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Development of a gRNA Expression and Processing Platform for Efficient CRISPR-Cas9-Based Gene Editing and Gene Silencing in Candida tropicalis.

ABSTRACT: Candida tropicalis, a nonmodel diploid microbe, has been applied in industry as a chassis cell. Metabolic engineering of C. tropicalis is challenging due to a lack of gene editing and regulation tools. Here, we report a tRNA:guide RNA (gRNA) platform for boosting gene editing and silencing efficiency in C. tropicalis. As the endogenous tRNA-processing system enables autocleavage for producing a large number of mature gRNAs, a tRNA^Gly sequence from the genome of C. tropicalis ATCC 20336 was selected for constructing the tRNA:gRNA platform. In the CRISPR-Cas9 system, the tRNA:gRNA platform proved to be efficient in single-gene and multi-gene editing. Furthermore, based on the tRNA:gRNA platform, a CRISPR interference (CRISPRi) system was developed to construct an efficient dCas9-mediated gene expression regulation system for C. tropicalis. The CRISPRi system was employed to regulate the expression of the exogenous gene GFP3 (green fluorescent protein) and the endogenous gene ADE2 (phosphoribosylaminoimidazole carboxylase). Different regions of GFP3 and ADE2 were targeted with the gRNAs processed by the tRNA^Gly, and the transcription levels of GFP3 and ADE2 were successfully downregulated to 23.9% ± 4.1% and 38.0% ± 7.4%, respectively. The effects of the target regions on gene regulation were also investigated. Additionally, the regulation system was applied to silence ERG9 (squalene synthase) to enhance β-carotene biosynthesis in a metabolically modified C. tropicalis strain. The results suggest that the endogenous tRNA^Gly and the CRISPRi system have great potential for metabolic engineering of C. tropicalis. IMPORTANCE In the nonmodel yeast Candida tropicalis, a lack of available RNA polymerase type III (Pol III) promoters hindered the development of guide RNA (gRNA) expression platforms for the establishment of CRISPR-Cas-mediated genome editing and silencing strategies. Here, a tRNA:gRNA platform was constructed. We show that this platform allows efficient and precise expression and processing of different gRNAs from a single polycistronic gene capable of mediating multi-gene editing in combination with CRISPR-Cas9. Furthermore, in combination with dCas9, the tRNA:gRNA platform was efficiently used for silencing of exogenous and endogenous genes, representing the first CRISPR interference tool (CRISPRi) in C. tropicalis. Importantly, the established CRISPRi-tRNA:gRNA tool was also used for metabolic engineering by regulating β-carotene biosynthesis in C. tropicalis. The results suggest that the tRNA:gRNA platform and the CRISPRi system will further advance the application of the CRISPR-Cas-based editing and CRISPRi systems for metabolic engineering in C. tropicalis.

SUBMITTER: Li Y

PROVIDER: S-EPMC9241840 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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 describe here an adaption of a previously developed CRISPR system in Candida parapsilosis that uses an autonomously replicating plasmid. Guide RNAs can be introduced in a single cloning step and are released by cleavage between a tRNA and a ribozyme. The plasmid also contains CAS9 and a selectable nourseothricin SAT1 marker. It can be used for markerless editing in C. parapsilosis, C. orthopsilosis, and C. metapsilosis We also show that CRISPR can easily be used to introduce molecular barcodes and to reintroduce wild-type sequences into edited strains. Heterozygous mutations can be generated, either by careful selection of the distance between the polymorphism and the Cas9 cut site or by providing two different repair templates at the same time. In addition, we have constructed a different autonomously replicating plasmid for CRISPR-Cas9 editing in Candida tropicalis We show that editing can easily be carried out in multiple C. tropicalis isolates. Nonhomologous end joining (NHEJ) repair occurs at a high level in C. metapsilosis and C. tropicalis IMPORTANCE Candida species are a major cause of infection worldwide. The species associated with infection vary with geographical location and with patient population. Infection with Candida tropicalis is particularly common in South America and Asia, and Candida parapsilosis infections are more common in the very young. Molecular methods for manipulating the genomes of these species are still lacking. We describe a simple and efficient CRISPR-based gene editing system that can be applied in the C. parapsilosis species group, including the sister species Candida orthopsilosis and Candida metapsilosis We have also constructed a separate system for gene editing in C. tropicalis.

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Development of a gRNA Expression and Processing Platform for Efficient CRISPR-Cas9-Based Gene Editing and Gene Silencing in Candida tropicalis.

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