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Short homology-directed repair using optimized Cas9 in the pathogen Cryptococcus neoformansenables rapid gene deletion and tagging
- Source :
- Genetics; January 2022, Vol. 220 Issue: 1
- Publication Year :
- 2022
-
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. pyogenesCas9 derivatives used in prior studies were not optimized for expression in C. neoformans, we designed, synthesized, and tested a fully C. neoformans-optimized (Cno) Cas9. We found that a Cas9 harboring only common C. neoformanscodons and a consensus C. neoformansintron together with a TEF1promoter and terminator and a nuclear localization signal (Cno CAS9or “CnoCAS9”) reliably enabled genome editing in the widely used KN99α C. neoformansstrain. 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 CnoCAS9further 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.
Details
- Language :
- English
- ISSN :
- 00166731 and 19432631
- Volume :
- 220
- Issue :
- 1
- Database :
- Supplemental Index
- Journal :
- Genetics
- Publication Type :
- Periodical
- Accession number :
- ejs60777945
- Full Text :
- https://doi.org/10.1093/genetics/iyab180