The document describes a new CRISPR system that allows for efficient genetic engineering of Candida albicans, an important human fungal pathogen. Key features of the CRISPR system include a Candida-compatible Cas9 nuclease and synthetic guide RNAs. The system permits homozygous mutations, mutations in gene families with multiple copies, and mutations in essential genes. Using this system, the researchers were able to simultaneously mutate three drug efflux pump genes and observe defects in filamentous growth and cold sensitivity from a mutation in an essential SNF1 gene. The CRISPR system provides a powerful new tool for characterizing clinically relevant drug-resistant C. albicans strains and developing new antifungal therapies.
1. A Candida albicans CRISPR system permits genetic
engineering of essential genes and gene families
Valmik K. Vyas, M. Inmaculada Barrasa, Gerald R. Fink
Francesca Savarese
2. Introduction
• Candida albicans: the main fungal pathogen of humans.
• Responsible for mucosal (oral and genital) and systematic infections => mortality in
immunocompromised individuals (e.g. HIV-infected patients)
• Analysis of C.albicans pathogenesis through genetic engineering : why such an issue?
Diploidy (recessive mutations => knock out of both alleles to obtain a phenotype)
No meiosis (loss-of-function mutations in essential genes => identified in gametes)
No plasmid system (functional copy of the gene=> loss-of-function mutations maintained in mutants)
Presence of gene families* (difficulty in obtaining multiple knockouts)
*more than 120 drug efflux pumps
Variation in chromosome number (difficulty in obtaining homozygous recessive mutations)
• C. albicans CRISPR system: efficient mutagenesis system (multiple genes, gene families, gene encoding
vital functions).
Candida-compatible Cas9 nuclease (CaCas9)
sgRNA (synthetic guide RNA)
3. Genome editing: CRISPR Cas9 system
HDR (homology directed repair)
*homozygous mutation (nonsense=knockout)Repair template created with 60-bp
oligonucleotide primers with 20-bp
overlap at its 3’ end centered on the
desired mutation point. Extended
with Ex Taq DNA Polymerase.
Guide (protospacer) designed to be
adjacent to or within 15 bp of the
desided mutagenesis point.
4. Duet system vs Solo system
Target gene: ADE2
Duet system
Solo System
NAT: nourseothricin resistance-gene used as resistance marker
SNR52p: RNA polymerase III promoter expressing sgRNA
ENO1p: constitutive promoter expressing Cas9
Note: Solo and Duet vectors linearized by digestion with Kpn1 and Sac1 to facilitate the integration
Transformations performed with lithium acetate method
5. Duet system vs Solo system
Target gene: ADE2
Mutants ade2/ade2=> red phenotype
Note: Duet system produced 20 to 40 % red colonies among
the transformants. Solo system (shown above) produced 60
to 80 % of the transformants.
* CRISPR - induced mutations : stop codon
(UAA) + mutation in PAM sequence
6. Gene families mutations
Target genes: CDR1 and CDR2 (members of the multigene drug efflux pump
encoding family)
Simultaneous mutation of three
genes in a single transformation
(Solo CDR system + ADE2 Duet
system)
Stop codon + restriction site
Fluconazole-resistance
7. Essential genes mutations
Target gene: SNF1
snf1-K81R: mutants with Lys81
instead of arginine in the ATP-
binding pocket.
Defective filamentous growth
Cold sensitivity
8. Discussion
Efficacy of CRISPR System:
More than 98% of the genes can be modified by the CRISPR system.
Mutations of both copies of a gene, several copies of a multigene
family (using a single guide) and multiple genes in a single
transformation.
High frequency of CRISPR-induced mutations: identification of
essential genes.
Discrimination among the functions of an essential gene.
9. Benefits of using CRISPR System:
Characterisation of clinical isolates of drug-resistant strains of
Candida albicans.
The development of new, more effective antifungal drugs.
Discussion
