CRISPR-Mediated Genome Editing in the Human Fungal Pathogen C. albicans.

Cas9-mediated genome editing is one tool investigators can use to study fungal pathogens. Such methodologies allow the investigator to examine how fungal cells differ from human cells and thus potentially identify novel therapeutic targets. In this chapter, we describe how CRISPR-mediated genome editing can be used to edit the genome of the most prevalent human fungal pathogen C. albicans. A cassette encoding a fungal optimized Cas9 nuclease and guide RNA is integrated into the C. albicans genome. The guide RNA targets Cas9 to the complementary genome sequence, and Cas9 cleaves the DNA. A repair template encoding whatever changes the investigator wished to make to the genome is co-transformed with the cassette and repairs the break via homologous recombination, thus introducing the change to the genome. The method we describe enables the researcher to edit the C. albicans genome and then efficiently remove the editing machinery and antibiotic resistance markers. This allows one to sequentially edit the C. albicans genome when multiple changes are desired. In addition, we provide notes that provide guidance on how the described protocols can be altered to meet the demands of the researcher. In these notes, we also describe the recent development of a more flexible CRISPR system that has a relaxed PAM site specificity. These and other advancements make CRISPR-mediated genome editing a practical approach when one needs to genetically alter C. albicans.

SpRY Cas9 Can Utilize a Variety of Protospacer Adjacent Motif Site Sequences To Edit the Candida albicans Genome.

Candida albicans is a human fungal pathogen capable of causing life-threatening infections. The ability to edit the C. albicans genome using CRISPR/Cas9 is an important tool investigators can leverage in their search for novel therapeutic targets. However, wild-type Cas9 requires an NGG protospacer adjacent motif (PAM), leaving many AT-rich regions of DNA inaccessible. A recently described near-PAMless CRISPR system that utilizes the SpRY Cas9 variant can target non-NGG PAM sequences. Using this system as a model, we developed C. albicans CRISPR/SpRY. We tested our system by mutating C. albicansADE2 and show that CRISPR/SpRY can utilize non-NGG PAM sequences in C. albicans Our CRISPR/SpRY system will allow researchers to efficiently modify C. albicans DNA that lacks NGG PAM sequences.IMPORTANCE Genetic modification of the human fungal pathogen Candida albicans allows us to better understand how fungi differ from humans at the molecular level and play essential roles in the development of therapeutics. CRISPR/Cas9-mediated genome editing systems can be used to introduce site-specific mutations to C. albicans However, wild-type Cas9 is limited by the requirement of an NGG PAM site. CRISPR/SpRY targets a variety of different PAM sequences. We modified the C. albicans CRISPR/Cas9 system using the CRISPR/SpRY as a guide. We tested CRISPR/SpRY on C. albicansADE2 and show that our SpRY system can facilitate genome editing independent of an NGG PAM sequence, thus allowing the investigator to target AT-rich sequences. Our system will potentially enable mutation of the 125 C. albicans genes which have been previously untargetable with CRISPR/Cas9. Additionally, our system will allow for precise targeting of many genomic locations that lack NGG PAM sites.

Stay-on-Task Exercises as a Tool To Maintain Focus during a CRISPR CURE.

Course-based undergraduate research experiences (CURE) offer the chance for students to experience authentic research investigation in a classroom setting. Such hands-on experiences afford unique opportunities work on a semi-independent research project in an efficient, structured environment. We have developed a CRISPR CURE in which undergraduate and graduate students use in silico, in vitro, and in vivo techniques to edit a fungal genome. During the development of this course, we have found that the asynchronous nature of the CRISPR CURE activities can be disruptive and lead to unproductive class time. To overcome this challenge, we have developed stay-on-task exercises (SOTEs). These short low-stakes assessments provide structured activities that are performed during these asynchronous incubation periods. SOTE activities leverage potentially unproductive class time and complement the CURE learning objectives. We have found SOTEs to be one method of maintaining classroom structure during a CURE. Furthermore, SOTE complexity, length, and subject can be easily modified to match course learning objectives.

CRISPR-mediated Genome Editing of the Human Fungal Pathogen Candida albicans.

This method describes the efficient CRISPR mediated genome editing of the diploid human fungal pathogen Candida albicans. CRISPR-mediated genome editing in C. albicans requires Cas9, guide RNA, and repair template. A plasmid expressing a yeast codon optimized Cas9 (CaCas9) has been generated. Guide sequences directly upstream from a PAM site (NGG) are cloned into the Cas9 expression vector. A repair template is then made by primer extension in vitro. Cotransformation of the repair template and vector into C. albicans leads to genome editing. Depending on the repair template used, the investigator can introduce nucleotide changes, insertions, or deletions. As C. albicans is a diploid, mutations are made in both alleles of a gene, provided that the A and B alleles do not harbor SNPs that interfere with guide targeting or repair template incorporation. Multimember gene families can be edited in parallel if suitable conserved sequences exist in all family members. The C. albicans CRISPR system described is flanked by FRT sites and encodes flippase. Upon induction of flippase, the antibiotic marker (CaCas9) and guide RNA are removed from the genome. This allows the investigator to perform subsequent edits to the genome. C. albicans CRISPR is a powerful fungal genetic engineering tool, and minor alterations to the described protocols permit the modification of other fungal species including C. glabrata, N. castellii, and S. cerevisiae.

Restriction digest screening facilitates efficient detection of site-directed mutations introduced by CRISPR in C. albicans UME6.

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 Zn2+-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.