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Rapid one-pot human single nucleotide polymorphism genotyping platform with Cas13a nuclease
1State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, 800 Dong-Chuan Road, Shanghai 200240, China.
Journal of Bioscience and Bioengineering
|September 20, 2024
Summary
A new CRISPR/Cas13a platform enables fast and simple genotyping of single nucleotide polymorphisms (SNPs) from saliva samples. This method improves disease detection and hereditary disease screening with high discrimination and reliability.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- Single nucleotide polymorphisms (SNPs) are key genetic factors in disease detection and hereditary disease screening.
- Existing SNP genotyping methods often require laboratory instruments and lengthy operating times, limiting their accessibility.
Purpose of the Study:
- To develop a simple, rapid, and reliable SNP genotyping platform for disease diagnosis.
- To enhance the discrimination specificity of SNP genotyping using CRISPR/Cas13a technology.
Main Methods:
- Developed a CRISPR/Cas13a-based platform integrating recombinase-aided amplification (RAA), T7 transcription, and Leptotrichia wadei Cas13a (LwaCas13a) detection.
- Introduced additional mismatches at position 2 of the guide RNA (gRNA) to improve SNP discrimination.
- Validated the platform using human saliva samples for one-pot testing within 60 minutes.
Main Results:
- Successfully genotyped 17 disease-related SNPs with improved discrimination specificity (3.0-35.1-fold) after gRNA modification.
- Demonstrated high specificity and reliability compared to traditional methods like TaqMan probe qPCR, RFLP, and AS-PCR.
- Achieved results within 60 minutes from saliva samples with reduced operational complexity and risk of cross-contamination.
Conclusions:
- The LwaCas13a-based SNP genotyping platform offers a simple, rapid, and reliable alternative for genetic analysis.
- This platform expands the application of CRISPR/Cas systems in nucleic acid detection and SNP genotyping for disease screening.
- The strategy of introducing mismatches in gRNA is effective for enhancing SNP discrimination in genotyping.
