An Efficient, Rapid, and Recyclable System for CRISPR-Mediated Genome Editing in Candida albicans

MSphere
Namkha NguyenAaron D Hernday

Abstract

Candida albicans is the most common fungal pathogen of humans. Historically, molecular genetic analysis of this important pathogen has been hampered by the lack of stable plasmids or meiotic cell division, limited selectable markers, and inefficient methods for generating gene knockouts. The recent development of clustered regularly interspaced short palindromic repeat(s) (CRISPR)-based tools for use with C. albicans has opened the door to more efficient genome editing; however, previously reported systems have specific limitations. We report the development of an optimized CRISPR-based genome editing system for use with C. albicans. Our system is highly efficient, does not require molecular cloning, does not leave permanent markers in the genome, and supports rapid, precise genome editing in C. albicans. We also demonstrate the utility of our system for generating two independent homozygous gene knockouts in a single transformation and present a method for generating homozygous wild-type gene addbacks at the native locus. Furthermore, each step of our protocol is compatible with high-throughput strain engineering approaches, thus opening the door to the generation of a complete C. albicans gene knockout library. IMPORTANCECand...Continue Reading

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Citations

Nov 2, 2017·Expert Review of Anti-infective Therapy·Marta TrevisanLuisa Barzon
Oct 25, 2017·Nature Microbiology·Rebecca S ShapiroJames J Collins
Feb 13, 2019·Current Protocols in Microbiology·Corey FrazerRichard J Bennett
Jan 17, 2020·PLoS Pathogens·Florent MorioGeraldine Butler
Apr 24, 2020·PLoS Pathogens·Virginia E Glazier, Damian J Krysan
Jul 12, 2019·PloS One·Encarnación Dueñas-SanteroCarlos R Vázquez de Aldana
Jun 4, 2020·G3 : Genes - Genomes - Genetics·Lucas R BrenesAlexander D Johnson
Aug 19, 2020·Molecular Microbiology·Edward M CulbertsonValeria C Culotta
May 28, 2019·F1000Research·Robert A Arkowitz, Martine Bassilana
Dec 7, 2019·The Journal of Biological Chemistry·Sabrina S SchatzmanValeria C Culotta
Jul 3, 2020·Frontiers in Microbiology·Stefanie WijnantsPatrick Van Dijck
Mar 15, 2019·MSphere·Lisa LombardiGeraldine Butler
Apr 4, 2019·Frontiers in Microbiology·Eugenio ManceraRichard J Bennett
Oct 19, 2019·Future Microbiology·Elvira RománJesús Pla
Aug 16, 2017·Scientific Reports·Lisa LombardiGeraldine Butler
Mar 1, 2019·Clinical Microbiology Reviews·Renáta TóthAttila Gácser
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Jan 26, 2021·Current Protocols·Manning Y HuangAaron P Mitchell
Mar 18, 2021·Virulence·Tibor NémethAttila Gacser
Apr 4, 2021·Journal of Fungi·Sanaz NargesiMohammad Taghi Hedayati
Mar 4, 2019·Trends in Genetics : TIG·Mélanie LegrandKaustuv Sanyal
Apr 20, 2018·Bio-protocol·Neta Dean, Henry Ng

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Methods Mentioned

BETA
gene knockouts
gene knockout
PCR
gene
PCRs

Software Mentioned

alb LEUpOUT
excel
Addgene
Benchling
LEUpOUT
ADD
TAG
mal LEUpOUT

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