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

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.

scholarly journals Migratory orientation in a narrow avian hybrid zone

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e3201 ◽  
Author(s):  
David P.L. Toews ◽  
Kira E. Delmore ◽  
Matthew M. Osmond ◽  
Philip D. Taylor ◽  
Darren E. Irwin
Keyword(s):  
Hybrid Zone ◽  
Genetic Background ◽  
Migratory Restlessness ◽  
Long Distance ◽  
Mountain Valley ◽  
Migratory Orientation ◽  
Isotopic Studies ◽  
Migratory Routes ◽  
Species Specific ◽  
Migratory Divide

Background Zones of contact between closely related taxa with divergent migratory routes, termed migratory divides, have been suggested as areas where hybrid offspring may have intermediate and inferior migratory routes, resulting in low fitness of hybrids and thereby promoting speciation. In the Rocky Mountains of Canada there is a narrow hybrid zone between Audubon’s and myrtle warblers that is likely maintained by selection against hybrids. Band recoveries and isotopic studies indicate that this hybrid zone broadly corresponds to the location of a possible migratory divide, with Audubon’s warblers migrating south-southwest and myrtle warblers migrating southeast. We tested a key prediction of the migratory divide hypothesis: that genetic background would be predictive of migratory orientation among warblers in the center of the hybrid zone. Methods We recorded fall migratory orientation of wild-caught migrating warblers in the center of the hybrid zone as measured by video-based monitoring of migratory restlessness in circular orientation chambers. We then tested whether there was a relationship between migratory orientation and genetic background, as measured using a set of species-specific diagnostic genetic markers. Results We did not detect a significant association between orientation and genetic background. There was large variation among individuals in orientation direction. Mean orientation was towards the NE, surprising for birds on fall migration, but aligned with the mountain valley in which the study took place. Conclusions Only one other study has directly analyzed migratory orientation among naturally-produced hybrids in a migratory divide. While the other study showed an association between genetic background and orientation, we did not observe such an association in yellow-rumped warblers. We discuss possible reasons, including the possibility of a lack of a strong migratory divide in this hybrid zone and/or methodological limitations that may have prevented accurate measurements of long-distance migratory orientation.

CRISPR/Cas9-mediated genome editing in Brassica

2021 ◽  
pp. 403-420
Author(s):  
Cheng Dai ◽  
◽  
Xia Tian ◽  
Chaozhi Ma ◽  
◽  
...  
Keyword(s):  
Brassica Napus ◽  
Genome Editing ◽  
Plant Species ◽  
Applied Research ◽  
Biotechnological Applications ◽  
Efficient Tool ◽  
Multiple Loci ◽  
Basic And Applied Research ◽  
Genome Modifications

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.

scholarly journals GEDpm-cg: Genome Editing Automated Design Platform for Point Mutation Construction in Corynebacterium glutamicum

2021 ◽  
Vol 9 ◽  
Author(s):  
Yi Yang ◽  
Yufeng Mao ◽  
Ye Liu ◽  
Ruoyu Wang ◽  
Hui Lu ◽  
...  
Keyword(s):  
Genome Editing ◽  
Point Mutation ◽  
In Silico ◽  
Computer Aided Design ◽  
Large Scale ◽  
Point Mutations ◽  
Dna Assembly ◽  
Cell Factories ◽  
Assembly Method ◽  
Counter Selection

