Short homology-directed repair using optimized Cas9 in the pathogen Cryptococcus neoformans enables rapid gene deletion and tagging

Abstract

Cryptococcus neoformans, the most common cause of fungal meningitis, is a basidiomycete haploid budding yeast with a complete sexual cycle. Genome modification by homologous recombination is feasible using biolistic transformation and long homology arms, but the method is arduous and unreliable. Recently, multiple groups have reported the use of CRISPR-Cas9 as an alternative to biolistics, but long homology arms are still necessary, limiting the utility of this method. Since the S. pyogenes Cas9 derivatives used in prior studies were not optimized for expression in C. neoformans, we designed, synthesized, and tested a fully C. neoformans-optimized Cas9. We found that a Cas9 harboring only common C. neoformans codons and a consensus C. neoformans intron together with a TEF1 promoter and terminator and a nuclear localization signal (C. neoformans-optimized CAS9 or 'CnoCAS9') reliably enabled genome editing in the widely-used KN99α C. neoformans strain. Furthermore, editing was accomplished using donors harboring short (50 bp) homology arms attached to marker DNAs produced with synthetic oligonucleotides and PCR amplification. We also demonstrated that prior stable integration of CnoCAS9 further enhances both transformation and homologous recombination efficiency; importantly, this manipulation does not impact virulence in animals. We also implemented a universal tagging module harboring a codon-optimized fluorescent protein (mNeonGreen) and a tandem Calmodulin Binding Peptide-2X FLAG Tag that allows for both localization and purification studies of proteins for which the corresponding genes are modified by short homology-directed recombination. These tools enable short-homology genome engineering in C. neoformans.

CRISPR-based tools for genetic manipulation in pathogenicSporothrixspecies

“…For targeted gene disruption and deletion, previous studies have reported that flanking homologies ranging in length from 30 to 2000 bp can yield efficient homology-directed repair after DNA double-strand breaks induced by Cas9 in aspergilli, Rhizopus microspores and C. neoformans (Huang et al, 2022;Lax et al, 2021;Nodvig et al, 2018). Therefore, based on the protocols used for gene deletion in Magnaporthe oryzae and Aspergillus species (Foster et al, 2018;Peres da Silva and Brock, 2022), we developed a CRISPR/Cas9-based protocol for gene disruption in S. brasiliensis and S. schenckii.…”

Distinct pathways of adaptive evolution inCryptococcus neoformansreveal a point mutation in adenylate cyclase with drastic tradeoffs for pathogenicity

“…Primers used in this work are listed in Supplemental File 2. All genetic manipulations were done using CRISPR-mediated gene editing and the TRACE system following previously published protocols 51,52 . Briefly, gRNAs were generated using guide-specific primers and amplification from the pBHM2329 plasmid (gift from H. Madhani).…”

Development and applications of a CRISPR activation system for facile genetic overexpression in Candida albicans

“…The application of similar CRISPRa systems to other fungal organisms would lend further insight into fungal biology across this kingdom. Many CRISPR tools have been developed in a diversity of fungal organisms (Muñoz et al 2019;Cai et (Fan and Lin 2018;Wang 2018;Huang et al 2021) and Aspergillus fumigatus (Fuller et al 2015;Zhang et al 2016;Al Abdallah et al 2018;van Rhijn et al 2020).…”

“…The application of similar CRISPRa systems to other fungal organisms would lend further insight into fungal biology across this kingdom. Many CRISPR tools have been developed in a diversity of fungal organisms (17,22,(135)(136)(137)(138)(139), including numerous nonalbicans Candida species (11,(140)(141)(142)(143)(144)(145)(146), and other prominent pathogens such as Cryptococcus neoformans (147)(148)(149) and Aspergillus fumigatus (150)(151)(152)(153). These CRISPR platforms could be adapted to similar CRISPRa tools in these organisms, with many possible applications for the analysis of pathogen biology or drug target identification (49)(50)(51)(52)154).…”