Short homology-directed repair using optimized Cas9 in the pathogen Cryptococcus neoformans enables rapid gene deletion and tagging.
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. Rec...
| Publicado en: | Genetics Vol. 220; no. 1; pp. 1 - 13 |
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| Autores principales: | , , , , , , , |
| Formato: | pictorial research tables/charts Journal Article |
| Publicado: |
Oxford University Press / USA
Jan2022
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| Acceso en línea: | Ver este registro en EBSCOhost |
| fields | @attributes: recordID: 1 pdfLink: plink: https://search.ebscohost.com/login.aspx?direct=true&db=ccm&AN=154500179&site=ehost-live header: @attributes: shortDbName: ccm uiTerm: 154500179 longDbName: CINAHL Complete uiTag: AN controlInfo: bkinfo: dissinfo: jinfo: jid: 00166731 GNT jtl: Genetics issn: 00166731 maglogo: N pubinfo: dt: Jan2022 vid: 220 iid: 1 pid: 622 pub: Oxford University Press / USA artinfo: ui: 154500179 154500179 154500179 10.1093/genetics/iyab180 154500179 ppf: 1 ppct: 12 formats: tig: atl: Short homology-directed repair using optimized Cas9 in the pathogen Cryptococcus neoformans enables rapid gene deletion and tagging. aug: au: Huang, Manning Y. Joshi, Meenakshi B. Boucher, Michael J. Sujin Lee Loza, Liza C. Gaylord, Elizabeth A. Doering, Tamara L. Madhani, Hiten D. affil: Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, CA 94158, USA sug: subj: Cryptococcus Mutation Clustered Regularly Interspaced Short Palindromic Repeats Genetic Techniques Sequence Analysis Technology, Medical Utilization Oligonucleotide Array Sequence Analysis DNA Polymerase Chain Reaction Genetic Engineering Gene Expression CRISPR-Associated Proteins Animal Studies Mice ab: 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 (Cno) 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 (Cno CAS9 or "CnoCAS9") reliably enabled genome editing in the widely used KN99a 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. pubtype: Academic Journal doctype: pictorial research tables/charts Journal Article ougenre: Article language: English refInfo: holdings: @attributes: islocal: N |
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