock-in. The designed genome-editing platform was easily and broadly applicable to other microorganisms. The novel platforms we constructed in this study provide a promising tool for efficient genome editing in diverse bacteria.

Development of a base editor for protein evolution via in situ mutation in vivo

“…The general sgRNAs were designed based on a previous study ( 35 ) via online software ( http://chopchop.cbu.uib.no/ ) ( 36 ). The 3′-terminal extended sgRNAs for the target region (N20) were designed by gradually adding the corresponding base.…”

“…To construct a multiplex base editing system for the modification of the SecYEG complex, plasmid pAD123 ( 35 ) was used as the donor vector for the expression of sgRNA driven by a strong constitutive promoter P veg . Using the pAD123, the plasmids pAD-T0, pAD-T3, pAD-secE, and pAD-secG that harboured sgRNAs targeting secY (T0), secY (T3), secE (ET) and secG (GT), respectively, were generated by reverse PCR using primers secYT0-F/secYT0-R, secYT3-F/secYT3-R, secEET-F/secEET-R and secGGT-F/secGGT-R, respectively.…”

“…For editing secY , three sgRNAs randomly targeting different positions on secY were designed. These sgRNAs harbored identical crRNA scaffold ( 35 ) with 20-nt targeting sequence downstream. These sgRNAs were integrated into the amyE site of CDA-nCas9-UGI-integrated cells, respectively.…”

CRISPR-Cas9-mediated Large Cluster Deletion and Multiplex Genome Editing in Paenibacillus polymyxa

“…Recently, it was reported that a chromosomally integrated Cas12a was able to facilitate large cluster deletion in B. subtilis by use of only one sgRNA. 18 Here, we demonstrated highly efficient Cas9-mediated large cluster deletion by using a one plasmid system with only one sgRNA, which has to be chosen carefully within the targeted sequence. In addition to the distance between cut site and homologous flanks, accessibility of the targeted region as well as the three-dimensional structure of the genome might also need to be considered especially when aiming for large genomic region deletions.…”

Advances in Enhanced Menaquinone-7 Production From Bacillus subtilis

“…In addition to the above technologies, B. subtilis gene-editing technology have been further developed based on CRISPR-Cas9 technology. Hao et al (2020) design a CRISPR-Cpf1-based toolkit employing a type V Cas protein, Cpf1 from F. novicida . Using this platform, they precisely delete single gene and gene cluster in B. subtilis with high editing efficiency, such as sacA, ganA, ligD,ligV , and bac operon.…”