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Universal and high-fidelity DNA single nucleotide polymorphism detection based on a CRISPR/Cas12a biochip

Single nucleotide polymorphisms (SNPs) are associated with many human diseases, so accurate and efficient SNP detection is of great significance for early diagnosis and clinical prognosis. This report proposes a universal and high-fidelity genotyping method in microfluidic point-of-care equipment ba...

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Autores principales: Chen, Yong, Mei, Yixin, Jiang, Xingyu
Formato: Online Artículo Texto
Lenguaje:English
Publicado: The Royal Society of Chemistry 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8179484/
https://www.ncbi.nlm.nih.gov/pubmed/34163711
http://dx.doi.org/10.1039/d0sc05717g
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author Chen, Yong
Mei, Yixin
Jiang, Xingyu
author_facet Chen, Yong
Mei, Yixin
Jiang, Xingyu
author_sort Chen, Yong
collection PubMed
description Single nucleotide polymorphisms (SNPs) are associated with many human diseases, so accurate and efficient SNP detection is of great significance for early diagnosis and clinical prognosis. This report proposes a universal and high-fidelity genotyping method in microfluidic point-of-care equipment based on the clustered regularly interspaced short palindromic repeat (CRISPR) system. Briefly, by systematically inserting the protospacer-adjacent-motif (PAM) sequence, we improved the universality of the CRISPR/Cas12a based SNP detection; by removing the complementary ssDNA and introducing an additional nucleotide mismatch, we improved the sensitivity and specificity. We preloaded the CRISPR/Cas12a reagents into the point-of-care biochip for automating the process, increasing the stability and long-term storage. This biochip enables us to rapidly and conveniently detect the genotypes within 20 min. In a practical application, the CRISPR/Cas12a biochip successfully distinguished three genotypes (homozygous wild type; the homozygous mutant type; and the heterozygous mutant type) of the CYP1A1*2 (A4889G, rs1048943), CYP2C19*2 (G681A, rs4244285), CYP2C9*3 (A1075C, rs1057910), and CYP2C19*3 (G636A, rs4986893) genes related to multiple cancers from 17 clinical blood samples. This CRISPR/Cas12a-based SNP genotyping method, being universal, accurate, and sensitive, will have broad applications in molecular diagnostics and clinical research.
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spelling pubmed-81794842021-06-22 Universal and high-fidelity DNA single nucleotide polymorphism detection based on a CRISPR/Cas12a biochip Chen, Yong Mei, Yixin Jiang, Xingyu Chem Sci Chemistry Single nucleotide polymorphisms (SNPs) are associated with many human diseases, so accurate and efficient SNP detection is of great significance for early diagnosis and clinical prognosis. This report proposes a universal and high-fidelity genotyping method in microfluidic point-of-care equipment based on the clustered regularly interspaced short palindromic repeat (CRISPR) system. Briefly, by systematically inserting the protospacer-adjacent-motif (PAM) sequence, we improved the universality of the CRISPR/Cas12a based SNP detection; by removing the complementary ssDNA and introducing an additional nucleotide mismatch, we improved the sensitivity and specificity. We preloaded the CRISPR/Cas12a reagents into the point-of-care biochip for automating the process, increasing the stability and long-term storage. This biochip enables us to rapidly and conveniently detect the genotypes within 20 min. In a practical application, the CRISPR/Cas12a biochip successfully distinguished three genotypes (homozygous wild type; the homozygous mutant type; and the heterozygous mutant type) of the CYP1A1*2 (A4889G, rs1048943), CYP2C19*2 (G681A, rs4244285), CYP2C9*3 (A1075C, rs1057910), and CYP2C19*3 (G636A, rs4986893) genes related to multiple cancers from 17 clinical blood samples. This CRISPR/Cas12a-based SNP genotyping method, being universal, accurate, and sensitive, will have broad applications in molecular diagnostics and clinical research. The Royal Society of Chemistry 2021-02-09 /pmc/articles/PMC8179484/ /pubmed/34163711 http://dx.doi.org/10.1039/d0sc05717g Text en This journal is © The Royal Society of Chemistry https://creativecommons.org/licenses/by-nc/3.0/
spellingShingle Chemistry
Chen, Yong
Mei, Yixin
Jiang, Xingyu
Universal and high-fidelity DNA single nucleotide polymorphism detection based on a CRISPR/Cas12a biochip
title Universal and high-fidelity DNA single nucleotide polymorphism detection based on a CRISPR/Cas12a biochip
title_full Universal and high-fidelity DNA single nucleotide polymorphism detection based on a CRISPR/Cas12a biochip
title_fullStr Universal and high-fidelity DNA single nucleotide polymorphism detection based on a CRISPR/Cas12a biochip
title_full_unstemmed Universal and high-fidelity DNA single nucleotide polymorphism detection based on a CRISPR/Cas12a biochip
title_short Universal and high-fidelity DNA single nucleotide polymorphism detection based on a CRISPR/Cas12a biochip
title_sort universal and high-fidelity dna single nucleotide polymorphism detection based on a crispr/cas12a biochip
topic Chemistry
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8179484/
https://www.ncbi.nlm.nih.gov/pubmed/34163711
http://dx.doi.org/10.1039/d0sc05717g
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