CRISPR-mediated genome editing in non-conventional yeasts for biotechnological applications

CRISPR/Cas9 is a valuable tool for both basic and applied research that has been widely applied to different plant species. In this chapter, we reviewed the application of CRISPR/Cas9 genome editing toolkit in Brassica crops. We also provided a case study in Brassica napus. Collectively, our results demonstrate that CRISPR/Cas9 is an efficient tool for creating targeted genome modifications at multiple loci in B. napus. These findings open many doors for biotechnological applications in oilseed crops.

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CRISPR/Cas12a mediated genome engineering in photosynthetic bacteria

10.1101/2020.10.05.327569 ◽

2020 ◽

Author(s):

Yang Zhang ◽

Jifeng Yuan

Keyword(s):

Genome Editing ◽

Transcriptional Repression ◽

Gene Editing ◽

Photosynthetic Bacteria ◽

Genome Engineering ◽

Biotechnological Applications ◽

End Joining ◽

Editing Efficiency ◽

Francisella Novicida ◽

Non Homologous End Joining

ABSTRACTPurple non-sulfur photosynthetic bacteria (PNSB) such as R. capsulatus serve as a versatile platform for fundamental studies and various biotechnological applications. In this study, we sought to develop the class II RNA-guided CRISPR/Cas12a system from Francisella novicida for both genome editing and gene down-regulation in R. capsulatus. About 90% editing efficiency was achieved by using CRISPR/Cas12a driven by a strong promoter Ppuc when targeting ccoO or nifH gene. When both genes were simultaneously targeted, the multiplex gene editing efficiency reached >63%. In addition, CRISPR interference using deactivated Cas12a was also evaluated using reporter genes gfp and lacZ, and the repression efficiency reached >80%. In summary, our work represents the first report to develop CRISPR/Cas12a mediated genome editing/transcriptional repression in R. capsulatus, which would greatly accelerate PNSB-related researches.IMPORTANCEPurple non-sulfur photosynthetic bacteria (PNSB) such as R. capsulatus serve as a versatile platform for fundamental studies and various biotechnological applications. However, lack of efficient gene editing tools remains a main obstacle for progressing in PNSB-related researches. Here, we developed CRISPR/Cas12a for genome editing via the non-homologous end joining (NHEJ) repair machinery in R. capsulatus. In addition, DNase-deactivated Cas12a was found to simultaneously suppress multiple targeted genes. Taken together, our work offers a new set of tools for efficient genome engineering in PNSB such as R. capsulatus.

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DNA targeting by Clostridium cellulolyticum CRISPR–Cas9 Type II-C system

Nucleic Acids Research ◽

10.1093/nar/gkz1225 ◽

2020 ◽

Vol 48 (4) ◽

pp. 2026-2034 ◽

Cited By ~ 1

Author(s):

Iana Fedorova ◽

Anatolii Arseniev ◽

Polina Selkova ◽

Georgii Pobegalov ◽

Ignatiy Goryanin ◽

...

Keyword(s):

Genome Editing ◽

Streptococcus Pyogenes ◽

Genome Engineering ◽

Bacterial Species ◽

Type Ii ◽

Biotechnological Applications ◽

Clostridium Cellulolyticum ◽

Dna Targeting ◽

Biochemical Diversity

Abstract Type II CRISPR–Cas9 RNA-guided nucleases are widely used for genome engineering. Type II-A SpCas9 protein from Streptococcus pyogenes is the most investigated and highly used enzyme of its class. Nevertheless, it has some drawbacks, including a relatively big size, imperfect specificity and restriction to DNA targets flanked by an NGG PAM sequence. Cas9 orthologs from other bacterial species may provide a rich and largely untapped source of biochemical diversity, which can help to overcome the limitations of SpCas9. Here, we characterize CcCas9, a Type II-C CRISPR nuclease from Clostridium cellulolyticum H10. We show that CcCas9 is an active endonuclease of comparatively small size that recognizes a novel two-nucleotide PAM sequence. The CcCas9 can potentially broaden the existing scope of biotechnological applications of Cas9 nucleases and may be particularly advantageous for genome editing of C. cellulolyticum H10, a bacterium considered to be a promising biofuel producer.

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UPGMA - analysis of type II CRISPR RNA-guided endonuclease Cas9 homologues from the compost metagenome

E3S Web of Conferences ◽

10.1051/e3sconf/202126504010 ◽

2021 ◽

Vol 265 ◽

pp. 04010

Author(s):

Andrei A. Zimin ◽

Alexandra N. Karmanova ◽

Yinhua Lu

Keyword(s):

Genetic Diversity ◽

Genome Editing ◽

Structural Modeling ◽

Type Ii ◽

Biotechnological Applications ◽

Catalytic Processes

Metagenomic approaches provide access to the genetic diversity of the environment for biotechnological applications, allowing the discovery of new enzymes and new pathways for numerous catalytic processes. Five new putative type II CRISPR-Cas9 DNA endonucleases were identified from the compost community using the DELTA-BLAST algorithm. It was determined using phylogenetic UPGMA analysis that four of these potential enzymes are similar to those of the Bacteroidetes. Protein structural modeling confirmed the data of DELTA-BLAST and UPGMA analysis. These new five proteins found may be promising for genome editing in termoresistant Actinomyces.

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Advances and Opportunities of CRISPR/Cas Technology in Bioengineering Non-conventional Yeasts

Frontiers in Bioengineering and Biotechnology ◽

10.3389/fbioe.2021.765396 ◽

2021 ◽

Vol 9 ◽

Author(s):

Lu Shan ◽

Zongjie Dai ◽

Qinhong Wang

Keyword(s):

Genome Editing ◽

Genetic Background ◽

Biotechnological Applications ◽

Cell Factories ◽

Species Specific

Non-conventional yeasts have attracted a growing interest on account of their excellent characteristics. In recent years, the emerging of CRISPR/Cas technology has improved the efficiency and accuracy of genome editing. Utilizing the advantages of CRISPR/Cas in bioengineering of non-conventional yeasts, quite a few advancements have been made. Due to the diversity in their genetic background, the ways for building a functional CRISPR/Cas system of various species non-conventional yeasts were also species-specific. Herein, we have summarized the different strategies for optimizing CRISPR/Cas systems in different non-conventional yeasts and their biotechnological applications in the construction of cell factories. In addition, we have proposed some potential directions for broadening and improving the application of CRISPR/Cas technology in non-conventional yeasts.

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