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Simplified All-In-One CRISPR-Cas9 Construction for Efficient Genome Editing in Cryptococcus Species
Cryptococcus neoformans and Cryptococcus deneoformans are opportunistic fungal pathogens found worldwide that are utilized to reveal mechanisms of fungal pathogenesis. However, their low homologous recombination frequency has greatly encumbered genetic studies. In preliminary work, we described a ‘s...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8303259/ https://www.ncbi.nlm.nih.gov/pubmed/34202664 http://dx.doi.org/10.3390/jof7070505 |
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author | Zhang, Ping Wang, Yu Li, Chenxi Ma, Xiaoyu Ma, Lan Zhu, Xudong |
author_facet | Zhang, Ping Wang, Yu Li, Chenxi Ma, Xiaoyu Ma, Lan Zhu, Xudong |
author_sort | Zhang, Ping |
collection | PubMed |
description | Cryptococcus neoformans and Cryptococcus deneoformans are opportunistic fungal pathogens found worldwide that are utilized to reveal mechanisms of fungal pathogenesis. However, their low homologous recombination frequency has greatly encumbered genetic studies. In preliminary work, we described a ‘suicide’ CRISPR-Cas9 system for use in the efficient gene editing of C. deneoformans, but this has not yet been used in the C. neoformans strain. The procedures involved in constructing vectors are time-consuming, whether they involve restriction enzyme-based cloning of donor DNA or the introduction of a target sequence into the gRNA expression cassette via overlap PCR, as are sophisticated, thus impeding their widespread application. Here, we report the optimized and simplified construction method for all-in-one CRISPR-Cas9 vectors that can be used in C. neoformans and C. deneoformans strains respectively, named pNK003 (Genbank: MW938321) and pRH003 (Genbank: KX977486). Taking several gene manipulations as examples, we also demonstrate the accuracy and efficiency of the new simplified all-in-one CRISPR-Cas9 genome editing tools in both Serotype A and Serotype D strains, as well as their ability to eliminate Cas9 and gDNA cassettes after gene editing. We anticipate that the availability of new vectors that can simplify and streamline the technical steps for all-in-one CRISPR-Cas9 construction could accelerate genetic studies of the Cryptococcus species. |
format | Online Article Text |
id | pubmed-8303259 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-83032592021-07-25 Simplified All-In-One CRISPR-Cas9 Construction for Efficient Genome Editing in Cryptococcus Species Zhang, Ping Wang, Yu Li, Chenxi Ma, Xiaoyu Ma, Lan Zhu, Xudong J Fungi (Basel) Article Cryptococcus neoformans and Cryptococcus deneoformans are opportunistic fungal pathogens found worldwide that are utilized to reveal mechanisms of fungal pathogenesis. However, their low homologous recombination frequency has greatly encumbered genetic studies. In preliminary work, we described a ‘suicide’ CRISPR-Cas9 system for use in the efficient gene editing of C. deneoformans, but this has not yet been used in the C. neoformans strain. The procedures involved in constructing vectors are time-consuming, whether they involve restriction enzyme-based cloning of donor DNA or the introduction of a target sequence into the gRNA expression cassette via overlap PCR, as are sophisticated, thus impeding their widespread application. Here, we report the optimized and simplified construction method for all-in-one CRISPR-Cas9 vectors that can be used in C. neoformans and C. deneoformans strains respectively, named pNK003 (Genbank: MW938321) and pRH003 (Genbank: KX977486). Taking several gene manipulations as examples, we also demonstrate the accuracy and efficiency of the new simplified all-in-one CRISPR-Cas9 genome editing tools in both Serotype A and Serotype D strains, as well as their ability to eliminate Cas9 and gDNA cassettes after gene editing. We anticipate that the availability of new vectors that can simplify and streamline the technical steps for all-in-one CRISPR-Cas9 construction could accelerate genetic studies of the Cryptococcus species. MDPI 2021-06-24 /pmc/articles/PMC8303259/ /pubmed/34202664 http://dx.doi.org/10.3390/jof7070505 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Zhang, Ping Wang, Yu Li, Chenxi Ma, Xiaoyu Ma, Lan Zhu, Xudong Simplified All-In-One CRISPR-Cas9 Construction for Efficient Genome Editing in Cryptococcus Species |
title | Simplified All-In-One CRISPR-Cas9 Construction for Efficient Genome Editing in Cryptococcus Species |
title_full | Simplified All-In-One CRISPR-Cas9 Construction for Efficient Genome Editing in Cryptococcus Species |
title_fullStr | Simplified All-In-One CRISPR-Cas9 Construction for Efficient Genome Editing in Cryptococcus Species |
title_full_unstemmed | Simplified All-In-One CRISPR-Cas9 Construction for Efficient Genome Editing in Cryptococcus Species |
title_short | Simplified All-In-One CRISPR-Cas9 Construction for Efficient Genome Editing in Cryptococcus Species |
title_sort | simplified all-in-one crispr-cas9 construction for efficient genome editing in cryptococcus species |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8303259/ https://www.ncbi.nlm.nih.gov/pubmed/34202664 http://dx.doi.org/10.3390/jof7070505 |
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