Nuclease dead Cas9 is a programmable roadblock for DNA replication

Limited experimental tools are available to study the consequences of collisions between DNA-bound molecular machines. Here, we repurpose a catalytically inactivated Cas9 (dCas9) construct as a generic, novel, targetable protein–DNA roadblock for studying mechanisms underlying enzymatic activities on DNA substrates in vitro. We illustrate the broad utility of this tool by demonstrating replication fork arrest by the specifically bound dCas9–guideRNA complex to arrest viral, bacterial and eukaryotic replication forks in vitro.


Assessment of diffusion limited binding kinetics of dCas9-dL5 binding to target containing DNA in SPR studies
The sensorgrams obtained during the association of dCas9-dL5-cgRNA at different concentrations of dCas9-dL5 (Fig. S1B) exhibited a distinct biphasic profile. The linear response (RU)time (s) relationship suggests that the association of the complex from solution could be a fast, diffusion limited process. To examine this possibility, solutions of dCas9-dL5 (10 nM) with cgRNA1 (50 nM) in SPR buffer supplemented with 150 mM NaCl and 10 mM MgCl2 were injected over immobilized template DNA for 120 s at three different flow rates accessible to the BIAcore T200 instrument: 20, 30 and 80 L/min (Fig. S1C). The observed increase in association rates with the increase in flow rates confirms that the interaction is indeed diffusion-limited, a situation that occurs when the diffusion of analyte from the bulk solution to the chip surface is slower than its binding to the ligand. Conversely, it suggests that the dissociation of analyte (dCas9-dL5cgRNA) from the ligand (83-mer DNA) must be also be a diffusion limited process, i.e. upon dissociation from the ligand, the analyte may not diffuse into bulk solution, allowing it to re-bind. This would result in an apparently slower dissociation rate; therefore, the dissociation half-life (t1/2) of dCas9-dL5-cgRNA1 from its target dsDNA is an over-estimate of the true dissociation half-life.

Measurement of position of bound dCas9-dL5-cgRNA on 18-kb template
Measurement of the position of the bound dCas9-dL5cgRNA complex on the 18-kb template was performed as follows: 1. Line profiles were manually drawn over all individual DNA molecules. The length of the individual molecules was defined as the distance between the maximum and minimum of the first derivative of the intensity along the drawn lines. Using these measurements, a length distribution was plotted, and values below the 25% and above the 75% percentile were classified as outliers. The resulting distribution was fit to a Gaussian distribution with a mean of 39.5 ± 0.1 pixels. This mean length was then assumed to correspond to the total length of 18,345 bp of the DNA substrate. This conversion resulted in a calibration factor of 466 ± 1 bp/pixel.

2.
Next, peaks were detected along the line profile in the MGE-channel. The position of the detected peaks, relative to the ends of the DNA-molecules was then calculated. The distances to both ends of the DNA-molecule were measured. The position in base-pairs was calculated using the calibration described above. The histogram shows the smaller of the two distances from the DNA ends for each molecule.

Description of cgRNAs used in studies of bacterial DNA replication
Exact positions of targeting gRNA on the rolling-circle DNA replication template are as follows; cgRNA1 targeted to nucleotides 1402-1421 of the lagging-strand, cgRNA3 targeted to nucleotides 177-196 of the leading-strand, and cgRNA4 targeted to nucleotides 1046-1065 of the leading-strand.

Description of cgRNAs used in studies of eukaryotic DNA replication
Exact positions of targeting gRNA on the eukaryotic linear DNA replication template are as follows: cgRNA0.6 targeted nucleotides 583-602 of the leading strand, cgRNA1.0 targeted nucleotides 1005-1024 of the lagging strand, cgRNA1.5 targeted nucleotides 1493-1512 of the lagging strand, and cgRNA2.2 targeted nucleotides 2196-2215 of the leading strand. Figure Fig. 2A and summary in Fig. 2H). N > 2 independent experiments. (e) Only dCas9-dL5 programmed with complementary gRNAs specifically arrests E. coli leadingand lagging-strand DNA synthesis. Reactions contained 50 nM dCas9-dL5 and 400 nM gRNAs. Reactions were initiated at 30°C and aliquots were removed and quenched at 0, 2, and 20 min time points. N > 3 independent experiments. (f) E. coli leading-and lagging-strand DNA synthesis arrest by target-bound dCas9-dL5 is not strand specific. Unless otherwise specified reactions contained 400 nM cgRNAs and 50 nM dCas9-dL5. Lg denotes cgRNA targeted to the lagging strand, and Ld denotes cgRNA targeted to the leading strand. N > 3 independent experiments. All panels show photographic negative images of gels that had been stained with SYBR-gold nucleic acid stain.