Two CRISPR/Cas12a-based methods for fast and accurate detection of single-base mutations

作者全名："Ling, Chao; Chang, Yanbin; Wang, Xingyue; Cao, Xiaoying; Tu, Qianrong; Liu, Bo; Huang, Shifeng"

作者地址："[Ling, Chao; Chang, Yanbin; Wang, Xingyue; Tu, Qianrong; Huang, Shifeng] Chongqing Med Univ, Affiliated Hosp 1, Dept Lab Med, Chongqing 400016, Peoples R China; [Cao, Xiaoying; Liu, Bo] Chongqing Med Univ, Affiliated Hosp 1, Dept Burn & Plast Surg, Chongqing 400016, Peoples R China"

通信作者："Huang, SF (通讯作者)，Chongqing Med Univ, Affiliated Hosp 1, Dept Lab Med, Chongqing 400016, Peoples R China.; Liu, B (通讯作者)，Chongqing Med Univ, Affiliated Hosp 1, Dept Burn & Plast Surg, Chongqing 400016, Peoples R China."

来源：ANALYTICA CHIMICA ACTA

ESI学科分类：CHEMISTRY

WOS号：WOS:000933058800001

JCR分区：Q1

影响因子：5.7

年份：2023

卷号：1247

期号：　

开始页：　

结束页：　

文献类型：Article

关键词：Clustered regularly interspaced short; palindromic repeats; Cas12a; Recombinase polymerase amplification; Polymerase chain reaction; Single -base mutation

摘要："Current single-base mutation detection approaches are time-consuming, labor-intensive, and costly. This high-lights the critical need for speedy and accurate technology capable of detecting single-base alterations. Using clustered regularly interspaced short palindromic repeats/associated protein 12a (CRISPR/Cas12a), two fundamental approaches for getting 100% differentiation of single-base mutations have been established, by which fluorescence signals could be detected for variants but not for wild strains. The first method required both polymerase chain reaction (PCR) and CRISPR/Cas12a cleavage: By introducing a mismatched base at the 3 ' end of the primers and adjusting the PCR settings, the wild strain strand amplifications were completely blocked prior to CRISPR/Cas12a cleavage. The parameters for Method 1 (PCR + CRISPR/Cas12a) could be easily controlled and adjusted to attain a sensitivity of one copy (about 6 copies mu L-1). The second method included isothermal recombinase polymerase amplification (RPA) and CRISPR/Cas12a cleavage: By introducing an extra mismatched base adjacent to the single-base mutant site by RPA (IMAS-RPA), the RPA products from the wild strains were rendered incapable of triggering the cleavage activity of CRISPR/Cas12a. Method 2 (IMAS-RPA) was rapid and easy to implement (can be finished within 1 h). Because each method has its own set of advantages, the labo-ratory environment-appropriate methods can be selected independently. Both approaches are expected to aid in clinical diagnosis to some extent in the near future."

基金机构："National Natural Science Founda- tion of China [82072346, 82072349]; Natural Science Foundation of Chongqing, China [cstc2020jcyj- msxmX0519]"

基金资助正文："This work was sponsored by the National Natural Science Founda- tion of China (Grant No. 82072346 and No. 82072349) and the Natural Science Foundation of Chongqing, China (No. cstc2020jcyj- msxmX0519) ."