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 

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

JOURNAL=Frontiers in Genetics

VOLUME=Volume 11 - 2020

YEAR=2021

URL=https://www.frontiersin.org/journals/genetics/articles/10.3389/fgene.2020.622671

DOI=10.3389/fgene.2020.622671

ISSN=1664-8021

ABSTRACT=The Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) associated endonuclease Cas13a can specifically bind and cleave RNA. Following nucleic acid pre-amplification, bacterial Cas13a has been used to detect genetic mutations. In our study, using transcription-mediated amplification (TMA) 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 (crDNA) with T7 promoter sequences and generated crRNA via TMA, instead of synthesizing and adding crRNA in a separate step. As a proof-of-concept, we showed that both m.1494 C>T and m.1555 A>G mutations were detected within 90 minutes. In addition, we explored various designs of crDNA 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 “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 screening of mitochondrial diseases.