Advances in robotic system-assisted genome editing techniques and computer-aided design tools have significantly facilitated the development of microbial cell factories. Although multiple separate software solutions are available for vector DNA assembly, genome editing, and verification, by far there is still a lack of complete tool which can provide a one-stop service for the entire genome modification process. This makes the design of numerous genetic modifications, especially the construction of mutations that require strictly precise genetic manipulation, a laborious, time-consuming and error-prone process. Here, we developed a free online tool called GEDpm-cg for the design of genomic point mutations in C. glutamicum. The suicide plasmid-mediated counter-selection point mutation editing method and the overlap-based DNA assembly method were selected to ensure the editability of any single nucleotide at any locus in the C. glutamicum chromosome. Primers required for both DNA assembly of the vector for genetic modification and sequencing verification were provided as design results to meet all the experimental needs. An in-silico design task of over 10,000 single point mutations can be completed in 5 min. Finally, three independent point mutations were successfully constructed in C. glutamicum guided by GEDpm-cg, which confirms that the in-silico design results could accurately and seamlessly be bridged with in vivo or in vitro experiments. We believe this platform will provide a user-friendly, powerful and flexible tool for large-scale mutation analysis in the industrial workhorse C. glutamicum via robotic/software-assisted systems.

scholarly journals CRISPR/Cas12a mediated genome engineering in photosynthetic bacteria

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.

scholarly journals Genome Editing of Saccharomyces Cerevisiae Using CRISPR-Cas9 System

2021 ◽  
Vol 2 (1) ◽  
pp. 20-28
Author(s):  
Yaseen Ismael Imran ◽  
Ibrahim Abdulla Ahmed ◽  
Ahmed Ali Muhawesh
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Immune System ◽  
Genome Editing ◽  
Genetically Modified ◽  
Food Additives ◽  
Genetically Engineered ◽  
Flexible Tool ◽  
Cell Factories ◽  
Pharmaceutical Proteins ◽  
Long Time

Saccharomyces cerevisiae is an important yeast has been exploited for a long time to produce alcohol or bread. Moreover, genetically engineered S. cerevisiae cells continue to be used as cell factories for production of biofuels, pharmaceutical proteins and food additives. Genetically modified strain of S. cerevisiae created using traditional methods is laborious and time consuming. Recently, originally an immune system in archaea and bacteria, Clustered regularly interspaced short palindromic repeats “CRISPR” and CRISPR-associated “Cas” have been used exploited  as a flexible tool for genome editing. Until now, this tool has been applied to many organisms including yeast. Here, we review the importance of S. cerevisiae as an industrial platform and the use of CRISPR/Cas system and its applications in research and industry of this yeast.  

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

2020 ◽  
Vol 48 (4) ◽  
pp. 2026-2034 ◽  
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.

scholarly journals Exploring the synthetic biology potential of bacteriophages for engineering non-model bacteria

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Eveline-Marie Lammens ◽  
Pablo Ivan Nikel ◽  
Rob Lavigne
Keyword(s):  
Synthetic Biology ◽  
Pseudomonas Putida ◽  
Genome Editing ◽  
Lactococcus Lactis ◽  
Large Scale ◽  
Limited Availability ◽  
Treasure Trove ◽  
Species Specific

Abstract Non-model bacteria like Pseudomonas putida, Lactococcus lactis and other species have unique and versatile metabolisms, offering unique opportunities for Synthetic Biology (SynBio). However, key genome editing and recombineering tools require optimization and large-scale multiplexing to unlock the full SynBio potential of these bacteria. In addition, the limited availability of a set of characterized, species-specific biological parts hampers the construction of reliable genetic circuitry. Mining of currently available, diverse bacteriophages could complete the SynBio toolbox, as they constitute an unexplored treasure trove for fully adapted metabolic modulators and orthogonally-functioning parts, driven by the longstanding co-evolution between phage and host.

scholarly journals Saccharomyces Cerevisiae—An Interesting Producer of Bioactive Plant Polyphenolic Metabolites

2020 ◽  
Vol 21 (19) ◽  
pp. 7343
Author(s):  
Grzegorz Chrzanowski
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Defense Mechanisms ◽  
Chemical Diversity ◽  
Industrial Biotechnology ◽  
Host Organism ◽  
Biotechnological Applications ◽  
Phenolic Metabolites ◽  
Cell Factories ◽  
Plant Genes ◽  
Agricultural Industries

