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

Abstract

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.

Fig 1. CRISPR–Cas9 methods in Candida albicans.

The vignettes illustrate four representative methods used for CRISPR–Cas9 genetic manipulation of C. albicans. The constructs expressing Cas9 and the sgRNA and the RT are depicted. Promoter sequences are in white, unless they constitute part of the upstream homology arm for the integration in the genomic locus, in which case they have the same color as the gene targeted for integration. The transformation method is also indicated. The targeted site on YFG is indicated by a dash. The ploidy of the alleles is indicated next to the DNA helix. (A) CAS9, the sgRNA, and the NAT cassette are stably integrated at the ENO1 locus, and HDR mediates the introduction of an RT carrying a stop codon following Cas9-induced cleavage [18]. (B) Transformation of linear DNA fragments into the cell results in transient expression of Cas9 and sgRNA, and HDR results in the integration of a NAT cassette into the targeted locus [33]. A further modification of this system allows for marker recycling [34]. (C) In the LEUpOUT approach, a split-marker strategy enables the in vivo reconstitution of the selectable cassette for the expression of the CRISPR elements. Integration of the reconstituted cassette disrupts the functional LEU2 locus of a LEU2/leu2 heterozygous strain. HDR between the targeted locus and the RT following Cas9 cut results in the deletion of the gene. HDR between directed repeats flanking the cassette (depicted as black lines on yellow background) mediates the removal of CRISPR elements and the restoration of the LEU2 ORF, which can be selected on leucine dropout medium [35]. Note that the leu2 deleted allele originally present in the LEU2/leu2 heterozygous strain remains untouched during the entire procedure, and so it is not depicted in the scheme. (D) An improved version of the method depicted in (A) allows for marker recycling. A cassette expressing the CRISPR elements and containing a FLP recombinase is flanked by FRT sequences (targets of Flp) and homology arms that mediate its integration in the Neut5L locus. HDR with an RT results in the target gene deletion. Activation of the inducible promoter driving the expression of Flp is followed by excision of the cassette, leaving only an FRT sequence in the Neut5L locus [30]. CRISPR, clustered regularly interspaced short palindromic repeats; DW, downstream; HDR, homology-directed repair; NTC R, Nourseothricin resistant; NTC S, Nourseothricin susceptible; ORF, open reading frame; RT, repair template; sgRNA, single-guide RNA; UP, upstream; YFG, your favorite gene.

Fig 2. CRISPR–Cas9 methods in non-albicans Candida species.

The vignettes illustrate representative methods for implementation of CRISPR–Cas9 in non-albicans species, including C. parapsilosis sensu lato, C. tropicalis, C. glabrata, C. lusitaniae, and C. auris. The ploidy of the species is indicated next to the DNA helices. (A) Two different selectable episomal plasmids allow the scarless introduction of premature stop codons by HDR in C. parapsilosis sensu lato and C. tropicalis. The relevant species-specific promoters and ARSs are depicted on the left side (blue dot: C. parapsilosis sensu lato; red dot: C. tropicalis). The sgRNA is expressed from an RNA pol II promoter, and the release of the mature molecule is mediated by the cleavage of the upstream tRNA molecule and the downstream HDV ribozyme. The plasmids are easily lost in the absence of selection [29]. (B) A similar approach can be used for gene editing and deletion in C. glabrata. In this case, the sgRNA is expressed from the Saccharomyces cerevisiae RNA pol III SNR52 promoter [30]. (C) The use of CRISPR RNPs, in which the crRNA and tracrRNA are assembled in vitro with Cas9 and then electroporated into the cells, results in the integration of a NAT cassette into the targeted gene in C. glabrata, C. lusitaniae, and C. auris [25]. Although this method does not require species-specific adaptation of the regulatory elements necessary for the expression of the CRISPR elements, marker recycling is not possible. Ag, Ashbya gossypii; ARS, autonomously replicating sequence; Ca, Candida albicans; Cp, C. parapsilosis; CRISPR, clustered regularly interspaced short palindromic repeats; crRNA, CRISPR-RNA; DW, downstream; HDR, homology-directed repair; HDV, human hepatitis delta virus; Mg, Meyerozyma guilliermondii; RNP, RNA–Cas9 protein complex; Sc, Saccharomyces cerevisiae; sgRNA, single-guide RNA; tracrRNA, trans-activating RNA; UP, upstream; YFG, your favorite gene.

Fig 3. CRISPR–Cas9 methods in Aspergillus fumigatus, Cryptococcus neoformans and C. deneoformans.

The vignettes illustrate representative methods for CRISPR–Cas9 genetic manipulation of A. fumigatus (A–B), C. neoformans, and C. deneoformans (C–E). All the species depicted here are haploid. (A) In A. fumigatus, the CRISPR elements are integrated randomly in the genome of protoplasts and selected due to the presence of an HPH cassette. NHEJ results in indels at the cleavage site [16]. (B) MMEJ can be used to integrate an HPH cassette into a desired locus by using short homology arms. The strain is first transformed with a plasmid expressing Cas9 and containing a PYR4 marker and then with an in vitro transcribed sgRNA and the HPH cassette [45]. (C) In this “suicide” system, a cassette containing both the CRISPR elements and a URA5 marker flanked by homology arms to the target site can be transformed into an uracil auxotrophic strain of C. deneoformans. The double-strand break induced by the transiently expressed Cas9 is repaired by HDR with the URA5 cassette, and the crossover results in the release of the DNA of the CRISPR elements that induced the cleavage in the first place, which may be degraded (hence the name “suicide” system) [17]. (D) An RNP method was developed for C. neoformans and C. deneoformans, similar to the one adapted to Candida species. The cells are transformed with a mixture of two RNPs, each targeting a different end of the target gene, thus maximizing the chances of gene deletion [27]. (E) CAS9 can be integrated in the Safe Haven on C. neoformans H99 and selected due to the presence of a HYGB cassette. This strain is then be transformed with two transient plasmids expressing two sgRNAs flanked by ribozymes from the native ACT1 promoter, targeting the beginning and the end of the target gene. A NEO cassette flanked by homology arms to the target gene is used for HDR [53]. Af, Aspergillus fumigatus; Cn, Cryptococcus neoformans; CRISPR, clustered regularly interspaced short palindromic repeats; crRNA, CRISPR-RNA; HDR, homology-directed repair; HDV, human hepatitis delta virus; HH, hammerhead; MMEJ, microhomology-mediated end joining; NHEJ, nonhomologous end joining RNP, RNA–Cas9 protein complex; Sc, Saccharomyces cerevisiae; sgRNA, single-guide RNA; tracrRNA, trans-activating RNA; YFG, your favorite gene.