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Application of different types of CRISPR/Cas-based systems in bacteria

Liu, Zhenquan, Dong, Huina, Cui, Yali, Cong, Lina, Zhang, Dawei·2020·Microbial Cell Factories
DOI 10.1186/s12934-020-01431-zPMC PMC7470686
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Abstract

As important genome editing tools, CRISPR/Cas systems, especially those based on type II Cas9 and type V Cas12a, are widely used in genetic and metabolic engineering of bacteria. However, the intrinsic toxicity of Cas9 and Cas12a-mediated CRISPR/Cas tools can lead to cell death in some strains, which led to the development of endogenous type I and III CRISPR/Cas systems. However, these systems are hindered by complicated development and limited applications. Thus, further development and optimization of CRISPR/Cas systems is needed. Here, we briefly summarize the mechanisms of different types of CRISPR/Cas systems as genetic manipulation tools and compare their features to provide a reference for selecting different CRISPR/Cas tools. Then, we show the use of CRISPR/Cas technology for bacterial strain evolution and metabolic engineering, including genome editing, gene expression regulation and the base editor tool. Finally, we offer a view of future directions for bacterial CRISPR/Cas technology.

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Fig. 1 Schematic of the mechanisms of different types of CRISPR systems. a The working principle of type II Cas9. In the presence of the PAM sequence (NGG), the targeting effect of sgRNA is used to guide Cas9 protein to cleave both the complementary and non-complementary strands, forming a blunt-ended nick. b The working principle of type V Cas12a protein. In the presence of the PAM sequence (NTTT), the targeting effect of crRNA is used to guide Cas12a protein to cleave both the complementary and non-complementary strands, forming a sticky-ended nick. c The working principle of type I Cas systems. In the presence of the PAM sequence, the targeting effect of crRNA is used to guide the Cas3 protein to cleave the non-complementary strand to form a large gap. d The working principle of type III Cas systems. In the absence of a PAM sequence, the targeting effect of sgRNA is used to guide Csm protein to cleave the non-complementary strand to form short nucleic acid fragments. The green transverse U represents sgRNA or crRNA, the nucleotide sequences marked in red represent the PAM sequence, and scissors represent the cleavage site of nucleases
Fig. 1 Schematic of the mechanisms of different types of CRISPR systems. a The working principle of type II Cas9. In the presence of the PAM sequence (NGG), the targeting effect of sgRNA is used to guide Cas9 protein to cleave both the complementary and non-complementary strands, forming a blunt-ended nick. b The working principle of type V Cas12a protein. In the presence of the PAM sequence (NTTT), the targeting effect of crRNA is used to guide Cas12a protein to cleave both the complementary and non-complementary strands, forming a sticky-ended nick. c The working principle of type I Cas systems. In the presence of the PAM sequence, the targeting effect of crRNA is used to guide the Cas3 protein to cleave the non-complementary strand to form a large gap. d The working principle of type III Cas systems. In the absence of a PAM sequence, the targeting effect of sgRNA is used to guide Csm protein to cleave the non-complementary strand to form short nucleic acid fragments. The green transverse U represents sgRNA or crRNA, the nucleotide sequences marked in red represent the PAM sequence, and scissors represent the cleavage site of nucleasesCC BY · PubMed Central
Fig. 2 Schematic of the gene tools based on Cas9, nCas9, and dCas9, respectively. a The HR system is used to accurately repair the DSBs introduced by Cas9 protein or the single-strand breaks introduced by nCas9 protein when donor DNA fragments are provided. The NHEJ system is used to inaccurately repair the DSBs introduced by Cas9 protein when Donor DNA fragments are not provided. b The combination of dCas9 and the target site inhibits the combination of RNAP and the DNA strand, which weakens the DNA transcription process and reduces the expression of the targeted gene. The expression of a fusion protein comprising dCas9 and a transactivator domain can recruit RNAP and enhance the process of DNA transcription, thus increasing targeted gene expression. The red dots in A/B represent gene mutations in the corresponding cleavage domains
Fig. 2 Schematic of the gene tools based on Cas9, nCas9, and dCas9, respectively. a The HR system is used to accurately repair the DSBs introduced by Cas9 protein or the single-strand breaks introduced by nCas9 protein when donor DNA fragments are provided. The NHEJ system is used to inaccurately repair the DSBs introduced by Cas9 protein when Donor DNA fragments are not provided. b The combination of dCas9 and the target site inhibits the combination of RNAP and the DNA strand, which weakens the DNA transcription process and reduces the expression of the targeted gene. The expression of a fusion protein comprising dCas9 and a transactivator domain can recruit RNAP and enhance the process of DNA transcription, thus increasing targeted gene expression. The red dots in A/B represent gene mutations in the corresponding cleavage domainsCC BY · PubMed Central

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Metadata

Authors
Liu, Z., Dong, H., Cui, Y., Cong, L., & Zhang, D.
ORCID
Zhang, Dawei
Venue
Microbial Cell Factories
Publisher
Springer Science and Business Media LLC
Published
2020-09-03
Volume
19
Issue
1
ISSN
1475-2859
DOI
10.1186/s12934-020-01431-z
PMC
PMC7470686
Cited by
185
References
8
Language
en
Licence
Funders
National Natural Science Foundation of China (NSFC 31800086)

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