A
            <i>Candida albicans</i>
            CRISPR system permits genetic engineering of essential genes and gene families

Vyas, Valmik K.; Barrasa, M. Inmaculada; Fink, Gerald R.

doi:10.1126/sciadv.1500248

Cited by 312 publications

(395 citation statements)

References 29 publications

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“…Ume6 guide RNA primers (Ume6 Guide 2 fr and rv) were subcloned into pv1093 as previously described (Vyas et al 2015). Repair templates were made by PCR using indicated primer pairs.…”

Section: Methodsmentioning

confidence: 99%

“…Furthermore, plasmids are not maintained consistently in C. albicans. The development of the Candida CRISPR system however has made introduction of mutations into the genome more efficient (Vyas et al 2015;Vyas et al 2018). During CRISPR-mediated genome editing, Cas9 nuclease bound to a guide RNA molecule targets a specific genomic sequence to be modified using base pairing.…”

Section: Introductionmentioning

confidence: 99%

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Peer Review #1 of "Restriction digest screening facilitates efficient detection of site-directed mutations introduced by CRISPR in C. albicans UME6 (v0.1)"

2018

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Introduction of point mutations to a gene of interest is a powerful tool when determining protein function. CRISPR-mediated genome editing allows for more efficient transfer of a desired mutation into a wide range of model organisms. Traditionally, PCR amplification and DNA sequencing is used to determine if isolates contain the intended mutation.However, mutation efficiency is highly variable, potentially making sequencing costly and time consuming. To more efficiently screen for correct transformants, we have identified restriction enzymes sites that encode for two identical amino acids or one or two stop codons. We used CRISPR to introduce these restriction sites directly upstream of the Candida albicans UME6 Zn 2+ -binding domain, a known regulator of C. albicans filamentation. While repair templates coding for different restriction sites were not equally successful at introducing mutations, restriction digest screening enabled us to rapidly identify isolates with the intended mutation in a cost-efficient manner. In addition, mutated isolates have clear defects in filamentation and virulence compared to wild type C. albicans. Our data suggest restriction digestion screening efficiently identifies point mutations introduced by CRISPR and streamlines the process of identifying residues important for a phenotype of interest. Manuscript to be reviewedIntroduction of point mutations to a gene of interest is a powerful tool when determining protein function. CRISPR-mediated genome editing allows for more efficient transfer of a desired mutation into a wide range of model organisms. Traditionally, PCR amplification and DNA sequencing is used to determine if isolates contain the intended mutation. However, mutation efficiency is highly variable, potentially making sequencing costly and time consuming. To more efficiently screen for correct transformants, we have identified restriction enzymes sites that encode for two identical amino acids or one or two stop codons. We used CRISPR to introduce these restriction sites directly upstream of the Candida albicans UME6 Zn 2+ -binding domain, a known regulator of C. albicans filamentation. While repair templates coding for different restriction sites were not equally successful at introducing mutations, restriction digest screening enabled us to rapidly identify isolates with the intended mutation in a cost-efficient manner. In addition, mutated isolates have clear defects in filamentation and virulence compared to wild type C. albicans. Our data suggest restriction digestion screening efficiently identifies point mutations introduced by CRISPR and streamlines the process of identifying residues important for a phenotype of interest.

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“…Ume6 guide RNA primers (Ume6 Guide 2 fr and rv) were subcloned into pv1093 as previously described (Vyas et al 2015). Repair templates were made by PCR using indicated primer pairs.…”

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Peer Review #1 of "Restriction digest screening facilitates efficient detection of site-directed mutations introduced by CRISPR in C. albicans UME6 (v0.1)"

2018

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“…Now CRISPR is making it possible to edit genes in many more organisms. In April, for example, researchers at the Whitehead Institute for Biomedical Research in Cambridge, Massachusetts, reported using CRISPR to study Candida albicans, a fungus that is particularly deadly in people with weakened immune systems, but had been difficult to genetically manipulate in the lab 3 . Jennifer Doudna, a CRISPR pioneer at the University of California, Berkeley, is keeping a list of CRISPR-altered creatures.…”

Section: Research Revolutionmentioning

confidence: 99%

CRISPR, the disruptor

Ledford¹

2015

Nature

320

189

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“…These features make the CRISPR/Cas9 system quite simple and easy to design, as well as being highly effective, as proven in human [38] and mouse [39] cells. Following successful site-directed mutagenesis in mammalian cells, the CRISPR/Cas9 system has been used widely in a variety organisms from bacteria to higher eukaryotes: Candida albicans [41], Caenorhabditis elegans [42], fruit fly [43], zebrafish [44], rodents [45], and cattle [46].…”

Section: Molecular Breeding: Genomics and Genetic Engineering In Woodmentioning

confidence: 99%

Genome engineering of woody plants: past, present and future

2016

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Engineered endonucleases that digest the specific sequences can be used to modify target genomes precisely. This is called ''genome editing'' and is used widely to modify the genome of various organisms. The DNA-binding domains of zinc finger (ZF) proteins were the first to be used as a genome editing tool, in the form of designed ZF nucleases and, more recently, transcription activator-like effector as well as the clustered, regularly interspaced, short palindromic repeats and CRISPR-associated protein 9 (CRISPR/Cas9) system that targets RNA-DNA rather than protein-DNA interactions have been used successfully. These are powerful tools with which targeted gene modifications can be introduced in various organisms, including various plant species. A key step in genome editing is the generation of a double-stranded DNA break that is specific to the target gene. This is achieved using custom-designed endonucleases, which enable site-directed mutagenesis via a non-homologous end joining repair pathway and/or gene targeting via homologous recombination to occur efficiently at specific sites in the genome. This review provides an overview of the current status of genomics and genetic engineering of woody plants, and recent advances in genome editing technologies in plants as well as fungi. We also discuss how these strategies can provide insights into molecular breeding technology for woody plants.

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A Candida albicans CRISPR system permits genetic engineering of essential genes and gene families

Cited by 312 publications

References 29 publications

Peer Review #1 of "Restriction digest screening facilitates efficient detection of site-directed mutations introduced by CRISPR in C. albicans UME6 (v0.1)"

Peer Review #1 of "Restriction digest screening facilitates efficient detection of site-directed mutations introduced by CRISPR in C. albicans UME6 (v0.1)"

CRISPR, the disruptor

Genome engineering of woody plants: past, present and future

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