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

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

来源：Analytica Chimica Acta, 2023, 1254: 341115.

DOI：10.1016/j.aca.2023.341115

• CRISPR
• Cas12a
• Fluorescence
• Collateral cleavage
• crRNA
• TdT activity

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.

• 单位
  南方医科大学; 1; 广东省人民医院