CRISPR/Cpf1 facilitated large fragment deletion in <i>Saccharomyces cerevisiae</i>

Li, Zhen-Hai; Liu, Min; Lyu, Xiaomei; Wang, Fengqing; Wei, Dongzhi

doi:10.1002/jobm.201800195

Cited by 15 publications

(14 citation statements)

References 15 publications

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“…The result of this work demonstrated that Cpf1 can broaden application sphere of the genome-editing toolbox available for research of S. cerevisiae [69]. Li et al firstly used the CRISPR/Cpf1 to delete large DNA fragment (the deletion of DNA fragment of ∼38 kb between the two genes of TRM10 and REX4) in S. cerevisiae, which demonstrating that the CRISPR/Cpf1 system can be used for genome simplification of S. cerevisiae, and to facilitate the laboratory evolution of the genome of S. cerevisiae [70]. Later in the year, Dank et multiple genomic defects in aroma metabolism is generated and activated to show an aroma composition comparable to wild type levels [71].…”

Section: Synthetic Genomics and The Use Of Crispr Technology In Synthetic Genomics Of S Cerevisiaementioning

confidence: 99%

Applications of CRISPR/Cas Technology to Research the Synthetic Genomics of Yeast

Lin¹,

Wang²,

Deng³

et al. 2022

Synthetic Genomics - From BioBricks to Synthetic Genomes

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The whole genome projects open the prelude to the diversity and complexity of biological genome by generating immense data. For the sake of exploring the riddle of the genome, scientists around the world have dedicated themselves in annotating for these massive data. However, searching for the exact and valuable information is like looking for a needle in a haystack. Advances in gene editing technology have allowed researchers to precisely manipulate the targeted functional genes in the genome by the state-of-the-art gene-editing tools, so as to facilitate the studies involving the fields of biology, agriculture, food industry, medicine, environment and healthcare in a more convenient way. As a sort of pioneer editing devices, the CRISPR/Cas systems having various versatile homologs and variants, now are rapidly giving impetus to the development of synthetic genomics and synthetic biology. Firstly, in the chapter, we will present the classification, structural and functional diversity of CRISPR/Cas systems. Then we will emphasize the applications in synthetic genome of yeast (Saccharomyces cerevisiae) using CRISPR/Cas technology based on year order. Finally, the summary and prospection of synthetic genomics as well as synthetic biotechnology based on CRISPR/Cas systems and their further utilizations in yeast are narrated.

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Section: Synthetic Genomics and The Use Of Crispr Technology In Synthetic Genomics Of S Cerevisiaementioning

confidence: 99%

Applications of CRISPR/Cas Technology to Research the Synthetic Genomics of Yeast

Lin¹,

Wang²,

Deng³

et al. 2022

Synthetic Genomics - From BioBricks to Synthetic Genomes

View full text Add to dashboard Cite

show abstract

“…Larger chromosomal deletions (>30 kb) and chromosome fusion have been achieved in S. cerevisiae through NHEJ repair between endpoints of the DNA breaks, which can facilitate the study of gene clusters or minimal genome research, as well as the study of replication, recombination, and segregation [70,71,72]. In principle, this may open up the possibility to generate CRISPR-Cas9-induced genome rearrangements in C. albicans, in which specific whole-chromosome aneuploidies have been associated with drug resistance and/or cross-adaptation to different drugs [73].…”

Section: Perspectives and Future Directionsmentioning

confidence: 99%

“…Study of the correlation existing between large chromosomal rearrangements and virulence and drug resistance (e.g., aneuploidy and LOH in C. albicans, aneuploidy in C. neoformans) [70][71][72] Directed evolution through CRISPRguided DNA polymerase…”

