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

Hua Jiang,Maoxiao Yan,Xiao Liu,Hongyan Liu,Xu Han,Yunxiu Wang,Chuan Ouyang,Chao Liu,Xinxin Zhang,Xue Fu,Kun Duan,Jun Wang,Xiaoqing Gao,Huiling Shi

doi:10.3389/fgene.2020.622671

Abstract

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.

Highlights

Since the invention of polymerase chain reaction (PCR) in the 1980s, nucleic acid test (NAT) has shown vast potential in the field of in vitro diagnostics
The assay was performed isothermally under non-denaturing conditions (41°C) in a single tube. We named it clustered regularly interspaced short palindromic repeat (CRISPR)-mediated nucleic acid detection (CNAD). Using both plasmid and genomic DNA, we showed that CNAD could detect target sequences or point mutations in
Transcription-mediated amplification (TMA) is an isothermal method for the amplification of RNA at 40–42°C (Figure 1A; Stary et al, 1998). It can be adapted for amplification of double-stranded DNA (dsDNA) by incorporating denaturing and annealing steps (Figure 1B; Kamisango et al, 1999)

Summary

Introduction

Since the invention of polymerase chain reaction (PCR) in the 1980s, nucleic acid test (NAT) has shown vast potential in the field of in vitro diagnostics. NAT enables the detection of genetic mutations and the clinical diagnosis of inherited diseases. Due to the limited amount of genetic materials in biological samples, NAT typically requires a nucleic acid amplification step, such as PCR (Mullis et al, 1986). PCR exponentially amplifies DNA through thermal cycling and is regularly performed in biomedical laboratories

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Results

Conclusion