Genome Engineering of Candida albicans

1. Service Overview

Candida albicans is a widely distributed opportunistic fungal pathogen. In terms of human health, it can cause a variety of diseases, ranging from superficial mucosal infections to severe systemic candidiasis. Systemic candidiasis is often associated with high morbidity and mortality, posing significant challenges to clinical treatment. In the field of scientific research, Candida albicans serves as an important model organism for studying fungal biology, pathogenic mechanisms, and host-pathogen interactions. Research on its gene functions helps to understand key biological processes of fungi, such as growth, reproduction, morphological transition, and drug resistance mechanisms, thereby providing a theoretical basis for the development of more effective antifungal drugs and therapeutic methods.

Based on the above research and application needs, the following genome engineering services for Candida albicans are provided:

(1) Gene Knockout Service: Enables single-gene or multi-gene knockout. Specific gRNA and donor DNA (dDNA) are designed for target genes to efficiently delete genes, laying a foundation for studying gene functions.

(2) Gene Complementation Service: Utilizes CRISPR-mediated gene complementation to accurately reintroduce wild-type genes into their native loci in mutant strains. This effectively avoids the impacts of copy number and position effects in traditional methods, ensuring accurate evaluation of gene functions.

(3) Customized Gene Editing Service: Provides personalized gene editing solutions according to specific customer needs, such as introducing point mutations, SNP exchanges, or constructing mutant strains with specific gene combinations.

2. Technical Principles

The genome engineering of Candida albicans mainly relies on the CRISPR-Cas9 system, and its specific operation process is as follows:

(1) Target Design: Professional bioinformatics tools (e.g., Design and Analyze Guides tool) are used to determine the appropriate 20bp gRNA target sequence based on the genome sequence of Candida albicans (e.g., the CA22 genome of the SC5314 strain), while ensuring that a suitable PAM sequence (e.g., NGG) exists near the target site.

(2) gRNA Expression Cassette Preparation: Two methods are mainly adopted: cloning-free overlap extension PCR assembly and single oligonucleotide circular polymerase extension cloning (soCPEC). The 20-mer gRNA target sequence is introduced via a 60-mer gRNA oligonucleotide, and assembly and transformation can be completed within one day.

(3) Donor DNA (dDNA) Preparation: For gene deletion or small modifications, annealed complementary oligonucleotides are used to synthesize dDNA fragments, with a length generally limited to less than 100nt. The upstream and downstream of the fragments contain approximately 50bp of homologous sequences flanking the target gene, respectively. For the integration of larger fragments such as gene complementation, PCR-amplified fragments are recommended, which contain approximately 500bp of upstream and downstream flanking homologous sequences.

(4) Transformation and Screening: The CRISPR components (Cas9 and gRNA expression cassettes) and linear dDNA fragments are co-transformed into Candida albicans via the lithium acetate method, and stable transformants are screened using nourseothricin. Subsequently, depending on different systems, the CRISPR components and markers are removed through screening on specific culture media.

3. Technical Flowchart

Fig.1 CRISPR-Mediated Genome Editing Strategy for Candida albicans

4. Technical Features

(1) High Efficiency: Multiple gene editing experiments have shown that high-frequency gene editing can be achieved whether using cloned gRNA cassettes or assembled gRNA cassettes. The efficiency of single-gene knockout can reach over 70%, and although the efficiency of simultaneous double-gene knockout is slightly lower (20%), it is still suitable for low-to-medium throughput applications, significantly shortening the experimental cycle.

(2) Marker-Free Editing: No permanent markers are left in the genome of the edited strain, avoiding potential impacts of marker genes on the physiological functions of the strain. This makes the research results more accurate and reliable, and the edited strain can be directly used in subsequent experiments without additional treatment.

(3) Rapid and Convenient: The optimized gRNA generation method does not require molecular cloning, greatly simplifying the experimental process. The entire process from reagent preparation to completion of transformation can be finished within one day, which significantly shortens the experimental time compared with traditional methods.

(4) Good Compatibility: The CRISPR plasmid system is compatible with various commonly used Candida albicans strains, providing users with diverse options for different research backgrounds.

(5) Standardized Process: The entire gene editing process has a standardized operation protocol, reducing the problem of unstable results caused by differences in experimental conditions and ensuring reliable experimental results in different laboratory environments.

5. Application Scenarios

(1) Basic Research on Fungal Biology: As a model organism, Candida albicans is widely used in studying the molecular mechanisms of fungal growth and reproduction, morphological transition (e.g., yeast-hypha transition), and stress response, providing insights into the evolution and physiological characteristics of fungi.

(2) Study on Fungal Pathogenic Mechanisms: By editing genes related to virulence (e.g., genes encoding adhesins or secreted proteases), researchers can explore the role of specific genes in the process of Candida albicans infecting hosts, clarifying the pathogenic mechanisms of opportunistic fungal infections.

(3) Development of Antifungal Drugs: Through genome engineering, drug-resistant related genes of Candida albicans can be modified to study the drug resistance mechanisms of fungi. This provides a theoretical basis for the development of new antifungal drugs targeting specific genes and the optimization of existing drug treatment regimens.

(4) Research on Host-Pathogen Interactions: Engineered Candida albicans strains (e.g., strains with fluorescently labeled target genes) can be used to observe the interaction process between fungi and host cells (e.g., immune cells) in real time, helping to understand the host's immune response mechanism against fungal infections.

6. Delivery Content and Standards

(1) PCR and DNA sequencing identification data of the target site (to verify the accuracy of genome editing at the target locus)

(2) 2 glycerol stocks of the engineered strain (for long-term preservation and subsequent experimental use of the strain)

(3) mRNA transcription level data (for overexpression projects, to verify the expression efficiency of the target gene after overexpression)

(4) Project final report (including detailed experimental procedures, raw data, result analysis, and conclusion summary)

7. References

[1] Nguyen N, Quail A M M F, Herndaya B A D. An Efficient, Rapid, and Recyclable System for CRISPR-Mediated Genome Editing in Candida albicans[J]. mSphere, 2017, 2(2): e00149-17. DOI: 10.1128/mSphereDirect.00149-17.