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 × 10⁻⁵ U μL⁻¹ in the concentration scope from 1 × 10⁻⁴ U μL⁻¹ to 1 × 10⁻¹ U μL⁻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.

提出了一种基于 CRISPR-Cas12a 侧链切割能力的等温单罐工具箱（称为 OPT-Cas），用于高度灵敏和选择性地测定末端脱氧核苷酸转移酶 (TdT) 活性。随机引入具有 3'-羟基 (OH) 末端的寡核苷酸引物用于 TdT 诱导的延伸。在 TdT 存在的情况下，dTTP 核苷酸在引物的 3' 末端聚合，产生丰富的 polyT 尾巴，作为 Cas12a 蛋白同步激活的触发器。最后，激活的 Cas12a反式-切割 FAM 和 BHQ1 双标记单链 DNA (ssDNA-FQ) 报告基因，产生显着放大的荧光信号。这种一锅法检测，即引物、crRNA、Cas12a 蛋白和 ssDNA-FQ 报告基因都在一个试管中，允许对 TdT 活性进行简单但高灵敏度的定量，检测限低至 6.16 × 10 −5 ^U μL ⁻¹在浓度范围从 1 × 10 ⁻⁴ U μL ⁻¹到 1 × 10 ⁻¹ U μL ⁻¹, 并与其他干扰蛋白实现非凡的选择性。此外，OPT-Cas已成功用于检测复杂基质中的TdT，并准确测定急性淋巴细胞白血病细胞中的TdT活性，这可能为TdT相关疾病的诊断和生物医学研究应用提供可靠的技术平台。