CRISPR-Cas9 is widely applied for genome engineering in various organisms. The assembly of single guide RNA (sgRNA) with the Cas9 protein may limit the Cas9/sgRNA effector complex function. We developed a FRET-based assay for detection of CRISPR–Cas9 complex binding to its targets and used this assay to investigate the kinetics of Cas9 assembly with a set of structurally distinct sgRNAs. We find that Cas9 and isolated sgRNAs form the effector complex efficiently and rapidly. Yet, the assembly process is sensitive to the presence of moderate concentrations of non-specific RNA competitors, which considerably delay the Cas9/sgRNA complex formation, while not significantly affecting already formed complexes. This observation suggests that the rate of sgRNA loading into Cas9 in cells can be determined by competition between sgRNA and intracellular RNA molecules for the binding to Cas9. Non-specific RNAs exerted particularly large inhibitory effects on formation of Cas9 complexes with sgRNAs bearing shortened 3′-terminal segments. This result implies that the 3′-terminal segment confers sgRNA the ability to withstand competition from non-specific RNA and at least in part may explain the fact that use of sgRNAs truncated for the 3′-terminal stem loops leads to reduced activity during genomic editing.

Significance: The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) systems have been widely used as tools for various genome-engineering applications. The most popular CRISPR-Cas system makes use of the Cas9 endonuclease from Streptococcus pyogenes (SpCas9) and a single-guide RNA (sgRNA). The SpCas9/sgRNA complex cleaves double-stranded DNA sequences specified by sgRNA and protospacer adjacent motif (PAM). This study explores the mechanism by which SpCas9/sgRNA efficiently locates the correct target DNA sequence. The results show that high efficiency of DNA interrogation can be explained, in part, by destabilization of double-stranded DNA conformation in the vicinity of PAM. The data also suggest that the local DNA destabilization is mediated by SpCas9/sgRNA affinity for a single-stranded DNA segment adjacent to PAM and by hindering of duplex DNA entry into the SpCas9/sgRNA interior.

Promoter recognition by RNA polymerase is a key point in gene expression and a target of regulation. Bacterial RNA polymerase binds promoters in the form of the holoenzyme, with the σ specificity subunit being primarily responsible for promoter recognition. Free σ, however, does not recognize promoter DNA, and it has been proposed that the intrinsic DNA binding ability is masked in free σ but becomes unmasked in the holoenzyme. Here, we use a newly developed fluorescent assay to quantitatively study the interactions of free σ⁷⁰ from Escherichia coli, the β′-σ complex, and the σ⁷⁰ RNA polymerase (RNAP) holoenzyme with non-template strand of the open promoter complex transcription bubble in the context of model non-template oligonucleotides and fork junction templates. We show that σ⁷⁰, free or in the context of the holoenzyme, recognizes the −10 promoter element with the same efficiency and specificity. The result implies that there is no need to invoke a conformational change in σ for recognition of the −10 element in the single-stranded form. In the holoenzyme, weak but specific interactions of σ are increased by contacts with DNA downstream of the −10 element. We further show that region 1 of σ⁷⁰ is required for stronger interaction with non-template oligonucleotides in the holoenzyme but not in free σ. Finally, we show that binding of the β′ RNAP subunit is sufficient to allow specific recognition of the TG motif of the extended −10 promoter element by σ⁷⁰. The new fluorescent assay, which we call a protein beacon assay, will be instrumental in quantitative dissection of fine details of RNAP interactions with promoters.

The CRISPR/Cas9 nucleases have been widely applied for genome editing in various organisms. Cas9 nucleases complexed with a guide RNA (Cas9/gRNA) find their targets by scanning and interrogating the genomic DNA for sequences complementary to the gRNA. Recognition of the DNA target sequence requires a short protospacer adjacent motif (PAM) located outside this sequence. Given that the efficiency of target location may depend on the strength of interactions that promote target recognition, here we sought to compare affinities of different Cas9 nucleases for their cognate PAM sequences. To this end, we measured affinities of Cas9 nucleases from Streptococcus pyogenes, Staphylococcus aureus, and Francisella novicida complexed with gRNAs (SpCas9/gRNA, SaCas9/gRNA, and FnCas9/gRNA, respectively) and of three engineered SpCas9/gRNA variants with altered PAM specificities for short, PAM-containing DNA probes. We used a “beacon” assay that measures the relative affinities of DNA probes by determining their ability to competitively affect the rate of Cas9/gRNA binding to fluorescently labeled target DNA derivatives called “Cas9 beacons.” We observed significant differences in the affinities for cognate PAM sequences among the studied Cas9 enzymes. The relative affinities of SpCas9/gRNA and its engineered variants for canonical and suboptimal PAMs correlated with previous findings on the efficiency of these PAM sequences in genome editing. These findings suggest that high affinity of a Cas9 nuclease for its cognate PAM promotes higher genome-editing efficiency.