CRISPR Genome Editing Systems in the Genus Clostridium: a Timely Advancement

Abstract

The genus Clostridium is composed of bioproducers, which are important for the industrial production of chemicals, as well as pathogens, which are a significant burden to the patients and on the health care industry. Historically, even though these bacteria are well known and are commonly studied, the genetic technologies to advance our understanding of these microbes have lagged behind other systems. New tools would continue the advancement of our understanding of clostridial physiology. The genetic modification systems available in several clostridia are not as refined as in other organisms and each exhibit their own drawbacks. With the advent of the repurposing of the CRISPR-Cas systems for genetic modification, the tools available for clostridia have improved significantly over the past four years. Several CRISPR-Cas systems such as using wild-type Cas9, Cas9n, dCas9/CRISPR interference (CRISPRi) and a newly studied Cpf1/Cas12a, are reported. These have the potential to greatly advance the study of clostridial species leading to future therapies or the enhanced production of industrially relevant compounds. Here we discuss the details of the CRISPR-Cas systems as well as the advances and current issues in the developed clostridial systems.

Keywords: CRISPR; CRISPRi; Clostridium; genome editing.

FIG 1

Comparison of CRISPR system plasmids. Representative sample mutagenesis plasmids for CRISPR editing systems are shown. Each contains the three regions: a homology region (denoted by homology arms [HA]), an endonuclease (Cas9 or Cpf1), and a gRNA. Plasmids for editing using Cas9 or Cas9n (A), dCas9 with no necessary homology region (B), Cpf1/Cas12a with one target (C), and Cpf1/Cas12a with two targets (D) are shown. Examples of CRISPR-Cas mutagenesis plasmids are listed inside each plasmid.

FIG 2

Review of CRISPR-Cas genetic modification systems. Shown are graphical representations of each CRISPR-Cas system discussed in this review. Each contains the different endonucleases: Cas9 (A), Cpf1/Cas12a (B), and dCas9 with an activator/repressor (C), as well as the sgRNA or crRNA, PAM site and sequence, and cleavage locations and their respective cleavage domains for each. A table is included in panel A to describe the different mutant alleles of Cas9. Panel C also shows the promoter region area and the start codon for reference.

FIG 3

Endogenous CRISPR genome editing in clostridia. Shown is a graphical representation of CRISPR-Cas genome editing in a clostridial vegetative cell. (1) The endogenous CRISPR region contains a cas operon which encodes a Cas endonuclease that is subsequently generated. (2) Meanwhile, a plasmid contains a synthesized CRISPR array under the control of an inducible promoter and a donor region for homologous recombination containing an upstream homology arm (HA) and a downstream HA. When induced, the plasmid transcribes the crRNAs to be used by the endogenous system to generate individual crRNAs. (3) The endogenous Cas endonuclease complexes with a crRNA to target and cleave the DNA. This is then repaired by the donor region located on the CRISPR plasmid.