Restriction digest screening facilitates efficient detection of site-directed mutations introduced by CRISPR in C. albicans UME6 .
| Autor: | Evans BA; Department of Biology, Ball State University, Muncie, IN, United States of America., Smith OL; Department of Biology, Ball State University, Muncie, IN, United States of America., Pickerill ES; Department of Biology, Ball State University, Muncie, IN, United States of America., York MK; Department of Biology, Ball State University, Muncie, IN, United States of America., Buenconsejo KJP; Department of Microbiology and Immunology, Drexel University, Philadelphia, PA, United States of America., Chambers AE; Department of Biology, Ball State University, Muncie, IN, United States of America., Bernstein DA; Department of Biology, Ball State University, Muncie, IN, United States of America. |
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| Jazyk: | angličtina |
| Zdroj: | PeerJ [PeerJ] 2018 Jun 06; Vol. 6, pp. e4920. Date of Electronic Publication: 2018 Jun 06 (Print Publication: 2018). |
| DOI: | 10.7717/peerj.4920 |
| Abstrakt: | Introduction of point mutations to a gene of interest is a powerful tool when determining protein function. CRISPR-mediated genome editing allows for more efficient transfer of a desired mutation into a wide range of model organisms. Traditionally, PCR amplification and DNA sequencing is used to determine if isolates contain the intended mutation. However, mutation efficiency is highly variable, potentially making sequencing costly and time consuming. To more efficiently screen for correct transformants, we have identified restriction enzymes sites that encode for two identical amino acids or one or two stop codons. We used CRISPR to introduce these restriction sites directly upstream of the Candida albicans UME6 Zn 2+ -binding domain, a known regulator of C. albicans filamentation. While repair templates coding for different restriction sites were not equally successful at introducing mutations, restriction digest screening enabled us to rapidly identify isolates with the intended mutation in a cost-efficient manner. In addition, mutated isolates have clear defects in filamentation and virulence compared to wild type C. albicans . Our data suggest restriction digestion screening efficiently identifies point mutations introduced by CRISPR and streamlines the process of identifying residues important for a phenotype of interest. Competing Interests: The authors declare there are no competing interests. |
| Databáze: | MEDLINE |
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