Rapid Hypothesis Testing in Candida albicans Clinical Isolates Using a Cloning-Free, Modular, and Recyclable System for CRISPR-Cas9 Mediated Mutant and Revertant Construction.

ABSTRACT: As increasing evidence emerges that interstrain genetic diversity among Candida albicans clinical isolates underpins phenotypic variation compared to the reference isolate SC5314, new genetic tools are required to interrogate gene function across strain backgrounds. Here, the SAT1-flipper plasmid was reengineered to contain a C. albicans codon optimized hygromycin B resistance gene (CaHygB). Cassettes were PCR-amplified from both SAT1-flipper and CaHygB-flipper plasmids using primers with homologous sequences flanking target genes of interest to serve as repair templates. Ribonucleoprotein (RNP) complexes containing proprietary CRISPR RNAs (crRNAs), universal transactivating CRISPR RNA (tracrRNA), and Cas9 protein were assembled in vitro and transformed,

SUBMITTER: Liu J 

PROVIDER: S-EPMC9241802 | biostudies-literature | 2022 Jun

REPOSITORIES: biostudies-literature