Secondary phenolic metabolites are defined as valuable natural products synthesized by different organisms that are not essential for growth and development. These compounds play an essential role in plant defense mechanisms and an important role in the pharmaceutical, cosmetics, food, and agricultural industries. Despite the vast chemical diversity of natural compounds, their content in plants is very low, and, as a consequence, this eliminates the possibility of the production of these interesting secondary metabolites from plants. Therefore, microorganisms are widely used as cell factories by industrial biotechnology, in the production of different non-native compounds. Among microorganisms commonly used in biotechnological applications, yeast are a prominent host for the diverse secondary metabolite biosynthetic pathways. Saccharomyces cerevisiae is often regarded as a better host organism for the heterologous production of phenolic compounds, particularly if the expression of different plant genes is necessary.

scholarly journals Development of a CRISPR-Cas9 System for Efficient Genome Editing of Candida lusitaniae

mSphere ◽  
2017 ◽  
Vol 2 (3) ◽  
Author(s):  
Emily L. Norton ◽  
Racquel K. Sherwood ◽  
Richard J. Bennett
Keyword(s):  
Genome Editing ◽  
Gene Targeting ◽  
Gene Deletion ◽  
Human Pathogen ◽  
Mechanistic Studies ◽  
Content Type ◽  
Dna Dsbs ◽  
Candida Lusitaniae ◽  
Species Specific ◽  
Genome Manipulation

ABSTRACT The ability to perform efficient genome editing is a key development for detailed mechanistic studies of a species. Candida lusitaniae is an important member of the Candida clade and is relevant both as an emerging human pathogen and as a model for understanding mechanisms of sexual reproduction. We highlight the development of a CRISPR-Cas9 system for efficient genome manipulation in C. lusitaniae and demonstrate the importance of species-specific promoters for expression of CRISPR components. We also demonstrate that the NHEJ pathway contributes to non-template-mediated repair of DNA DSBs and that removal of this pathway enhances efficiencies of gene targeting by CRISPR-Cas9. These results therefore establish important genetic tools for further exploration of C. lusitaniae biology. Candida lusitaniae is a member of the Candida clade that includes a diverse group of fungal species relevant to both human health and biotechnology. This species exhibits a full sexual cycle to undergo interconversion between haploid and diploid forms. C. lusitaniae is also an emerging opportunistic pathogen that can cause serious bloodstream infections in the clinic and yet has often proven to be refractory to facile genetic manipulations. In this work, we develop a clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated gene 9 (Cas9) system to enable genome editing of C. lusitaniae. We demonstrate that expression of CRISPR-Cas9 components under species-specific promoters is necessary for efficient gene targeting and can be successfully applied to multiple genes in both haploid and diploid isolates. Gene deletion efficiencies with CRISPR-Cas9 were further enhanced in C. lusitaniae strains lacking the established nonhomologous end joining (NHEJ) factors Ku70 and DNA ligase 4. These results indicate that NHEJ plays an important role in directing the repair of DNA double-strand breaks (DSBs) in C. lusitaniae and that removal of this pathway increases integration of gene deletion templates by homologous recombination. The described approaches significantly enhance the ability to perform genetic studies in, and promote understanding of, this emerging human pathogen and model sexual species. IMPORTANCE The ability to perform efficient genome editing is a key development for detailed mechanistic studies of a species. Candida lusitaniae is an important member of the Candida clade and is relevant both as an emerging human pathogen and as a model for understanding mechanisms of sexual reproduction. We highlight the development of a CRISPR-Cas9 system for efficient genome manipulation in C. lusitaniae and demonstrate the importance of species-specific promoters for expression of CRISPR components. We also demonstrate that the NHEJ pathway contributes to non-template-mediated repair of DNA DSBs and that removal of this pathway enhances efficiencies of gene targeting by CRISPR-Cas9. These results therefore establish important genetic tools for further exploration of C. lusitaniae biology.

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