A one-pot CRISPR-Cas12a-based toolbox enables determination of terminal deoxynucleotidyl transferase activity for acute leukemia screening

作者全名："Yi, Ming; Gong, Yao; Zhan, Qian; Dai, Yulian; Yang, Tiantian; Cheng, Xiaoxue; Ding, Shijia; Gu, Bing; Cheng, Wei; Zhang, Decai"

作者地址："[Yi, Ming; Gong, Yao; Zhan, Qian; Dai, Yulian; Yang, Tiantian; Cheng, Xiaoxue; Cheng, Wei; Zhang, Decai] Chongqing Med Univ, Affiliated Hosp 1, Ctr Clin Mol Med Detect, Chongqing 400016, Peoples R China; [Ding, Shijia] Chongqing Med Univ, Coll Lab Med, Key Lab Clin Lab Diagnost, Minist Educ, Chongqing 400016, Peoples R China; [Gu, Bing; Zhang, Decai] Southern Med Univ, Guangdong Prov Peoples Hosp, Guangdong Acad Med Sci, Lab Med, Guangzhou 510000, Peoples R China"

通信作者："Cheng, W; Zhang, DC (通讯作者)，Chongqing Med Univ, Affiliated Hosp 1, Ctr Clin Mol Med Detect, Chongqing 400016, Peoples R China.; Gu, B; Zhang, DC (通讯作者)，Southern Med Univ, Guangdong Prov Peoples Hosp, Guangdong Acad Med Sci, Lab Med, Guangzhou 510000, Peoples R China."

来源：ANALYTICA CHIMICA ACTA

ESI学科分类：CHEMISTRY

WOS号：WOS:000958001800001

JCR分区：Q1

影响因子：5.7

年份：2023

卷号：1254

期号：　

开始页：　

结束页：　

文献类型：Article

关键词：CRISPR; Cas12a; Fluorescence; Collateral cleavage; crRNA; TdT activity

摘要："An isothermal, one-pot toolbox (called OPT-Cas) based on CRISPR-Cas12a collateral cleavage capability is proposed for highly sensitive and selective determination of terminal deoxynucleotidyl transferase (TdT) activity. Oligonucleotide primers with 3 '-hydroxyl (OH) terminal were randomly introduced for TdT-induced elongation. In the presence of TdT, dTTP nucleotides polymerized at the 3 ' terminals of the primers to generate abundant polyT-tails, which function as triggers for the synchronous activation of Cas12a proteins. Finally, the activated Cas12a trans-cleaved FAM and BHQ1 dual-labeled single-stranded DNA (ssDNA-FQ) reporters, producing significantly amplified fluorescence signals. This one-pot assay, that is primer, crRNA, Cas12a protein and ssDNA-FQ reporter are all in one tube, allows simple but high-sensitive quantification of TdT activity with a low detection limit of 6.16 x 10-5 U mu L-1 in the concentration scope from 1 x 10-4 U mu L-1 to 1 x 10-1 U mu L-1, and achieves extraordinary selectivity with other interfering proteins. Furthermore, the OPT-Cas was successfully used to detect TdT in complex matrices and accurate determination of TdT activity in acute lymphoblastic leukemia cells, which might be a reliable technique platform for the diagnosis of TdT-related diseases and biomedical research applications."

基金机构："National Natural Science Foundation of China [81873972, 82102505]; Foundation for Innovative Research Groups of Chongqing Higher Education Institutions [CXQT20013]; Chongqing Science Fund for Distinguished Young Scholars [cstc2019jcyjjqX0028]; Training Program for Advanced Young Medical Personnel of Chongqing [2017HBRC003]"

基金资助正文："This work was funded by the National Natural Science Foundation of China (81873972 and 82102505) , the Foundation for Innovative Research Groups of Chongqing Higher Education Institutions (CXQT20013) , the Chongqing Science Fund for Distinguished Young Scholars (cstc2019jcyjjqX0028) , and the Training Program for Advanced Young Medical Personnel of Chongqing (2017HBRC003) ."