Method for CRISPR/Cas9 Mutagenesis in Candida albicans.

  title={Method for CRISPR/Cas9 Mutagenesis in Candida albicans.},
  author={Neta Dean and Henry Ng},
  volume={8 8},
Candida albicans is the most prevalent and important human fungal pathogen. The advent of CRISPR as a means of gene editing has greatly facilitated genetic analysis in C. albicans. Here, we describe a detailed step-by-step procedure to construct and analyze C. albicans deletion mutants. This protocol uses plasmids that allow simple ligation of synthetic duplex 23mer guide oligodeoxynucleotides for high copy gRNA expression in C. albicans strains that express codon-optimized Cas9. This protocol… 

Figures from this paper


Candida albicans Gene Deletion with a Transient CRISPR-Cas9 System
It is shown here that the CRISPR-Cas9 genetic elements can function transiently, without stable integration into the genome, to enable the introduction of a gene deletion construct.
Dramatic Improvement of CRISPR/Cas9 Editing in Candida albicans by Increased Single Guide RNA Expression
The model that gRNA expression can be rate limiting for efficient CRISPR/Cas mutagenesis in C. albicans is supported, and a new means of sgRNA delivery that improves previously described methods by ~10-fold is described.
Rapid Hypothesis Testing with Candida albicans through Gene Disruption with Short Homology Regions
It is shown that three independent hrm101/hrm101 mutants and two independent enx3/enx3 mutants are defective in filamentation on Spider medium, arguing that HRM101 and ENX3 sequences are indeed portions of genes and that the respective gene products have related functions.
A recyclable Candida albicans URA3 cassette for PCR product‐directed gene disruptions
A gene disruption cassette with 200 bp flanking repeats that permit efficient PCR amplification of the entire cassette is described and used to create rim101::dpl200/rim101::URA3‐dpl 200 mutants exclusively through PCR product‐directed disruption.
Multiplex Genome Engineering Using CRISPR/Cas Systems
Two different type II CRISPR/Cas systems are engineered and it is demonstrated that Cas9 nucleases can be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells, demonstrating easy programmability and wide applicability of the RNA-guided nuclease technology.
RNA-Guided Human Genome Engineering via Cas9
The type II bacterial CRISPR system is engineer to function with custom guide RNA (gRNA) in human cells to establish an RNA-guided editing tool for facile, robust, and multiplexable human genome engineering.
A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity
This study reveals a family of endonucleases that use dual-RNAs for site-specific DNA cleavage and highlights the potential to exploit the system for RNA-programmable genome editing.
CHOPCHOP v2: a web tool for the next generation of CRISPR genome engineering
This major update of CHOPCHOP introduces tools for the next generation of CRISPR advances, including Cpf1 and Cas9 nickases, and provides support for custom length sgRNAs and evaluates the sequence composition of the whole sgRNA and its surrounding region using models compiled from multiple large-scale studies.
Optimizing sgRNA position markedly improves the efficiency of CRISPR/dCas9-mediated transcriptional repression
This work provides a framework for efficient CRISPRi assay design based on functionally defined TSSs and features of the target site chromatin, with high efficiency focused in the regions of open chromatin.
Cas9–crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria
It is demonstrated that the Cas9–crRNA complex of the Streptococcus thermophilus CRISPR3/Cas system introduces in vitro a double-strand break at a specific site in DNA containing a sequence complementary to crRNA, paving the way for engineering of universal programmable RNA-guided DNA endonucleases.