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

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. 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. [Display omitted] • An isothermal, one-pot toolbox (called OPT-Cas) for TdT activity detection was proposed. • TdT-induced DNA elongation and CRISPR/Cas12a-based signal amplification were achieved. • OPT-Cas can detect TdT down to 6.8 × 10−5 U μL−1 with a wide linear range from 1.0 × 10−4 to 1.0 × 10−1 U μL−1. • The biosensor exhibited good performance for the monitoring of TdT activity in clinical real biological samples. [ABSTRACT FROM AUTHOR]