Dramatic Improvement of CRISPR/Cas9 Editing in <named-content content-type="genus-species">Candida albicans</named-content> by Increased Single Guide RNA Expression

ABSTRACT The clustered regularly interspaced short palindromic repeat system with CRISPR-associated protein 9 nuclease (CRISPR/Cas9) has emerged as a versatile tool for genome editing in Candida albicans. Mounting evidence from other model systems suggests that the intracellular levels of single gui...

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Autores principales:	Henry Ng, Neta Dean
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2017
Materias:	CRISPR Candida albicans RFP double-strand-break repair Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/8f2e2829062f40ec87ede26aa3660356
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spelling	oai:doaj.org-article:8f2e2829062f40ec87ede26aa36603562021-11-15T15:21:46ZDramatic Improvement of CRISPR/Cas9 Editing in <named-content content-type="genus-species">Candida albicans</named-content> by Increased Single Guide RNA Expression10.1128/mSphere.00385-162379-5042https://doaj.org/article/8f2e2829062f40ec87ede26aa36603562017-04-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSphere.00385-16https://doaj.org/toc/2379-5042ABSTRACT The clustered regularly interspaced short palindromic repeat system with CRISPR-associated protein 9 nuclease (CRISPR/Cas9) has emerged as a versatile tool for genome editing in Candida albicans. Mounting evidence from other model systems suggests that the intracellular levels of single guide RNA (sgRNA) limit the efficiency of Cas9-dependent DNA cleavage. Here, we tested this idea and describe a new means of sgRNA delivery that improves previously described methods by ~10-fold. The efficiency of Cas9/sgRNA-dependent cleavage and repair of a single-copy yeast enhanced monomeric red fluorescent protein (RFP) gene was measured as a function of various parameters that are hypothesized to affect sgRNA accumulation, including transcriptional and posttranscriptional processing. We analyzed different promoters (SNR52, ADH1, and tRNA), as well as different posttranscriptional RNA processing schemes that serve to generate or stabilize mature sgRNA with precise 5′ and 3′ ends. We compared the effects of flanking sgRNA with self-cleaving ribozymes or by tRNA, which is processed by endogenous RNases. These studies demonstrated that sgRNA flanked by a 5′ tRNA and transcribed by a strong RNA polymerase II ADH1 promoter increased Cas9-dependent RFP mutations by 10-fold. Examination of double-strand-break (DSB) repair in strains hemizygous for RFP demonstrated that both homology-directed and nonhomologous end-joining pathways were used to repair breaks. Together, these results support the model that gRNA expression can be rate limiting for efficient CRISPR/Cas mutagenesis in C. albicans. IMPORTANCE Candida albicans is an important human fungal pathogen. An understanding of fungal virulence factors has been slow because C. albicans is genetically intractable. The recent development of CRISPR/Cas in C. albicans (V. K. Vyas, M. I. Barrasa, G. R. Fink, Sci Adv 1:e1500248, 2015, https://doi.org/10.1126/sciadv.1500248 ) has the potential to circumvent this problem. However, as has been found in other organisms, CRISPR/Cas mutagenesis efficiency can be frustratingly variable. Here, we systematically examined parameters hypothesized to alter sgRNA intracellular levels in order to optimize CRISPR/Cas in C. albicans. Our most important conclusion is that increased sgRNA expression and maturation dramatically improve efficiency of CRISPR/Cas mutagenesis in C. albicans by ~10-fold. Thus, we anticipate that the modifications described here will further advance the application of CRISPR/Cas for genome editing in C. albicans.Henry NgNeta DeanAmerican Society for MicrobiologyarticleCRISPRCandida albicansRFPdouble-strand-break repairMicrobiologyQR1-502ENmSphere, Vol 2, Iss 2 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	CRISPR Candida albicans RFP double-strand-break repair Microbiology QR1-502
spellingShingle	CRISPR Candida albicans RFP double-strand-break repair Microbiology QR1-502 Henry Ng Neta Dean Dramatic Improvement of CRISPR/Cas9 Editing in <named-content content-type="genus-species">Candida albicans</named-content> by Increased Single Guide RNA Expression
