Recent advances of Cas12a applications in bacteria

Meliawati, Meliawati; Schilling, Christoph; Schmid, Jochen

doi:10.1007/s00253-021-11243-9

Cited by 26 publications

(19 citation statements)

References 85 publications

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“…Traditional genetic manipulation for S. chattanoogensis L10 was proved to be intricate and time-consuming (Liu et al, 2015 ). The CRISPR-Cpf1 system has been widely used in many bacterial species (Meliawati et al, 2021 ). In the genus Streptomyces , this system has been applied successfully to conduct precise genome editing by homology-directed repair (Li et al, 2018 ).…”

Section: Resultsmentioning

confidence: 99%

Activation and Characterization of Lanthomicins A–C by Promoter Engineering in Streptomyces chattanoogensis L10

et al. 2022

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The emergence of drug resistance highlights the importance of new drug discovery. Microbial secondary metabolites encoded in biosynthetic gene clusters (BGCs) are a prolific source of drugs, whereas most of these BGCs are cryptic. Thus, taking strategies to activate these cryptic BGCs is of great importance for potential drug discovery. In this work, three novel pentangular polyphenols lanthomicin A–C were identified by activating a cryptic aromatic polyketide BGC through promoter engineering combined with optimization of fermentation conditions. We further confirmed the involvement of lanthomicin (ltm) BGC in biosynthesis by CRISPR-Cpf1-assisted gene editing. Based on functional analysis of homologous genes, a putative biosynthetic pathway was proposed for the three lanthomicins. Particularly, lanthomicin A showed antiproliferative activity with IC50 0.17 μM for lung cancer cell line A-549. The discovery of lanthomicins brings new members to the pentangular polyphenol subclade of aromatic polyketide and demonstrates the potential of Streptomyces as a source for drug discovery.

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Section: Resultsmentioning

confidence: 99%

Activation and Characterization of Lanthomicins A–C by Promoter Engineering in Streptomyces chattanoogensis L10

et al. 2022

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“…25,26 Previous studies have reported CRISPR-Cas-based multiplex genetic engineering in different microorganisms. [27][28][29] However, only a few demonstrated multiplex gene integration, which was mainly performed in yeast. 30,31 While it has been reported in E. coli, 32 to the best of our knowledge, our study is the first to utilize a Cas9-based system to achieve multiplex gene integrations in Gram-positive bacteria.…”

Section: Resultsmentioning

confidence: 99%

CRISPR-Cas9-mediated Large Cluster Deletion and Multiplex Genome Editing in Paenibacillus polymyxa

Meliawati

Teckentrup

Schmid

2021

Preprint

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Clustered regularly interspaced short palindromic repeats (CRISPR) system has rapidly advanced genetic engineering research. The system has been applied for different genetic engineering purposes in multiple organisms including the quite rarely explored Paenibacillus polymyxa. Only limited studies on CRISPR-based system have been described for this highly interesting and versatile bacterium. Here, we demonstrated the utilization of a Cas9-based system to realize 32.8 kb deletion of genomic region by using a single targeting sgRNA. Large cluster deletion was successfully performed with remarkable efficiency of 97 %. Furthermore, we also exploited the system for multiplexing by editing of two distantly located genes at once. We investigated double gene knockouts as well as simultaneous gene integrations and reached editing efficiencies of 78 % and 50 %, respectively. The findings reported in this study are anticipated to accelerate future research in P. polymyxa and related species.

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“…Furthermore, the CRISPR system is varied and suitable for upgrades to the genetic operating system [36,37]. The fusion of various protein motifs to Cas effector proteins has facilitated a diverse set of genetic manipulations, such as base editing, transposition, recombination, and epigenetic regulation [38][39][40][41][42][43].…”

Section: Discussionmentioning

confidence: 99%

An Optimized and Efficient CRISPR/Cas9 System for the Endophytic Fungus Pestalotiopsis fici

Huang

Yin

2021

JoF

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Endophytic fungi are emerging as attractive producers of natural products with diverse bioactivities and novel structures. However, difficulties in the genetic manipulation of endophytic fungi limit the search of novel secondary metabolites. In this study, we improved the polyethylene glycol (PEG)-mediated protoplast transformation method by introducing the CRISPR/Cas9 system into endophytic fungus Pestalotiopsis fici. Using this approach, we performed genome editing such as site-specific gene insertion, dual-locus mutations, and long DNA fragment deletions in P. fici efficiently. The average efficiency for site-specific gene insertion and two-site gene editing was up to 48.0% and 44.4%, respectively. In addition, the genetic manipulation time with long DNA fragment (5–10 kb) deletion was greatly shortened to one week in comparison with traditional methods such as Agrobacterium tumefaciens-mediated transformation (ATMT). Taken together, the development of the CRISPR/Cas9 system in the endophytic fungus will accelerate the discovery of novel natural products and further biological study.

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Recent advances of Cas12a applications in bacteria

Cited by 26 publications

References 85 publications

Activation and Characterization of Lanthomicins A–C by Promoter Engineering in Streptomyces chattanoogensis L10

Activation and Characterization of Lanthomicins A–C by Promoter Engineering in Streptomyces chattanoogensis L10

CRISPR-Cas9-mediated Large Cluster Deletion and Multiplex Genome Editing in Paenibacillus polymyxa

An Optimized and Efficient CRISPR/Cas9 System for the Endophytic Fungus Pestalotiopsis fici

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