Kinetics of dCas9 target search in Escherichia coli

Abstract

How fast can a cell locate a specific chromosomal DNA sequence specified by a single-stranded oligonucleotide? To address this question, we investigate the intracellular search processes of the Cas9 protein, which can be programmed by a guide RNA to bind essentially any DNA sequence. This targeting flexibility requires Cas9 to unwind the DNA double helix to test for correct base pairing to the guide RNA. Here we study the search mechanisms of the catalytically inactive Cas9 (dCas9) in living Escherichia coli by combining single-molecule fluorescence microscopy and bulk restriction-protection assays. We find that it takes a single fluorescently labeled dCas9 6 hours to find the correct target sequence, which implies that each potential target is bound for less than 30 milliseconds. Once bound, dCas9 remains associated until replication. To achieve fast targeting, both Cas9 and its guide RNA have to be present at high concentrations.

Fig. 1. Comparison of Cas9 and transcription factor search

Cas9 (in yellow) must unwind dsDNA at every PAM (red dots) to test for sgRNA complementarity to the protospacer (detail, lower right). In contrast, a transcription factor (in blue) scans the dsDNA by sliding in the grooves (detail, lower left).

Fig. 2. Association

(A) Upper: Schematic illustration of the single molecule assay, where dCas9-YPet is expressed at low copy number in a strain containing pSMART plasmids each with 36 lacO1-binding sites. In the absence of IPTG, lacO1 sites are occupied by LacI, preventing dCas9-YPet from binding. LacI dissociates after addition of IPTG, and subsequently dCas9-YPet binds lacO1, enabling specific fluorescent spots to be detected using exposure times of 5 s. Lower: fluorescence images acquired before (left) and 10 min after (right) addition of IPTG (strain PL42F9). Scale-bar 2µm. (B) Fraction of cells containing at least one spot (y-axis) as a function of time after addition of IPTG (x-axis). The curve is fitted to a single exponential function. Left inset distributions of number of fluorescent dCas-YPet (strain PL42F2) Right inset Distribution of accessible pSMART (strain PL41D2) per cell, which is based on the pSMART copy number distribution (fig. S4) corrected for the fact that 50% of the pSMARTs are available for binding (fig. S5). In total 63382 cells are analyzed. (C) Upper: Schematic illustration of the bulk assay, in which dCas9 or dCas9-YPet is expressed at a high copy number in the presence of only one target site per chromosome. LacI dissociates from lacO1 after addition of IPTG, permitting dCas9-YPet to bind. Lower: Schematic of experimental procedure. IPTG is added at t=0, freeing the lacO1 site. At later times, here time 1 or time 2, different batches of cells are fixed and bound dCas9 are cross-linked to DNA; the chromatin is subsequently purified and digested with BsrBI. The fraction of cut and uncut DNA is quantified by qPCR with two sets of primers, one upstream and one amplifying across the cut site. (D) Measured bulk association to lacO1-b in the intC locus for dCas9-YPet (strain DJ3F6) is compared to dCas9 (strain DJ3I3) (see table S1 for sequences). Please note that the concentrations are slightly different in the two strains which is why the rates should not be directly compared (see main text and supplementary text section 2.1.3.) (E) Measured bulk association to lacO1-a in the intC locus for dCas9-YPet with (strain DJ3F5) or without (strain DJ3F8) a PAM adjacent to the protospacer. (D+E) Each data point is derived from three biological replicates (each consisting of three technical replicates). r represents the rate at which any of the dCas9 proteins binds to the single target site. See fig. S6 and table S1 for strains and sequences.

Fig. 3. Dissociation and repression

(A) Fluorescence images of cells with dCas9-YPet at three different time points (t in min) after removing IPTG. Scale bar: 2µm. (B) Two examples from a set of dissociation curves acquired at different growth rates resulting from different carbon sources and temperatures. The red dashed lines are a smoothed versions of the curves. The time range when the curve has dropped to within 5%-15% of the plateau is indicated by horizontal dashed lines. (C) The time for dissociation (defined as reaching 10% from the plateau) is plotted (y-axis) as a function of generation time (x-axis) for individual experiments. The error bars represent the 5%-15% range shown in B. Different colors represent different growth conditions (legend), in which only the temperature or the carbon source of the M9 media is changing (see supplementary material and methods section 1.6.3.2 for details). The generation times for data points with filled symbols are estimated in the same microfluidic experiment as the corresponding dissociation time estimates. The generation times for the data points with open symbols are averages of other experiments (filled symbols) under the same growth conditions. Strains: PL42F9 (circles) PL42H2 (triangles). All individual dissociation curves are shown in fig. S10 (D) The repression ratio (red dashed line) as predicted by Eq. 1 using the measured binding rate (rC=0.34 min^-1, strain DJ3D5) and the generation time (T=107 min) is compared to the measured repression ratio of the regulated lacZ gene (markers). If the binding rate instead would be calculated from the repression ratio for DJ3D5 it would be rC=0.29 min^-1. The expression level of dCas9-YPet is measured using Western blots (fig. S1) and plotted relative to the strain DJ3D5 (black marker). The other strains are, in order of increasing expression, PL41E1, PL42B6, PL42C1, and PL42C9. X-axis error bars are based on three different Western blots; y-axis error bars on at least three repression ratio measurements.

Fig. 4. Non-specific Binding

Cells containing dCas9-YPet but no specific chromosomal target are imaged in the presence (strain:PL42H9) or absence (strain:PL42H8) of sgRNA-a, using exposure times ranging from 2ms to 1s while keeping the laser power times exposure-time constant. A. Examples of fluorescence images for different laser exposure times in absence (-sgRNA) and presence (+sgRNA) sgRNA-a. Automatically detected fluorescent dots are indicated by red circles. Each row has the same camera counts to grayscale mapping as indicated by the scale bar in between the example images. B. The average number of dots detected per cell as function of laser exposure time (dots). Error bars are the standard error of the mean of three technical repeats using the same microfluidic device. Solid lines are smoothing spline fits, which are extrapolated until 5 seconds laser exposure time. C. Distribution of residence times estimated using the fitted lines, <N(t)>, in (A). The distribution is given by p(t) = - D (1/t) d<N(t)>/dt, where D is chosen such that integral of p(t) = 1 and t is the laser exposure time. (see SI for details on calculation). The average residence times are <30ms in presence of sgRNA-a and <20 ms in absence of sgRNA-a.