description	ABSTRACT The clustered regularly interspaced short palindromic repeat system with CRISPR-associated protein 9 nuclease (CRISPR/Cas9) has emerged as a versatile tool for genome editing in Candida albicans. Mounting evidence from other model systems suggests that the intracellular levels of single guide RNA (sgRNA) limit the efficiency of Cas9-dependent DNA cleavage. Here, we tested this idea and describe a new means of sgRNA delivery that improves previously described methods by ~10-fold. The efficiency of Cas9/sgRNA-dependent cleavage and repair of a single-copy yeast enhanced monomeric red fluorescent protein (RFP) gene was measured as a function of various parameters that are hypothesized to affect sgRNA accumulation, including transcriptional and posttranscriptional processing. We analyzed different promoters (SNR52, ADH1, and tRNA), as well as different posttranscriptional RNA processing schemes that serve to generate or stabilize mature sgRNA with precise 5′ and 3′ ends. We compared the effects of flanking sgRNA with self-cleaving ribozymes or by tRNA, which is processed by endogenous RNases. These studies demonstrated that sgRNA flanked by a 5′ tRNA and transcribed by a strong RNA polymerase II ADH1 promoter increased Cas9-dependent RFP mutations by 10-fold. Examination of double-strand-break (DSB) repair in strains hemizygous for RFP demonstrated that both homology-directed and nonhomologous end-joining pathways were used to repair breaks. Together, these results support the model that gRNA expression can be rate limiting for efficient CRISPR/Cas mutagenesis in C. albicans. IMPORTANCE Candida albicans is an important human fungal pathogen. An understanding of fungal virulence factors has been slow because C. albicans is genetically intractable. The recent development of CRISPR/Cas in C. albicans (V. K. Vyas, M. I. Barrasa, G. R. Fink, Sci Adv 1:e1500248, 2015, https://doi.org/10.1126/sciadv.1500248 ) has the potential to circumvent this problem. However, as has been found in other organisms, CRISPR/Cas mutagenesis efficiency can be frustratingly variable. Here, we systematically examined parameters hypothesized to alter sgRNA intracellular levels in order to optimize CRISPR/Cas in C. albicans. Our most important conclusion is that increased sgRNA expression and maturation dramatically improve efficiency of CRISPR/Cas mutagenesis in C. albicans by ~10-fold. Thus, we anticipate that the modifications described here will further advance the application of CRISPR/Cas for genome editing in C. albicans.
format	article
author	Henry Ng Neta Dean
author_facet	Henry Ng Neta Dean
author_sort	Henry Ng
title	Dramatic Improvement of CRISPR/Cas9 Editing in <named-content content-type="genus-species">Candida albicans</named-content> by Increased Single Guide RNA Expression
title_short	Dramatic Improvement of CRISPR/Cas9 Editing in <named-content content-type="genus-species">Candida albicans</named-content> by Increased Single Guide RNA Expression
title_full	Dramatic Improvement of CRISPR/Cas9 Editing in <named-content content-type="genus-species">Candida albicans</named-content> by Increased Single Guide RNA Expression
title_fullStr	Dramatic Improvement of CRISPR/Cas9 Editing in <named-content content-type="genus-species">Candida albicans</named-content> by Increased Single Guide RNA Expression
title_full_unstemmed	Dramatic Improvement of CRISPR/Cas9 Editing in <named-content content-type="genus-species">Candida albicans</named-content> by Increased Single Guide RNA Expression
title_sort	dramatic improvement of crispr/cas9 editing in <named-content content-type="genus-species">candida albicans</named-content> by increased single guide rna expression
publisher	American Society for Microbiology
publishDate	2017
url	https://doaj.org/article/8f2e2829062f40ec87ede26aa3660356
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Dramatic Improvement of CRISPR/Cas9 Editing in <named-content content-type="genus-species">Candida albicans</named-content> by Increased Single Guide RNA Expression

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