Editing of the Bacillus subtilis Genome by the CRISPR-Cas9 System

Abstract

The clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) systems are adaptive immune systems of bacteria. A type II CRISPR-Cas9 system from Streptococcus pyogenes has recently been developed into a genome engineering tool for prokaryotes and eukaryotes. Here, we present a single-plasmid system which allows efficient genome editing of Bacillus subtilis The plasmid pJOE8999 is a shuttle vector that has a pUC minimal origin of replication for Escherichia coli, the temperature-sensitive replication origin of plasmid pE194(ts) for B. subtilis, and a kanamycin resistance gene working in both organisms. For genome editing, it carries the cas9 gene under the control of the B. subtilis mannose-inducible promoter PmanP and a single guide RNA (sgRNA)-encoding sequence transcribed via a strong promoter. This sgRNA guides the Cas9 nuclease to its target. The 20-nucleotide spacer sequence at the 5' end of the sgRNA sequence, responsible for target specificity, is located between BsaI sites. Thus, the target specificity is altered by changing the spacer sequences via oligonucleotides fitted between the BsaI sites. Cas9 in complex with the sgRNA induces double-strand breaks (DSBs) at its target site. Repair of the DSBs and the required modification of the genome are achieved by adding homology templates, usually two PCR fragments obtained from both sides of the target sequence. Two adjacent SfiI sites enable the ordered integration of these homology templates into the vector. The function of the CRISPR-Cas9 vector was demonstrated by introducing two large deletions in the B. subtilis chromosome and by repair of the trpC2 mutation of B. subtilis 168.

Importance: In prokaryotes, most methods used for scarless genome engineering are based on selection-counterselection systems. The disadvantages are often the lack of a suitable counterselection marker, the toxicity of the compounds needed for counterselection, and the requirement of certain mutations in the target strain. CRISPR-Cas systems were recently developed as important tools for genome editing. The single-plasmid system constructed for the genome editing of B. subtilis overcomes the problems of counterselection methods. It allows deletions and introduction of point mutations. It is easy to handle and very efficient, and it may be adapted for use in other firmicutes.

FIG 1

CRISPR-Cas9 vector pJOE8999. (A) Physical map of vector pJOE8999 containing the pUC18 minimal origin, the temperature-sensitive replication origin from pE194^ts, a kanamycin resistance gene (kanR), cas9 under the control of the P_manP promoter, the sgRNA transcribed from the semisynthetic promoter P_vanP* interrupted by the lacZ α fragment (lacPOZ'), the λ oop terminator, and the T7 promoter (T7P). (B) Cloning sites in the vector pJOE8999. (C) Promoter sequence (P_vanP*) upstream of the sgRNA with the −35 and −10 sequences and the BsaI restriction sites (bold) for insertion of the spacer sequence.

FIG 2

B. subtilis chromosomal region carrying the pulcherrimin biosynthesis genes. The bars on the top indicate the fragments amplified by PCR and integrated between the SfiI sites of pJOE8999 to delete the genes cypX and yvmA. The sequence in cypX was selected as a target for the Cas9/sgRNA complex, and the oligonucleotides inserted into pJOE8999 are shown below. Lowercase letters indicate the deoxynucleotides fitting to the BsaI sites.

FIG 3

Nucleotide and amino acid sequences of the trpC2 gene of B. subtilis 168 around the missing ATT codon (middle), corrected sequence (top) with the added codon (lowercase letters), and new modified sequence (bottom, lowercase and bold letters). The spacer sequence used in the CRISPR-Cas9 vectors to correct the mutation is aligned (vertical lines). wt, wild type.