Simplified All-In-One CRISPR-Cas9 Construction for Efficient Genome Editing in <i>Cryptococcus</i> Species
<i>Cryptococcus neoformans</i> and <i>Cryptococcus deneoformans</i> 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....
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MDPI AG
2021-06-01
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| Series: | Journal of Fungi |
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| Online Access: | https://www.mdpi.com/2309-608X/7/7/505 |
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| author | Ping Zhang Yu Wang Chenxi Li Xiaoyu Ma Lan Ma Xudong Zhu |
| author_facet | Ping Zhang Yu Wang Chenxi Li Xiaoyu Ma Lan Ma Xudong Zhu |
| author_sort | Ping Zhang |
| collection | DOAJ |
| description | <i>Cryptococcus neoformans</i> and <i>Cryptococcus deneoformans</i> 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 <i>C. deneoformans</i>, but this has not yet been used in the <i>C. neoformans</i> 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 <i>C. neoformans</i> and <i>C. deneoformans</i> 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 <i>Cryptococcus</i> species. |
| first_indexed | 2024-03-10T10:05:35Z |
| format | Article |
| id | doaj.art-6c467d3dd67e46c89d2164a379586f46 |
| institution | Directory Open Access Journal |
| issn | 2309-608X |
| language | English |
| last_indexed | 2024-03-10T10:05:35Z |
| publishDate | 2021-06-01 |
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| series | Journal of Fungi |
| spelling | doaj.art-6c467d3dd67e46c89d2164a379586f462023-11-22T01:34:42ZengMDPI AGJournal of Fungi2309-608X2021-06-017750510.3390/jof7070505Simplified All-In-One CRISPR-Cas9 Construction for Efficient Genome Editing in <i>Cryptococcus</i> SpeciesPing Zhang0Yu Wang1Chenxi Li2Xiaoyu Ma3Lan Ma4Xudong Zhu5Beijing Key Laboratory of Genetic Engineering Drug and Biotechnology, College of Life Sciences, Beijing Normal University, Beijing 100875, ChinaCollege of Life Sciences, Jiangxi Agricultural University, Nanchang 330045, ChinaBeijing Key Laboratory of Genetic Engineering Drug and Biotechnology, College of Life Sciences, Beijing Normal University, Beijing 100875, ChinaBeijing Key Laboratory of Genetic Engineering Drug and Biotechnology, College of Life Sciences, Beijing Normal University, Beijing 100875, ChinaBeijing Key Laboratory of Genetic Engineering Drug and Biotechnology, College of Life Sciences, Beijing Normal University, Beijing 100875, ChinaBeijing Key Laboratory of Genetic Engineering Drug and Biotechnology, College of Life Sciences, Beijing Normal University, Beijing 100875, China<i>Cryptococcus neoformans</i> and <i>Cryptococcus deneoformans</i> 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 <i>C. deneoformans</i>, but this has not yet been used in the <i>C. neoformans</i> 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 <i>C. neoformans</i> and <i>C. deneoformans</i> 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 <i>Cryptococcus</i> species.https://www.mdpi.com/2309-608X/7/7/505<i>Cryptococcus neoformans</i><i>Cryptococcus deneoformans</i>CRISPR-Cas9 systemBspQI |
| spellingShingle | Ping Zhang Yu Wang Chenxi Li Xiaoyu Ma Lan Ma Xudong Zhu Simplified All-In-One CRISPR-Cas9 Construction for Efficient Genome Editing in <i>Cryptococcus</i> Species Journal of Fungi <i>Cryptococcus neoformans</i> <i>Cryptococcus deneoformans</i> CRISPR-Cas9 system BspQI |
| title | Simplified All-In-One CRISPR-Cas9 Construction for Efficient Genome Editing in <i>Cryptococcus</i> Species |
| title_full | Simplified All-In-One CRISPR-Cas9 Construction for Efficient Genome Editing in <i>Cryptococcus</i> Species |
| title_fullStr | Simplified All-In-One CRISPR-Cas9 Construction for Efficient Genome Editing in <i>Cryptococcus</i> Species |
| title_full_unstemmed | Simplified All-In-One CRISPR-Cas9 Construction for Efficient Genome Editing in <i>Cryptococcus</i> Species |
| title_short | Simplified All-In-One CRISPR-Cas9 Construction for Efficient Genome Editing in <i>Cryptococcus</i> Species |
| title_sort | simplified all in one crispr cas9 construction for efficient genome editing in i cryptococcus i species |
| topic | <i>Cryptococcus neoformans</i> <i>Cryptococcus deneoformans</i> CRISPR-Cas9 system BspQI |
| url | https://www.mdpi.com/2309-608X/7/7/505 |
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