A Simplified Method for CRISPR-Cas9 Engineering of Bacillus subtilis.

John D Helmann,Tino Polen,Alexander J Alfonso,Ankita J Sachla

doi:10.1128/spectrum.00754-21

Abstract

ABSTRACTThe clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system from Streptococcus pyogenes has been widely deployed as a tool for bacterial strain construction. Conventional CRISPR-Cas9 editing strategies require design and molecular cloning of an appropriate guide RNA (gRNA) to target genome cleavage and a repair template for introduction of the desired site-specific genome modification. Here, we present a streamlined method that leverages the existing collection of nearly 4,000 Bacillus subtilis strains (the BKE collection) with individual genes replaced by an integrated erythromycin (erm) resistance cassette. A single plasmid (pAJS23) with a gRNA targeted to erm allows cleavage of the genome at any nonessential gene and at sites nearby to many essential genes. This plasmid can be engineered to include a repair template, or the repair template can be cotransformed with the plasmid as either a PCR product or genomic DNA. We demonstrate the utility of this system for generating gene replacements, site-specific mutations, modification of intergenic regions, and introduction of gene-reporter fusions. In sum, this strategy bypasses the need for gRNA design and allows the facile transfer of mutations and genetic constructions with no requirement for intermediate cloning steps.IMPORTANCE Bacillus subtilis is a well-characterized Gram-positive model organism and a popular platform for biotechnology. Although many different CRISPR-based genome editing strategies have been developed for B. subtilis, they generally involve the design and cloning of a specific guide RNA (gRNA) and repair template for each application. By targeting the erm resistance cassette with an anti-erm gRNA, genome editing can be directed to any of nearly 4,000 gene disruptants within the existing BKE collection of strains. Repair templates can be engineered as PCR products, or specific alleles and constructions can be transformed as chromosomal DNA, thereby bypassing the need for plasmid construction. The described method is rapid and facilitates a wide range of genome manipulations.

Highlights

Introduction of an epitope tagAs a second application, we incorporated a FLAG epitope tag by modification of the B. subtilis sodA gene encoding superoxide dismutase
Simplified clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 Engineering of B. subtilis designing a unique guide RNA (gRNA) for each application, we sought to leverage the large BKE collection of single-gene erm replacements by design of an anti-erm gRNA
76 13 (5/38)e yceF::erm!yceF(Ile206Thr)c aNA, not applicable. bLength of UP and DO homology is depicted in parenthesis. crsgA of 876 bp was amplified from S. aureus Newman strain. d715 bp of green fluorescent protein (GFP) coding sequence was amplified from pGFP-star plasmid DNA. eRepresents the fact that only 5 out of 28 clones and 5 out of 38 clones showed PCR products. fCotransformation had different size transformants after 2 days on Kan 1 0.2% mannose plates, and we found large colonies that appeared rapidly and usually did not show a successful CRISPR event

Summary

Introduction

We incorporated a FLAG epitope tag by modification of the B. subtilis sodA gene encoding superoxide dismutase. We generated plasmid pAJS25 to include a 3.9-kb repair template containing the desired sodA-FLAG gene flanked by upstream (yqgB-yqgC) and downstream (yqgE) genes (Fig. 2B). In this case, recombinational repair could occur downstream of sodA, leading to successful integration of the sodA-FLAG gene, or within the sodA gene. After curing of the plasmid, most of the recovered clones (66%, Table 1) were MLSs, consistent with loss of the erm cassette. All 10 of the MLSs transformants characterized encoded

Methods

Results

Conclusion