Site-Specific Labeling Reveals Cas9 Induces Partial Unwinding Without RNA/DNA Pairing in Sequences Distal to the PAM

CRISPR-Cas9 Activities with Truncated 16-Nucleotide RNA Guides Are Tuned by Target Duplex Stability Beyond the RNA/DNA Hybrid

“…sgRNAs with the same 80-nt core but varying in guide sequence and length were synthesized by T7 in vitro transcription. The double-stranded DNA templates for transcription were prepared by overlapping PCR as previously described . Transcriptions and sgRNA purifications were carried out following previously reported procedures .…”

“…The fraction of products, f pro , at each time point of a reaction was calculated as f normalp normalr normalo = I normalC 1 + I normalC 2 I normalU + I normalC 1 + I normalC 2 with I U being the uncleaved “precursor” band intensity and I C1 and I C2 being intensities of cleaved products 1 (top band) and 2 (bottom band), respectively. It has been established that the active Cas9 concentrations used in this study were at saturating conditions . The reaction rate constant, which represents k cat , was determined by fitting the time dependence of f pro to a single-exponential using Origin 2018: f normalp normalr normalo = a · ( 1 − e − k c a t · t ) with a being the active fraction of the target DNA.…”

“…Absorbance was obtained immediately after the fluorescence measurement on a LAMBDA UV/vis/NIR Spectrophotometer (PerkinElmer). The normalized 2AP quantum yield of a tested sample with respect to the corresponding DNA duplex, ratio(φ), was obtained using the concentration-normalized background-corrected 2AP emission ( F / A ) as described in our previous study normalr normala normalt normali normalo ( φ ) = φ φ normald normalu normalp normall normale normalx = ε ε normald normalu normalp normall normale normalx · false( true F 370 A 260 false) false( true F 370 A 260 false) normald normalu normalp normall normale normalx where ε and ε duplex are the extinction coefficients at 260 nm for the sample and the duplex, respectively; F 370 is the background-corrected 2AP emission of the sample at 370 nm; A 260 is the absorbance of the same sample at 260 nm; ( F 370 / A 260 ) duplex is the F / A value for the corresponding duplex.…”

“…With both substrates, the linearized duplexed precursor was cleaved completely into two shorter fragments (Figure 1B), and the time course of product generation was fit nicely with a single-exponential model (eq 2) to yield the cleavage reaction rate (Figure 1C), which represents k cat of the reaction. 25 The measurements yielded k cat = (0.08 ± 0.01 min −1 ) for S GCGG , which is ∼16 times slower than that of S TATA (1.29 ± 0.05 min −1 ) (Figure 1C, Table S2). Note that ΔG NN(17−20) 0 is 10.00 kcal/mol for S GCGG and 3.57 kcal/mol for S TATA (Figure 1B, Table S2).…”

“…23,24 Recently, we reported that, for RNA guides that are longer than 15-nt, the Cas9 ribonucleoprotein complex partially unwinds PAM-distal DNA beyond the RNA/DNA hybrid, and the rate of double-stranded DNA cleavage correlates with the degree of PAM-distal unwinding. 25 This would imply that, with truncated guides, intrinsic properties of the DNA protospacer beyond the RNA/DNA hybrid could modulate Cas9 cleavage rates by tuning the PAM-distal unwinding. Specifically, we hypothesize that the duplex dissociation free energy, which measures the stand-alone DNA duplex stability, can serve as an indicator of the ability of DNA to unwind upon interacting (less stable) were shown to unwind to a larger degree by a 2-amino-purine (2AP) fluorescence assay and indeed yielded larger k cat , i.e., faster cleavage.…”