Cargando…
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...
Autores principales: | , , , , , , , , , , , , , |
---|---|
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 |
_version_ | 1783642610191040512 |
---|---|
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. |
format | Online Article Text |
id | pubmed-7835798 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
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 |
work_keys_str_mv | AT jianghua detectionofmitochondrialmutationsthroughisothermalnucleicacidamplificationcoupledwithclusteredregularlyinterspacedshortpalindromicrepeatassociatedendonucleasecas13a AT duankun detectionofmitochondrialmutationsthroughisothermalnucleicacidamplificationcoupledwithclusteredregularlyinterspacedshortpalindromicrepeatassociatedendonucleasecas13a AT hanxu detectionofmitochondrialmutationsthroughisothermalnucleicacidamplificationcoupledwithclusteredregularlyinterspacedshortpalindromicrepeatassociatedendonucleasecas13a AT wangjun detectionofmitochondrialmutationsthroughisothermalnucleicacidamplificationcoupledwithclusteredregularlyinterspacedshortpalindromicrepeatassociatedendonucleasecas13a AT liuxiao detectionofmitochondrialmutationsthroughisothermalnucleicacidamplificationcoupledwithclusteredregularlyinterspacedshortpalindromicrepeatassociatedendonucleasecas13a AT yanmaoxiao detectionofmitochondrialmutationsthroughisothermalnucleicacidamplificationcoupledwithclusteredregularlyinterspacedshortpalindromicrepeatassociatedendonucleasecas13a AT wangyunxiu detectionofmitochondrialmutationsthroughisothermalnucleicacidamplificationcoupledwithclusteredregularlyinterspacedshortpalindromicrepeatassociatedendonucleasecas13a AT liuhongyan detectionofmitochondrialmutationsthroughisothermalnucleicacidamplificationcoupledwithclusteredregularlyinterspacedshortpalindromicrepeatassociatedendonucleasecas13a AT shihuiling detectionofmitochondrialmutationsthroughisothermalnucleicacidamplificationcoupledwithclusteredregularlyinterspacedshortpalindromicrepeatassociatedendonucleasecas13a AT gaoxiaoqing detectionofmitochondrialmutationsthroughisothermalnucleicacidamplificationcoupledwithclusteredregularlyinterspacedshortpalindromicrepeatassociatedendonucleasecas13a AT ouyangchuan detectionofmitochondrialmutationsthroughisothermalnucleicacidamplificationcoupledwithclusteredregularlyinterspacedshortpalindromicrepeatassociatedendonucleasecas13a AT fuxue detectionofmitochondrialmutationsthroughisothermalnucleicacidamplificationcoupledwithclusteredregularlyinterspacedshortpalindromicrepeatassociatedendonucleasecas13a AT zhangxinxin detectionofmitochondrialmutationsthroughisothermalnucleicacidamplificationcoupledwithclusteredregularlyinterspacedshortpalindromicrepeatassociatedendonucleasecas13a AT liuchao detectionofmitochondrialmutationsthroughisothermalnucleicacidamplificationcoupledwithclusteredregularlyinterspacedshortpalindromicrepeatassociatedendonucleasecas13a |