Review of knockout technology approaches in bacterial drug resistance research

Abstract

Gene knockout is a widely used method in biology for investigating gene function. Several technologies are available for gene knockout, including zinc-finger nuclease technology (ZFN), suicide plasmid vector systems, transcription activator-like effector protein nuclease technology (TALEN), Red homologous recombination technology, CRISPR/Cas, and others. Of these, Red homologous recombination technology, CRISPR/Cas9 technology, and suicide plasmid vector systems have been the most extensively used for knocking out bacterial drug resistance genes. These three technologies have been shown to yield significant results in researching bacterial gene functions in numerous studies. This study provides an overview of current gene knockout methods that are effective for genetic drug resistance testing in bacteria. The study aims to serve as a reference for selecting appropriate techniques.

Keywords: Bacterial drug resistance; CRISPR/Cas9; Gene knockout; Red homologous recombination; Suicide plasmid.

Figure 1. λ-Red homologous recombination.

(A) The targeting fragment contains homologous arms (H1, H2), FRT sites and resistance genes for screening; (B) the homologous arms (H1, H2) on both sides of the target gene are used to undergo homologous recombination; (C) the targeting fragment successfully displaces the target gene under the mediation of Red homologous recombination; (D) the pcp20 plasmid expresses FLP recombinase, recognizes the FRT sites and eliminates the resistance genes.

Figure 2. CRISPR/Cas9 for gene knockout.

crRNA binds to tracrRNA through base pairing to form Guide RNA, which forms a complex with the nuclease Cas9 and guides Cas9 to shear ds DNA at the sequence target site paired with crRNA. After shearing down the target gene, the cell can use its own non-homologous end-joining repair to repair the broken chromosome, or it can use Red homologous recombination, which artificially introduces homologous fragments for homologous end-joining repair.

Figure 3. Suicide plasmid for gene knockout.

Suicide vectors were integrated into the bacterial genome after undergoing the first homologous recombination, after which they underwent the second homologous recombination to obtain two results, strains with the same sequence as the wild type (A), and strains without trace knockout (B).