DNA curtain assay for dCas12a/CS10B colocalization

A self-assembling polypeptide (C-S10-B, labeled in green) binds to a DNA that has been pre-decorated with CRISPR-dCas12a (labeled in magenta) via multiple CRISPR-RNAs (crRNAs). C-S10-B binds and diffuses along DNA but cannot move beyond stably bound dCas12a. During self-assembly, large clusters of C-S10-B commonly form and colocalize with dCas12a.

Bacteriophage λ DNA (λDNA) (NEB, N3011S) was mixed in T4 DNA ligase (NEB, M0202S) reaction buffer with biotinylated oligos complementary to λDNA cohesive ends, for 15 min at 70°C, followed by a cool down to 15 °C for over 2 h. Ligation took place overnight at room temperature. After T4 DNA ligase inactivation with 2 M NaCl, the biotinylated DNA was purified on a Sephacryl S-1000 size exclusion column (GE Healthcare).

Using a lipid solution (1.954% DOPC, 0.04% DOPE-mPEG2k and 0.006% DOPE-biotin) in buffer (10 mM Tris-HCL pH 8, 100 mM NaCl), the flowcell was passivated at room temperature for 30 min. Next, BSA buffer (40 mM Tris-HCl pH 8, 2 mM MgCl2, 0.2 mg/mL BSA) was used to wash the flowcell, followed by incubation for 10 min. BSA buffer containing biotinylated DNA was injected into the flowcell, before washing out all the non-tethered DNA material. Imaging was done in BSA buffer supplemented with 100 mM NaCl, 5 mM MgCl2, 2 mM DTT.

dCas12a was mixed with the crRNA pool at a 1:10 molar ratio in buffer (20 mM Tris-HCl pH 8.0, 100 mM NaCl, 5 mM MgCl2, 2% glycerol, 2 mM DTT) for 30 min at 37°C. The formed ribonucleoprotein particle complexes (10 nM) were injected into the flowcell to induce DNA binding for 30 min at room temperature. Monoclonal ANTI-FLAG ® BioM2-Biotin (Sigma-Aldrich, F9291) conjugated to quantum dots (Thermo, Q21361MP) were used to label dCas12a. C-S10-B was labeled at a single N-terminal cysteine with maleimide-Alexa-488.

Imaging was carried out in an inverted Nikon Ti-E microscope at 60X magnification. Excitation of the sample was provided by a 488 nm laser. Emission light was split with a 638 nm dichroic beam splitter (Chroma) and registered by two EM-CCD cameras (Andor iXon DU897). Image processing was done in FIJI.