Section: S Cerevisiaementioning

confidence: 99%

The CRISPR toolbox in medical mycology: State of the art and perspectives

2020

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Fungal pathogens represent a major human threat affecting more than a billion people worldwide. Invasive infections are on the rise, which is of considerable concern because they are accompanied by an escalation of antifungal resistance. Deciphering the mechanisms underlying virulence traits and drug resistance strongly relies on genetic manipulation techniques such as generating mutant strains carrying specific mutations, or gene deletions. However, these processes have often been time-consuming and cumbersome in fungi due to a number of complications, depending on the species (e.g., diploid genomes, lack of a sexual cycle, low efficiency of transformation and/or homologous recombination, lack of cloning vectors, nonconventional codon usage, and paucity of dominant selectable markers). These issues are increasingly being addressed by applying clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 mediated genetic manipulation to medically relevant fungi. Here, we summarize the state of the art of CRISPR-Cas9 applications in four major human fungal pathogen lineages: Candida spp., Cryptococcus neoformans, Aspergillus fumigatus, and Mucorales. We highlight the different ways in which CRISPR has been customized to address the critical issues in different species, including different strategies to deliver the CRISPR-Cas9 elements, their transient or permanent expression, use of codon-optimized CAS9, and methods of marker recycling and scarless editing. Some approaches facilitate a more efficient use of homology-directed repair in fungi in which nonhomologous end joining is more commonly used to repair double-strand breaks (DSBs). Moreover, we highlight the most promising future perspectives, including gene drives, programmable base editors, and nonediting applications, some of which are currently available only in model fungi but may be adapted for future applications in pathogenic species. Finally, this review discusses how the further evolution of CRISPR technology will allow mycologists to tackle the multifaceted issue of fungal pathogenesis.

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“…However, this approach is limited by the number of available marker-based plasmid backbones (4,(8)(9)(10)(11)(12). More recently, several successful examples have shown that several gRNAs can be expressed from a single gRNA-array, using different tricks to release the mature gRNAs (3,5,6,(13)(14)(15)(16)(17). However, complexity of these gRNA expression cassettes and their tailored sequence design may be difficult to synthesize and requires laborious and time-consuming cloning steps, therefore hindering the workflow for strain construction.…”

Section: Introductionmentioning

confidence: 99%

gEL DNA, a cloning- and PCR-free method for CRISPR-based multiplexed genome editing

Randazzo

Daran

Daran‐Lapujade

2020

Preprint

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Even for the genetically accessible yeast Saccharomyces cerevisiae, the CRISPR/Cas DNA editing technology has strongly accelerated and facilitated strain construction. Several methods have been validated for fast and highly efficient single editing events and diverse approaches for multiplex genome editing have been described in literature by means of Cas9 or Cas12a endonucleases and their associated gRNAs. The gRNAs used to guide the Cas endonuclease to the editing site are typically expressed from plasmids using native PolII or PolIII RNA polymerases. These gRNA-expression plasmids require laborious, time-consuming cloning steps, which hampers their implementation for academic and applied purposes. In this study, we explore the potential of expressing gRNA from linear DNA fragments using the T7 RNA polymerase (T7RNAP) for single and multiplex genome editing in S. cerevisiae. Using Cas12a, this work demonstrates that transforming short, linear DNA fragments encoding gRNAs in yeast strains expressing T7RNAP promotes highly efficient single DNA editing. These DNA fragments can be custom-ordered, which makes this approach highly suitable for high-throughput strain construction. This work expands the CRISPR-toolbox for large-scale strain construction programs in S. cerevisiae and promises to be relevant for other, less genetically accessible yeast species.

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CRISPR/Cpf1 facilitated large fragment deletion in Saccharomyces cerevisiae

Cited by 15 publications

References 15 publications

Applications of CRISPR/Cas Technology to Research the Synthetic Genomics of Yeast

Applications of CRISPR/Cas Technology to Research the Synthetic Genomics of Yeast

The CRISPR toolbox in medical mycology: State of the art and perspectives

gEL DNA, a cloning- and PCR-free method for CRISPR-based multiplexed genome editing

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