ArticleScientific reports2016
A 'suicide' CRISPR-Cas9 system to promote gene deletion and restoration by electroporation in Cryptococcus neoformans.
Article in Scientific reports, 2016. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 48 papers.
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Who cites it
48 citing papers in PubMed, 101 citations in OpenAlex.
- Advancements in CRISPR-Cas Systems for Genome Editing towards Eradication of Human Microbial Pathogens.Molecular biotechnology · 2026Review
- CRISPR/Cas9-compatible plasmids enabling seven dominant genetic selection methods for the human fungal pathogenMicrobiology spectrum · 2025Article
- Near 100% efficient homology-dependent genome engineering in the human fungal pathogen Cryptococcus neoformans.G3 (Bethesda, Md.) · 2025Article
- Article
- Temporal restriction of Cas9 expression improves CRISPR-mediated deletion efficacy and fidelity.Molecular therapy. Nucleic acids · 2024Article
- Advanced genetic techniques in fungal pathogen research.mSphere · 2024Review
- Biolistic Transformation of Cryptococcus neoformans.Methods in molecular biology (Clifton, N.J.) · 2024Article
- Article
- CRISPR-Cas9 System: A Prospective Pathway toward Combatting Antibiotic Resistance.Antibiotics (Basel, Switzerland) · 2023Review
- Advances and Challenges in CRISPR/Cas-Based Fungal Genome Engineering for Secondary Metabolite Production: A Review.Journal of fungi (Basel, Switzerland) · 2023Review
- Combating increased antifungal drug resistance inActa biochimica et biophysica Sinica · 2023Review
- Article
- Bioengineering of fungal endophytes through the CRISPR/Cas9 system.Frontiers in microbiology · 2023Review
- Cryptococcus neoformans Database in Synthetic Biology Open Language.Microbiology resource announcements · 2022Article
- Genomic Approaches to Antifungal Drug Target Identification and Validation.Annual review of microbiology · 2022Review
- Ten decadal advances in fungal biology leading towards human well-being.Fungal diversity · 2022Review
- Deletion of a Rare Fungal PKS CgPKS11 Promotes Chaetoglobosin A Biosynthesis, Yet Defers the Growth and Development ofJournal of fungi (Basel, Switzerland) · 2021Article
- Simplified All-In-One CRISPR-Cas9 Construction for Efficient Genome Editing inJournal of fungi (Basel, Switzerland) · 2021Article
- Recent Advances in Genome Editing Tools in Medical Mycology Research.Journal of fungi (Basel, Switzerland) · 2021Review
- Novel CRISPR-Cas Systems: An Updated Review of the Current Achievements, Applications, and Future Research Perspectives.International journal of molecular sciences · 2021Review
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Authors and funding
7 authors at 2 institutions in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Loss-of-function mutagenesis is an important tool used to characterize gene functions, and the CRISPR-Cas9 system is a powerful method for performing targeted mutagenesis in organisms that present low recombination frequencies, such as the serotype D strains of Cryptococcus neoformans. However, when the CRISPR-Cas9 system persists in the host cells, off-target effects and Cas9 cytotoxicity may occur, which might block subsequent genetic manipulation. Here, we report a method of spontaneously eliminating the CRISPR-Cas9 system without impairing its robust editing function. We successfully expressed single guide RNA under the driver of an endogenous U6 promoter and the human codon-optimized Cas9 endonuclease with an ACT1 promoter. This system can effectively generate an indel mutation and efficiently perform targeted gene disruption via homology-directed repair by electroporation in yeast. We then demonstrated the spontaneous elimination of the system via a cis arrangement of the CRISPR-Cas9 expression cassettes to the recombination construct. After a system-mediated double crossover, the CRISPR-Cas9 cassettes were cleaved and degraded, which was validated by Southern blotting. This 'suicide' CRISPR-Cas9 system enables the validation of gene functions by subsequent complementation and has the potential to minimize off-target effects. Thus, this technique has the potential for use in functional genomics studies of C. neoformans.
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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.