New CRISPR Mutagenesis Strategies Reveal Variation in Repair Mechanisms among Fungi
We have created new vectors for clustered regularly interspaced short palindromic repeat (CRISPR) mutagenesis in Candida albicans, Saccharomyces cerevisiae, Candida glabrata, and Naumovozyma castellii These new vectors permit a comparison of the requirements for CRISPR mutagenesis in each of these s...
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American Society for Microbiology
2018
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| Online Access: | http://hdl.handle.net/1721.1/116421 https://orcid.org/0000-0002-4172-2187 https://orcid.org/0000-0002-3872-2856 https://orcid.org/0000-0003-3704-2899 |
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| author | Vyas, Valmik K. Bushkin, G. Guy Bernstein, Douglas A. Sewastianik, Magdalena Barrasa, M. Inmaculada Getz, Matthew Aaron Bartel, David Fink, Gerald R |
| author2 | Massachusetts Institute of Technology. Department of Biology |
| author_facet | Massachusetts Institute of Technology. Department of Biology Vyas, Valmik K. Bushkin, G. Guy Bernstein, Douglas A. Sewastianik, Magdalena Barrasa, M. Inmaculada Getz, Matthew Aaron Bartel, David Fink, Gerald R |
| author_sort | Vyas, Valmik K. |
| collection | MIT |
| description | We have created new vectors for clustered regularly interspaced short palindromic repeat (CRISPR) mutagenesis in Candida albicans, Saccharomyces cerevisiae, Candida glabrata, and Naumovozyma castellii These new vectors permit a comparison of the requirements for CRISPR mutagenesis in each of these species and reveal different dependencies for repair of the Cas9 double-stranded break. Both C. albicans and S. cerevisiae rely heavily on homology-directed repair, whereas C. glabrata and N. castellii use both homology-directed and nonhomologous end-joining pathways. The high efficiency of these vectors permits the creation of unmarked deletions in each of these species and the recycling of the dominant selection marker for serial mutagenesis in prototrophs. A further refinement, represented by the "Unified" Solo vectors, incorporates Cas9, guide RNA, and repair template into a single vector, thus enabling the creation of vector libraries for pooled screens. To facilitate the design of such libraries, we have identified guide sequences for each of these species with updated guide selection algorithms.IMPORTANCE CRISPR-mediated genome engineering technologies have revolutionized genetic studies in a wide range of organisms. Here we describe new vectors and guide sequences for CRISPR mutagenesis in the important human fungal pathogens C. albicans and C. glabrata, as well as in the related yeasts S. cerevisiae and N. castellii The design of these vectors enables efficient serial mutagenesis in each of these species by leaving few, if any, exogenous sequences in the genome. In addition, we describe strategies for the creation of unmarked deletions in each of these species and vector designs that permit the creation of vector libraries for pooled screens. These tools and strategies promise to advance genetic engineering of these medically and industrially important species. |
| first_indexed | 2024-09-23T14:54:00Z |
| format | Article |
| id | mit-1721.1/116421 |
| institution | Massachusetts Institute of Technology |
| last_indexed | 2024-09-23T14:54:00Z |
| publishDate | 2018 |
| publisher | American Society for Microbiology |
| record_format | dspace |
| spelling | mit-1721.1/1164212022-09-29T11:19:35Z New CRISPR Mutagenesis Strategies Reveal Variation in Repair Mechanisms among Fungi Vyas, Valmik K. Bushkin, G. Guy Bernstein, Douglas A. Sewastianik, Magdalena Barrasa, M. Inmaculada Getz, Matthew Aaron Bartel, David Fink, Gerald R Massachusetts Institute of Technology. Department of Biology Getz, Matthew Aaron Bartel, David Fink, Gerald R We have created new vectors for clustered regularly interspaced short palindromic repeat (CRISPR) mutagenesis in Candida albicans, Saccharomyces cerevisiae, Candida glabrata, and Naumovozyma castellii These new vectors permit a comparison of the requirements for CRISPR mutagenesis in each of these species and reveal different dependencies for repair of the Cas9 double-stranded break. Both C. albicans and S. cerevisiae rely heavily on homology-directed repair, whereas C. glabrata and N. castellii use both homology-directed and nonhomologous end-joining pathways. The high efficiency of these vectors permits the creation of unmarked deletions in each of these species and the recycling of the dominant selection marker for serial mutagenesis in prototrophs. A further refinement, represented by the "Unified" Solo vectors, incorporates Cas9, guide RNA, and repair template into a single vector, thus enabling the creation of vector libraries for pooled screens. To facilitate the design of such libraries, we have identified guide sequences for each of these species with updated guide selection algorithms.IMPORTANCE CRISPR-mediated genome engineering technologies have revolutionized genetic studies in a wide range of organisms. Here we describe new vectors and guide sequences for CRISPR mutagenesis in the important human fungal pathogens C. albicans and C. glabrata, as well as in the related yeasts S. cerevisiae and N. castellii The design of these vectors enables efficient serial mutagenesis in each of these species by leaving few, if any, exogenous sequences in the genome. In addition, we describe strategies for the creation of unmarked deletions in each of these species and vector designs that permit the creation of vector libraries for pooled screens. These tools and strategies promise to advance genetic engineering of these medically and industrially important species. National Institutes of Health (U.S.) (Grant GM035010) National Institutes of Health (U.S.) (Grant GM118135) National Institutes of Health (U.S.) (Grant R15AI130950) 2018-06-19T18:11:34Z 2018-06-19T18:11:34Z 2018-04 2018-03 2018-06-15T18:44:43Z Article http://purl.org/eprint/type/JournalArticle 2379-5042 http://hdl.handle.net/1721.1/116421 Vyas, Valmik K. et al. “New CRISPR Mutagenesis Strategies Reveal Variation in Repair Mechanisms Among Fungi.” Edited by Aaron P. Mitchell. mSphere 3, 2 (April 2018): e00154–18 © 2018 Vyas et al https://orcid.org/0000-0002-4172-2187 https://orcid.org/0000-0002-3872-2856 https://orcid.org/0000-0003-3704-2899 http://dx.doi.org/10.1128/mSphere.00154-18 mSphere Creative Commons Attribution 4.0 International License http://creativecommons.org/licenses/by/4.0/ application/pdf American Society for Microbiology American Society for Microbiology |
| spellingShingle | Vyas, Valmik K. Bushkin, G. Guy Bernstein, Douglas A. Sewastianik, Magdalena Barrasa, M. Inmaculada Getz, Matthew Aaron Bartel, David Fink, Gerald R New CRISPR Mutagenesis Strategies Reveal Variation in Repair Mechanisms among Fungi |
| title | New CRISPR Mutagenesis Strategies Reveal Variation in Repair Mechanisms among Fungi |
| title_full | New CRISPR Mutagenesis Strategies Reveal Variation in Repair Mechanisms among Fungi |
| title_fullStr | New CRISPR Mutagenesis Strategies Reveal Variation in Repair Mechanisms among Fungi |
| title_full_unstemmed | New CRISPR Mutagenesis Strategies Reveal Variation in Repair Mechanisms among Fungi |
| title_short | New CRISPR Mutagenesis Strategies Reveal Variation in Repair Mechanisms among Fungi |
| title_sort | new crispr mutagenesis strategies reveal variation in repair mechanisms among fungi |
| url | http://hdl.handle.net/1721.1/116421 https://orcid.org/0000-0002-4172-2187 https://orcid.org/0000-0002-3872-2856 https://orcid.org/0000-0003-3704-2899 |
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