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Review
. 2022 Oct 25;23(21):12852.
doi: 10.3390/ijms232112852.

Development and Applications of CRISPR/Cas9-Based Genome Editing in Lactobacillus

Yulin Mu  1 , Chengxiao Zhang  1 , Taihua Li  1 , Feng-Jie Jin  1 , Yun-Ju Sung  2 , Hee-Mock Oh  3 , Hyung-Gwan Lee  3 , Long Jin  1
Affiliations
Review

Development and Applications of CRISPR/Cas9-Based Genome Editing in Lactobacillus

Yulin Mu et al. Int J Mol Sci. .

Abstract

Lactobacillus, a genus of lactic acid bacteria, plays a crucial function in food production preservation, and probiotics. It is particularly important to develop new Lactobacillus strains with superior performance by gene editing. Currently, the identification of its functional genes and the mining of excellent functional genes mainly rely on the traditional gene homologous recombination technology. CRISPR/Cas9-based genome editing is a rapidly developing technology in recent years. It has been widely applied in mammalian cells, plants, yeast, and other eukaryotes, but less in prokaryotes, especially Lactobacillus. Compared with the traditional strain improvement methods, CRISPR/Cas9-based genome editing can greatly improve the accuracy of Lactobacillus target sites and achieve traceless genome modification. The strains obtained by this technology may even be more efficient than the traditional random mutation methods. This review examines the application and current issues of CRISPR/Cas9-based genome editing in Lactobacillus, as well as the development trend of CRISPR/Cas9-based genome editing in Lactobacillus. In addition, the fundamental mechanisms of CRISPR/Cas9-based genome editing are also presented and summarized.

Keywords: CRISPR/Cas9; Cas9 protein; Lactobacillus; genome editing.

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Conflict of interest statement

The authors here declare that they have no known competing financial interests or personal relationships that could have influenced this research.

Figures

Figure 1
Figure 1
CRISPR/Cas9 system in bacterial adaptive immunity. The functional driving of CRISPR/Cas9 system is divided into three stages. Acquisition: After phage infection, the Cas1-Cas2 protein complex will scan the invading DNA and identify the PAM region, and integrate the new spacer into the CRISPR sequence through the collection mechanism (cas1, cas2, csn2). Transcription: CRISPR sequence is transcribed to produce pre-crRNA under the control of the leading region, and tracrRNA is also transcribed. Pre-crRNA is further processed to form mature crRNA, which forms double-stranded RNA with tracrRNA through base complementary pairing. (3) Interference: The crRNA-tracrRNA duplex binds to Cas9 and guides Cas9 to cleave foreign DNA containing a 20-nt crRNA complementary sequence preceding PAM.
Figure 2
Figure 2
Composition of CRISPR/Cas9 genome editing system. (A) Natural CRISPR/Cas9 genome editing system, crRNA and tracrRNA form guide RNA (gRNA) through local base pairing. (B) Engineered CRISPR/Cas9 genome editing system; the crRNA-tracrRNA chimera was modified to form sgRNA by creating a connecting loop.
Figure 3
Figure 3
Mechanism of CRISPR/Cas9 genome editing system. The guide RNA directs a Cas9 endonuclease to cleavage target DNA. The DSB (double-strand break) generated by Cas9 nuclease domains is repaired by host-mediated DNA repair mechanisms, i.e., homology-directed repair (HDR) and non-homologous end joining (NHEJ).
Figure 4
Figure 4
Modifications of Cas9 in genome editing. (A) The Cas9 nuclease cleaves the strands of the target DNA via HNH and RuvC domains. (B) Cas9n, containing the inactivating mutation D10A, inhibits the activity of the RuvC domain. (C) Cas9n, containing the inactivating mutation H840A, inhibits the activity of the RuvC domain. (D) Dcas9, inactivating the mutation of HNH and RuvC domains.
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Grants and funding

This research was funded by the Korea Environment Industry & Technology Institute (KEITI) through a project to develop new, eco-friendly materials and processing technology derived from wildlife, funded by the Korea Ministry of Environment (MOE), grant number 2021003240004, as well as by the National Research Foundation of Korea (NRF), with a grant funded by the Korean government, grant number NRF-2018R1C1B3009513. The APC was funded by 2021003240004.

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