Restriction digest screening facilitates efficient detection of site-directed mutations introduced by CRISPR in C. albicans UME6
| Autor: | Mary K. York, Ethan S. Pickerill, Antonio E. Chambers, Ben A. Evans, Kristen J.P. Buenconsejo, Douglas A. Bernstein, Olivia L. Smith |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
030103 biophysics lcsh:Medicine Computational biology Biology medicine.disease_cause Microbiology General Biochemistry Genetics and Molecular Biology 03 medical and health sciences Genome editing C. albicans medicine Genetics CRISPR Molecular Biology Mutation Site-directed mutagenesis General Neuroscience Point mutation lcsh:R General Medicine Cell Biology Stop codon Restriction digestion Restriction enzyme Restriction site 030104 developmental biology Filamentation Restriction digest General Agricultural and Biological Sciences |
| Zdroj: | PeerJ PeerJ, Vol 6, p e4920 (2018) |
| ISSN: | 2167-8359 |
| Popis: | 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 theCandida albicans UME6Zn2+-binding domain, a known regulator ofC. albicansfilamentation. 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 typeC. 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. |
| Databáze: | OpenAIRE |
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