Meyerozyma guilliermondii CRISPR-Cas9 vector季也蒙畢赤酵母菌株基因編輯質(zhì)粒載體

Many Candida species that cause infection have diploid genomes and do not undergo classical meiosis. The application of clustered regularly interspaced short palindromic repeat-Cas9 (CRISPR-Cas9) gene editing systems has therefore greatly facilitated the generation of gene disruptions and the introduction of specific polymorphisms. However, CRISPR methods are not yet available for all Candida species.
We first replaced the GFP gene in pAYCU268 from Defosse et al. with CAS9 from Vyas et al. , placing CAS9 under the control of the TEF1 promoter from Meyerozyma guilliermondii. pAYCU268 expresses SAT1 from the C. dubliniensis TEF1 promoter and is designed to integrate randomly into the C. tropicalis genome. We identified an autonomously replicating sequence (CaARS2) which is reported to promote replication in C. tropicalis and introduced it into the pAYCU268 backbone. Finally, a tRNA cassette similar to that in pCP-tRNA was synthesized and inserted into the plasmid, generating pCT-tRNA. Guide RNAs were then able to be cloned between the tRNA and a ribozyme and were expressed from an Ashbya gossypii TEF1 promoter, followed by the Saccharomyces cerevisiae CYC1 terminator. An important feature of this plasmid is that, apart from CaARS2, all the DNA parts can be changed thanks to the presence of specific restriction sites.

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