Single Molecule FRET Analysis of CRISPR Cas9 Single Guide RNA Folding Dynamics.

CRISPR Cas9 is an RNA guided endonuclease that is part of a bacterial adaptive immune system. Single guide RNA (sgRNA) can be designed to target genomic DNA, making Cas9 a programmable DNA binding/cutting enzyme and allowing applications such as epigenome editing, controlling transcription, and targeted DNA insertion. Some of the main hurdles against an even wider adoption are off-target effects and variability in Cas9 editing outcomes. Most studies that aim to understand the mechanisms that underlie these two areas have focused on Cas9 DNA binding, DNA unwinding, and target cleavage. The assembly of Cas9 RNA ribonucleoprotein complex (RNP) precedes all these steps and includes sgRNA folding and Cas9 binding to sgRNA. We know from the crystal structure of the Cas9 RNP what the final sgRNA conformation is. However, the assembly dynamics has not been studied in detail and a better understanding of RNP assembly could lead to better-designed sgRNAs and better editing outcomes. To study this process, we developed a single molecule FRET assay to monitor the conformation of the sgRNA and the binding of Cas9 to sgRNA. We labeled the sgRNA with a donor fluorophore and an acceptor fluorophore such that when the sgRNA folds, there are changes in FRET efficiency. We measured sgRNA folding dynamics under different ion conditions, under various methods of folding (refolding vs vectorial), and with or without Cas9. sgRNA that closely mimics the sgRNA construct used for high resolution structural analysis of the Cas9-gRNA complex showed two main FRET states without Cas9, and Cas9 addition shifted the distribution toward the higher FRET state attributed to the properly assembled complex. Even in the absence of Cas9, folding the sgRNA vectorially using a superhelicase-dependent release of the sgRNA in the direction of transcription resulted in almost exclusively high FRET state. An addition of Cas9 during vectorial folding greatly reduced a slow-folding fraction. Our studies shed light on the heterogeneous folding dynamics of sgRNA and the impact of co-transcriptional folding and Cas9 binding in sgRNA folding. Further studies of sequence dependence may inform rational design of sgRNAs for optimal function.

Single molecule methods for studying CRISPR Cas9-induced DNA unwinding.

Like helicases, CRISPR proteins such as Cas9 and Cas12a unwind DNA, but unlike helicases, these CRISPR proteins do not use ATP. Instead, they use binding energy to melt DNA locally and then utilize basepairing between guide (g) RNA and target strand to continue to unwind the DNA. CRISPR Cas9 is the most widely used tool for genome editing applications. The Cas9 endonuclease forms a complex with gRNA that can be programmed to bind a specific 20 bp segment of DNA, the protospacer. If there is enough of a sequence match between sgRNA and protospacer, Cas9 undergoes a conformational change, which activates the two nuclease domains, causing a double strand break in the DNA. We can use single-molecule FRET (smFRET) to probe the state of DNA unwinding as a function of mismatches between sgRNA and DNA. This approach can also be used to probe the position of Cas9's HNH domain before and after cleavage.

Single molecule analysis of effects of non-canonical guide RNAs and specificity-enhancing mutations on Cas9-induced DNA unwinding.

Cas9 has made a wide range of genomic manipulation possible. However, its specificity continues to be a challenge. Non-canonical gRNAs and new engineered variants of Cas9 have been developed to improve specificity, but at the cost of the on-target activity. DNA unwinding is a checkpoint before cleavage by Cas9, and was shown to be made more sensitive to sequence mismatches by specificity-enhancing mutations in engineered Cas9s. Here we performed single-molecule FRET-based DNA unwinding experiments using various combinations of non-canonical gRNAs and different Cas9s. All engineered Cas9s were less promiscuous than wild type when canonical gRNA was used, but HypaCas9 had much-reduced on-target unwinding. Cas9-HF1 and eCas9 showed the best balance between low promiscuity and high on-target activity with canonical gRNA. When extended gRNAs with one or two non-matching guanines added to the 5' end were used, Sniper1-Cas9 showed the lowest promiscuity while maintaining high on-target activity. Truncated gRNA generally reduced unwinding and adding a non-matching guanine to the 5' end of gRNA influenced unwinding in a sequence-context dependent manner. Our results are consistent with cell-based cleavage data and provide a mechanistic understanding of how various Cas9/gRNA combinations perform in genome engineering.