Emerging technologies for genetic modification of solventogenic clostridia: From tool to strategy development

Bai, Sheng-Kai; Hong, Ying; Wu, Yi-Rui

doi:10.1016/j.biortech.2021.125222

Cited by 12 publications

(2 citation statements)

References 112 publications

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“…Clostridium offers a broad range of potential applications in biotechnology, whether for medical or industrial applications. However, genetic manipulation in clostridia is still challenging due to species-specific nuances ( 43 ), and the CRISPR-Cas tools in clostridia are not as refined as in model organisms ( 7 ). In this study, we achieved successful implementation of the CRISPR-Cas12a system in two Clostridium sensu stricto species, C. butyricum , and C. sporogenes , utilizing tetracycline-inducible systems.…”

Section: Discussionmentioning

confidence: 99%

Development of a CRISPR-Cas12a system for efficient genome engineering in clostridia

Zhang,

Kubiak,

Bailey

et al. 2023

Microbiol Spectr

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Clostridium species have gained attention in industrial and medical applications, and the development of genetic tools has enabled the advancement of the CRISPR-Cas systems for these bacteria. Compared to the primarily used Cas9 from Streptococcus pyogenes , the utilization of Cas12a (previously known as Cpf1) proteins remains incomplete in clostridia, although they exhibit potential advantages, including T-rich recognition for Clostridium genomes and lower toxicity. In this study, we expanded the CRISPR-Cas tools in clostridia by establishing a CRISPR-Cas12a system with two different cas12a genes ( Ascas12a from Acidaminococcus and Fncas12a from Francisella novicida ). The optimized tetracycline-inducible systems were initially determined by the glucuronidase reporter and were used to drive expression of the cas12a genes and crRNAs. Our results demonstrate that the CRISPR-FnCas12a system offers flexible target selection in clostridia, and a specific folding pattern of the precursor crRNA is important to enable high mutation generation efficiency. By using sacB (encoding levansucrase) as a negative marker for plasmid curing and determining the optimal size of the donor DNA template for gene integration in the CRISPR-FnCas12a system, we achieved highly efficient and rapid genome modification, exemplified by the successful engineering of clostridia ( Clostridium butyricum and Clostridium sporogenes ) to produce near-infrared fluorescence from biliverdin and hemin. IMPORTANCE Continued efforts in developing the CRISPR-Cas systems will further enhance our understanding and utilization of Clostridium species. This study demonstrates the development and application of a genome-engineering tool in two Clostridium strains, Clostridium butyricum and Clostridium sporogenes , which have promising potential as probiotics and oncolytic agents. Particular attention was given to the folding of precursor crRNA and the role of this process in off-target DNA cleavage by Cas12a. The results provide the guidelines necessary for efficient genome engineering using this system in clostridia. Our findings not only expand our fundamental understanding of genome-engineering tools in clostridia but also improve this technology to allow use of its full potential in a plethora of biotechnological applications.

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Section: Discussionmentioning

confidence: 99%

Development of a CRISPR-Cas12a system for efficient genome engineering in clostridia

Zhang,

Kubiak,

Bailey

et al. 2023

Microbiol Spectr

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“…The CRISPR system is broadly classified into two major classes: the class I system, which needs multiple effector proteins, while the class II system needs only single effector proteins. The class II system has either Cas9 or Cpf1 endonuclease [ 71 ]. The class II CRISPR system of Streptococcus thermophilus and Streptococcus pyogenes utilize a simple tactic using specific Cas9 nucleases dependent on the species-oriented RNA-based adaptive immunity mechanism [ 72 ].…”

Section: Genetic Manipulation For Intensified Cellulase Productionmentioning

confidence: 99%

Tailoring in fungi for next generation cellulase production with special reference to CRISPR/CAS system

Mondal

Halder

Mondal

2021

Syst Microbiol and Biomanuf

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Cellulose is the utmost plenteous source of biopolymer in our earth, and fungi are the most efficient and ubiquitous organism in degrading the cellulosic biomass by synthesizing cellulases. Tailoring through genetic manipulation has played a substantial role in constructing novel fungal strains towards improved cellulase production of desired traits. However, the traditional methods of genetic manipulation of fungi are time-consuming and tedious. With the availability of the full-genome sequences of several industrially relevant filamentous fungi, CRISPR-CAS (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein) technology has come into the focus for the proficient development of manipulated strains of filamentous fungi. This review summarizes the mode of action of cellulases, transcription level regulation for cellulase expression, various traditional strategies of genetic manipulation with CRISPR-CAS technology to develop modified fungal strains for a preferred level of cellulase production, and the futuristic trend in this arena of research.

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Utilization of Food Waste for Bioenergy Production

Rout,

Gupta,

Karunanithi

et al. 2024

Applied Biotechnology and Bioinformatics

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Emerging technologies for genetic modification of solventogenic clostridia: From tool to strategy development

Cited by 12 publications

References 112 publications

Development of a CRISPR-Cas12a system for efficient genome engineering in clostridia

Development of a CRISPR-Cas12a system for efficient genome engineering in clostridia

Tailoring in fungi for next generation cellulase production with special reference to CRISPR/CAS system

Utilization of Food Waste for Bioenergy Production

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