Temperature-boosted PAM-less activation of CRISPR-Cas12a combined with selective inhibitors enhances detection of SNVs with VAFs below 0.01%

作者全名："Chen, Kena; Dai, Ling; Zhao, Jie; Deng, Mengjun; Song, Lin; Bai, Dan; Wu, You; Zhou, Xi; Yang, Yujun; Yang, Shuangshuang; Zhao, Lin; Chen, Xueping; Xie, Guoming; Li, Junjie"

作者地址："[Chen, Kena; Dai, Ling; Deng, Mengjun; Song, Lin; Bai, Dan; Wu, You; Zhou, Xi; Yang, Yujun; Xie, Guoming; Li, Junjie] Chongqing Med Univ, Coll Lab Med, Key Lab Clin Lab Diagnost, Chongqing Med Lab Microfluid,Chinese Minist Educ, Chongqing 400016, Peoples R China; [Chen, Kena; Dai, Ling; Deng, Mengjun; Song, Lin; Bai, Dan; Wu, You; Zhou, Xi; Yang, Yujun; Xie, Guoming; Li, Junjie] Chongqing Med Univ, SPRi Engn Res Ctr, Chongqing 400016, Peoples R China; [Zhao, Jie; Chen, Xueping] Chongqing Med Univ, Affiliated Hosp 1, Ctr Clin Mol Med Detect, Chongqing 400016, Peoples R China; [Yang, Shuangshuang] Chongqing Med Univ, Affiliated Hosp 1, Dept Lab Med, Chongqing 400016, Peoples R China; [Zhao, Lin] Chongqing Med Univ, Affiliated Hosp 1, Dept Emergency & Crit Care Med, Chongqing 400016, Peoples R China"

通信作者："Xie, GM; Li, JJ (通讯作者)，Chongqing Med Univ, Coll Lab Med, Key Lab Clin Lab Diagnost, Chongqing Med Lab Microfluid,Chinese Minist Educ, Chongqing 400016, Peoples R China.; Xie, GM; Li, JJ (通讯作者)，Chongqing Med Univ, SPRi Engn Res Ctr, Chongqing 400016, Peoples R China.; Chen, XP (通讯作者)，Chongqing Med Univ, Affiliated Hosp 1, Ctr Clin Mol Med Detect, Chongqing 400016, Peoples R China."

来源：TALANTA

ESI学科分类：CHEMISTRY

WOS号：WOS:001001936700001

JCR分区：Q1

影响因子：5.6

年份：2023

卷号：261

期号：　

开始页：　

结束页：　

文献类型：Article

关键词：Single nucleotide variations; CRISPR-Cas12a; PAM-Less activation; PCR additives; Blocker displacement amplification

摘要："The precise identification of rare single nucleotide variations (SNVs) concomitant with excess wild-type DNA is a valuable method for minimally invasive disease diagnosis and early prediction of drug responsiveness. Selective enrichment of mutant variants via strand displacement reaction offers an ideal approach of SNVs analysis but fails to differentiate wildtype from mutants with variant allele fraction (VAF) < 0.01%. Here, we demonstrate that integration of PAM-less CRISPR-Cas12a and adjacent mutation-enhanced inhibition of wild-type alleles enables highly sensitive measurement of SNVs well below the 0.01% VAF threshold. Raising the reaction temperature to the upper limit of LbaCas12a helps to boost PAM-less activation of collateral DNase activity, which can be further enhanced using PCR additives, leading to ideal discriminative performance for single point mutations. Along with selective inhibitors bearing additional adjacent mutation, it allowed detection of model EGFR L858R mutants down to 0.001% with high sensitivity and specificity. Preliminary investigation on adulterated genomic samples prepared in two different ways also suggests that it can accurately measure ultralow-abundance SNVs extracted directly from clinical samples. We believe that our design, which combines the superior SNV enrichment capability of strand displacement reaction and unparalleled programmability of CRISPR-Cas12a, has the potential to significantly advance current SNV profiling technologies."

基金机构："CQMU Program for Youth Innovation in Future Medicine, China [W0104]; National Natural Science Foundation of China [82002255]; Basic Science and Cuttingedge Technology Research Projects of Chongqing Science and Technology Commission, China [cstc2019jcyj-msxmX0848]; Chongqing Medical Scientific Research Project (Chongqing Health Commission) [2022QNXM053]; Chongqing Medical Scientific Research Project (Science and Technology Bureau) [2022QNXM053]; Top Talent Project of Chongqing Medical University, China [BJRC202123]"

基金资助正文："This research was supported by CQMU Program for Youth Innovation in Future Medicine, China (No. W0104), the National Natural Science Foundation of China (No. 82002255), the Basic Science and Cuttingedge Technology Research Projects of Chongqing Science and Technology Commission, China (No. cstc2019jcyj-msxmX0848), the Chongqing Medical Scientific Research Project (Joint project of Chongqing Health Commission and Science and Technology Bureau, No. 2022QNXM053), and Top Talent Project of Chongqing Medical University, China (No. BJRC202123)."