A Universal, Genomewide GuideFinder for CRISPR/Cas9 Targeting in Microbial Genomes

ABSTRACT The CRISPR/Cas system has significant potential to facilitate gene editing in a variety of bacterial species. CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) represent modifications of the CRISPR/Cas9 system utilizing a catalytically inactive Cas9 protein for transcription rep...

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Autores principales:	Michelle Spoto, Changhui Guan, Elizabeth Fleming, Julia Oh
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2020
Materias:	CRISPR Cas9 genomewide knockdown microbiome Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/9d416f30bed54a0f8705132a8fd21858
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spelling	oai:doaj.org-article:9d416f30bed54a0f8705132a8fd218582021-11-15T15:27:52ZA Universal, Genomewide GuideFinder for CRISPR/Cas9 Targeting in Microbial Genomes10.1128/mSphere.00086-202379-5042https://doaj.org/article/9d416f30bed54a0f8705132a8fd218582020-02-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSphere.00086-20https://doaj.org/toc/2379-5042ABSTRACT The CRISPR/Cas system has significant potential to facilitate gene editing in a variety of bacterial species. CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) represent modifications of the CRISPR/Cas9 system utilizing a catalytically inactive Cas9 protein for transcription repression and activation, respectively. While CRISPRi and CRISPRa have tremendous potential to systematically investigate gene function in bacteria, few programs are specifically tailored to identify guides in draft bacterial genomes genomewide. Furthermore, few programs offer open-source code with flexible design parameters for bacterial targeting. To address these limitations, we created GuideFinder, a customizable, user-friendly program that can design guides for any annotated bacterial genome. GuideFinder designs guides from NGG protospacer-adjacent motif (PAM) sites for any number of genes by the use of an annotated genome and FASTA file input by the user. Guides are filtered according to user-defined design parameters and removed if they contain any off-target matches. Iteration with lowered parameter thresholds allows the program to design guides for genes that did not produce guides with the more stringent parameters, one of several features unique to GuideFinder. GuideFinder can also identify paired guides for targeting multiplicity, whose validity we tested experimentally. GuideFinder has been tested on a variety of diverse bacterial genomes, finding guides for 95% of genes on average. Moreover, guides designed by the program are functionally useful—focusing on CRISPRi as a potential application—as demonstrated by essential gene knockdown in two staphylococcal species. Through the large-scale generation of guides, this open-access software will improve accessibility to CRISPR/Cas studies of a variety of bacterial species. IMPORTANCE With the explosion in our understanding of human and environmental microbial diversity, corresponding efforts to understand gene function in these organisms are strongly needed. CRISPR/Cas9 technology has revolutionized interrogation of gene function in a wide variety of model organisms. Efficient CRISPR guide design is required for systematic gene targeting. However, existing tools are not adapted for the broad needs of microbial targeting, which include extraordinary species and subspecies genetic diversity, the overwhelming majority of which is characterized by draft genomes. In addition, flexibility in guide design parameters is important to consider the wide range of factors that can affect guide efficacy, many of which can be species and strain specific. We designed GuideFinder, a customizable, user-friendly program that addresses the limitations of existing software and that can design guides for any annotated bacterial genome with numerous features that facilitate guide design in a wide variety of microorganisms.Michelle SpotoChanghui GuanElizabeth FlemingJulia OhAmerican Society for MicrobiologyarticleCRISPRCas9genomewide knockdownmicrobiomeMicrobiologyQR1-502ENmSphere, Vol 5, Iss 1 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	CRISPR Cas9 genomewide knockdown microbiome Microbiology QR1-502
spellingShingle	CRISPR Cas9 genomewide knockdown microbiome Microbiology QR1-502 Michelle Spoto Changhui Guan Elizabeth Fleming Julia Oh A Universal, Genomewide GuideFinder for CRISPR/Cas9 Targeting in Microbial Genomes
description	ABSTRACT The CRISPR/Cas system has significant potential to facilitate gene editing in a variety of bacterial species. CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) represent modifications of the CRISPR/Cas9 system utilizing a catalytically inactive Cas9 protein for transcription repression and activation, respectively. While CRISPRi and CRISPRa have tremendous potential to systematically investigate gene function in bacteria, few programs are specifically tailored to identify guides in draft bacterial genomes genomewide. Furthermore, few programs offer open-source code with flexible design parameters for bacterial targeting. To address these limitations, we created GuideFinder, a customizable, user-friendly program that can design guides for any annotated bacterial genome. GuideFinder designs guides from NGG protospacer-adjacent motif (PAM) sites for any number of genes by the use of an annotated genome and FASTA file input by the user. Guides are filtered according to user-defined design parameters and removed if they contain any off-target matches. Iteration with lowered parameter thresholds allows the program to design guides for genes that did not produce guides with the more stringent parameters, one of several features unique to GuideFinder. GuideFinder can also identify paired guides for targeting multiplicity, whose validity we tested experimentally. GuideFinder has been tested on a variety of diverse bacterial genomes, finding guides for 95% of genes on average. Moreover, guides designed by the program are functionally useful—focusing on CRISPRi as a potential application—as demonstrated by essential gene knockdown in two staphylococcal species. Through the large-scale generation of guides, this open-access software will improve accessibility to CRISPR/Cas studies of a variety of bacterial species. IMPORTANCE With the explosion in our understanding of human and environmental microbial diversity, corresponding efforts to understand gene function in these organisms are strongly needed. CRISPR/Cas9 technology has revolutionized interrogation of gene function in a wide variety of model organisms. Efficient CRISPR guide design is required for systematic gene targeting. However, existing tools are not adapted for the broad needs of microbial targeting, which include extraordinary species and subspecies genetic diversity, the overwhelming majority of which is characterized by draft genomes. In addition, flexibility in guide design parameters is important to consider the wide range of factors that can affect guide efficacy, many of which can be species and strain specific. We designed GuideFinder, a customizable, user-friendly program that addresses the limitations of existing software and that can design guides for any annotated bacterial genome with numerous features that facilitate guide design in a wide variety of microorganisms.
format	article
author	Michelle Spoto Changhui Guan Elizabeth Fleming Julia Oh
author_facet	Michelle Spoto Changhui Guan Elizabeth Fleming Julia Oh
author_sort	Michelle Spoto
title	A Universal, Genomewide GuideFinder for CRISPR/Cas9 Targeting in Microbial Genomes
title_short	A Universal, Genomewide GuideFinder for CRISPR/Cas9 Targeting in Microbial Genomes
title_full	A Universal, Genomewide GuideFinder for CRISPR/Cas9 Targeting in Microbial Genomes
title_fullStr	A Universal, Genomewide GuideFinder for CRISPR/Cas9 Targeting in Microbial Genomes
title_full_unstemmed	A Universal, Genomewide GuideFinder for CRISPR/Cas9 Targeting in Microbial Genomes
title_sort	universal, genomewide guidefinder for crispr/cas9 targeting in microbial genomes
publisher	American Society for Microbiology
publishDate	2020
url	https://doaj.org/article/9d416f30bed54a0f8705132a8fd21858
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A Universal, Genomewide GuideFinder for CRISPR/Cas9 Targeting in Microbial Genomes

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