Fig. 1: Overview and validation of Af-CUT&Tag.

a, b. Schematic diagram of the protein-protein interactions between HiBiT (yellow) binding with LgBiT (dark blue) and ALFA-tag (light blue) binding with NbALFA (green). c. The binding kinetics and affinity between the recombinant protein ExoⅢ-HiBiT and LgBiT (left) or LgBiT-Tn5 (right) were determined using Bio-Layer Interferometry (BLI). The ExoⅢ-HiBiT protein was immobilized on the sensor surface, and the sensors were dipped into wells containing different concentrations of LgBiT (1-25 nM) or LgBiT-Tn5 (1-20 nM). d. The binding kinetics and affinity between the recombinant protein GFP-ALFA-tag and NbALFA (left) or NbALFA-Tn5 (right) were determined using Bio-Layer Interferometry (BLI). The GFP-ALFA-tag protein was immobilized on the sensor surface, and the sensors were dipped into wells containing different concentrations of NbALFA (1-8 nM) or NbALFA-Tn5 (1-8 nM). e. Schematic representation of the assembly of the fusion protein LgBiT/NbALFA-Tn5 with the transposon adapter. f. Construction of Nano-Luciferase reporter gene knock in system based on CRISPR/Cas9 (Left). 100,000 SW480 RPB1-HiBiT and SW480 CTCF-HiBiT gene knock-in cells were separately lysed using Nano-Glo® Lytic Buffer (Promega). Excess substrate furimazine and 1 μL (26 μM) of LgBiT-Tn5 were added for chemiluminescent detection (Right). The detection parameters were set as follows: LUM-EndPoint, Single label, 700 nm Blocker, Measurement direction: top, Measurement time: 1,000 ms, Default Z focus: 8.5 mm. Each sample was measured in three technical replicates, and data are presented as mean ± SD (unpaired t-test, ****p < 0.0001). g. The assembled transposome LgBiT-Tn5 (0.5 μM) and NbALFA-Tn5 (0.5 μM) were incubated with 200 ng gDNA at 55 °C for 10 min. The cleavage of gDNA was analyzed using agarose gel electrophoresis. The gel image shown is representative of results obtained from multiple independent experiments. h. Comparison of strategies between antibody-dependent CUT&Tag, nano-CUT&Tag and Af-CUT&Tag. Adding LgBiT-Tn5 can directly target HiBiT/ALFA-tag at the C-terminus of the target transcription factor and bind with high affinity. After multiple rounds of washing to remove excess transposons, add Mg²⁺ to activate transposons, insert adapters into transcription factor binding sites, and construct DNA sequencing libraries. In the CUT&Tag strategy, primary antibody against the target protein, secondary antibody, and pA/G-Tn5 are required, with washing steps after each incubation. In nano-CUT&Tag, primary antibody and nanobody-Tn5 are added, followed by washing. The library construction steps are consistent across all strategies.