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Detection of Mitochondrial Mutations Through Isothermal Nucleic Acid Amplification Coupled With Clustered Regularly Interspaced Short Palindromic Repeat-Associated Endonuclease Cas13a

The clustered regularly interspaced short palindromic repeat (CRISPR)-associated endonuclease Cas13a can specifically bind and cleave RNA. After nucleic acid pre-amplification, bacterial Cas13a has been used to detect genetic mutations. In our study, using a transcription-mediated amplification toge...

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Autores principales: Jiang, Hua, Duan, Kun, Han, Xu, Wang, Jun, Liu, Xiao, Yan, Maoxiao, Wang, Yunxiu, Liu, Hongyan, Shi, Huiling, Gao, Xiaoqing, Ouyang, Chuan, Fu, Xue, Zhang, Xinxin, Liu, Chao
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7835798/
https://www.ncbi.nlm.nih.gov/pubmed/33510775
http://dx.doi.org/10.3389/fgene.2020.622671
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author Jiang, Hua
Duan, Kun
Han, Xu
Wang, Jun
Liu, Xiao
Yan, Maoxiao
Wang, Yunxiu
Liu, Hongyan
Shi, Huiling
Gao, Xiaoqing
Ouyang, Chuan
Fu, Xue
Zhang, Xinxin
Liu, Chao
author_facet Jiang, Hua
Duan, Kun
Han, Xu
Wang, Jun
Liu, Xiao
Yan, Maoxiao
Wang, Yunxiu
Liu, Hongyan
Shi, Huiling
Gao, Xiaoqing
Ouyang, Chuan
Fu, Xue
Zhang, Xinxin
Liu, Chao
author_sort Jiang, Hua
collection PubMed
description The clustered regularly interspaced short palindromic repeat (CRISPR)-associated endonuclease Cas13a can specifically bind and cleave RNA. After nucleic acid pre-amplification, bacterial Cas13a has been used to detect genetic mutations. In our study, using a transcription-mediated amplification together with Cas13a, we can isothermally amplify and detect mitochondrial point mutations under non-denaturing conditions from human genomic DNA. Unlike previous reports, we prepared CRISPR DNA with T7 promoter sequences and generated CRISPR RNA via transcription-mediated amplification instead of synthesizing and adding CRISPR RNA in a separate step. As a proof-of-concept, we showed that both m.1494C > T and m.1555A > G mutations were detected within 90 min. In addition, we explored various designs of CRISPR DNA to improve assay specificity, including the location and number of nucleotide mismatches, length of protospacer sequence, and different buffering conditions. We also confirmed the possibility of a “one-step single-tube” reaction for mutation detection. This assay can robustly distinguish circular DNA templates that differ by a single nucleotide. It has the potential to be adapted for automated applications, such as the screening of mitochondrial diseases.
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spelling pubmed-78357982021-01-27 Detection of Mitochondrial Mutations Through Isothermal Nucleic Acid Amplification Coupled With Clustered Regularly Interspaced Short Palindromic Repeat-Associated Endonuclease Cas13a Jiang, Hua Duan, Kun Han, Xu Wang, Jun Liu, Xiao Yan, Maoxiao Wang, Yunxiu Liu, Hongyan Shi, Huiling Gao, Xiaoqing Ouyang, Chuan Fu, Xue Zhang, Xinxin Liu, Chao Front Genet Genetics The clustered regularly interspaced short palindromic repeat (CRISPR)-associated endonuclease Cas13a can specifically bind and cleave RNA. After nucleic acid pre-amplification, bacterial Cas13a has been used to detect genetic mutations. In our study, using a transcription-mediated amplification together with Cas13a, we can isothermally amplify and detect mitochondrial point mutations under non-denaturing conditions from human genomic DNA. Unlike previous reports, we prepared CRISPR DNA with T7 promoter sequences and generated CRISPR RNA via transcription-mediated amplification instead of synthesizing and adding CRISPR RNA in a separate step. As a proof-of-concept, we showed that both m.1494C > T and m.1555A > G mutations were detected within 90 min. In addition, we explored various designs of CRISPR DNA to improve assay specificity, including the location and number of nucleotide mismatches, length of protospacer sequence, and different buffering conditions. We also confirmed the possibility of a “one-step single-tube” reaction for mutation detection. This assay can robustly distinguish circular DNA templates that differ by a single nucleotide. It has the potential to be adapted for automated applications, such as the screening of mitochondrial diseases. Frontiers Media S.A. 2021-01-12 /pmc/articles/PMC7835798/ /pubmed/33510775 http://dx.doi.org/10.3389/fgene.2020.622671 Text en Copyright © 2021 Jiang, Duan, Han, Wang, Liu, Yan, Wang, Liu, Shi, Gao, Ouyang, Fu, Zhang and Liu. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Genetics
Jiang, Hua
Duan, Kun
Han, Xu
Wang, Jun
Liu, Xiao
Yan, Maoxiao
Wang, Yunxiu
Liu, Hongyan
Shi, Huiling
Gao, Xiaoqing
Ouyang, Chuan
Fu, Xue
Zhang, Xinxin
Liu, Chao
Detection of Mitochondrial Mutations Through Isothermal Nucleic Acid Amplification Coupled With Clustered Regularly Interspaced Short Palindromic Repeat-Associated Endonuclease Cas13a
title Detection of Mitochondrial Mutations Through Isothermal Nucleic Acid Amplification Coupled With Clustered Regularly Interspaced Short Palindromic Repeat-Associated Endonuclease Cas13a
title_full Detection of Mitochondrial Mutations Through Isothermal Nucleic Acid Amplification Coupled With Clustered Regularly Interspaced Short Palindromic Repeat-Associated Endonuclease Cas13a
title_fullStr Detection of Mitochondrial Mutations Through Isothermal Nucleic Acid Amplification Coupled With Clustered Regularly Interspaced Short Palindromic Repeat-Associated Endonuclease Cas13a
title_full_unstemmed Detection of Mitochondrial Mutations Through Isothermal Nucleic Acid Amplification Coupled With Clustered Regularly Interspaced Short Palindromic Repeat-Associated Endonuclease Cas13a
title_short Detection of Mitochondrial Mutations Through Isothermal Nucleic Acid Amplification Coupled With Clustered Regularly Interspaced Short Palindromic Repeat-Associated Endonuclease Cas13a
title_sort detection of mitochondrial mutations through isothermal nucleic acid amplification coupled with clustered regularly interspaced short palindromic repeat-associated endonuclease cas13a
topic Genetics
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7835798/
https://www.ncbi.nlm.nih.gov/pubmed/33510775
http://dx.doi.org/10.3389/fgene.2020.622671
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