The helicase unwinding experiments were conducted as follows (35). First, the dCas9/sgRNA complex was added to the chamber and incubated for 5 min. Second, 25 μl of 100 nM BLM helicase in unwinding buffer [25 mM tris-HCl (pH 7.5), 100 mM NaCl, 1 mM MgCl2, 0.1 g/ml BSA, 2 mM adenosine triphosphate, and 3 mM DTT] was added to the chamber before data acquisition. Last, the DNA tether was stretched until the force reached 12 pN; the force was maintained at a constant level, while the helicase unwound the dsDNA. The DNA length was recorded in real time.

A Phi29 DNAP strand displacement replication assay was conducted similarly. In brief, 25 μl of 30 nM Phi29 DNAP in the replication buffer [50 mM tris-HCl (pH 7.5), 10 mM MgCl2, 10 mM (NH4)2SO4, and 4 mM DTT] was added to the chamber before data acquisition. During the experiment, several hundred base pairs of dsDNA were mechanically unzipped to produce a priming template for the polymerase. The DNA length was maintained until the force dropped below a threshold, indicating that the polymerase was unwinding the DNA fork. Last, a constant force of 15 pN was maintained, while the polymerase unwound and replicated the dsDNA.

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