Authors
Marcia Brady
Summary
As B. subtilis was selected as a model bacterium, simple and efficient genetic tools have been developed in the past decades. Classical genome modification relies on the insertion of a selectable marker, usually an antibiotic resistance gene, into the chromosome of the target strain. The most commonly used scarless genetic manipulations systems for B. subtilis rely on counter-selectable markers (CSM), while other methods include site-specific recombination systems (SSR), and CRISPR-Cas9 system.
Introduction
CSM is often used for the markerless construction of engineered strains and have been used to construct Bacillus cell factories for various industrial applications. Selectable markers can generally be divided into positive and negative selection markers, whereby the former are most commonly antibiotic-resistance markers. However, CSM-based strategies require host pre-modification and have a low success rate due to the leaky expression of the CSM.
Materials
Site-specific recombination (SSR) systems are powerful tools for the precise excision of DNA fragments. These systems, such as FLP/FRT and Cre/loxP, have much higher recombination efficiency than the endogenous recombination systems, making them an ideal tool for many genetic manipulations. By combining a mutated Cre/lox system with the long segment fusion PCR method has been developed as a rapid and accurate B. subtilis genome engineering tool that allows operations such as targeted gene inactivation, long-fragment deletion, and in-frame deletion of target genes.
Procedure
The application of the CRISPR-related (Cas) system in B. subtilis has further enriched the gene-editing toolbox in recent years. The CRISPR locus is first transcribed into a precursor CRISPR ribonucleic acid (pre-crRNA), which is then cut into small RNA units under the action of Cas protein or endonuclease. These small RNA units are mature crRNAs that contain spacer sequences and partial repeat sequences. Maturation of the crRNAs of type II CRISPR/Cas systems requires not only the participation of Cas9 and RNase, but also the guidance of a tracrRNA. Mature crRNAs and tracrRNA form double-stranded RNA structures through complementary base pairing. The resulting duplexes bind Cas9 protein to form a targeted cutting complex, specifically cutting foreign sequences to achieve the goal of identifying and eliminating invading foreign genes such as plasmids and viruses.
At present, there are three kinds of CRISPR/Cas9-based genome editing strategies widely used in B. subtilis.
(1) The single-plasmid based system, in which Cas9, a single guide RNA (gRNA), donor DNA, and other elements are assembled into the same carrier skeleton, wherein Cas9 protein and gRNA are respectively expressed from inducible or strong constitutive promoters.
(2) The two-plasmid-based system is more flexible than the single plasmid system. In this system, Cas9, gRNA, and donor DNA are assembled on two different plasmids, which are respectively used to produce Cas9 protein and deliver the gRNA transcription module and donor DNA template.
(3) The chromosomally integrated system is more stable and effective than the first two systems, but it requires the use of engineered strains. The Cas9 was integrated into the genome, and then araE/R initiation subsystem was used to construct a multi-gRNA delivery vector.
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