biostudies-literatureOkafor ICHoward Hughes Medical InstituteNational Institute of General Medical SciencesNIGMS NIH HHSNational Science Foundation45-51https://www.ebi.ac.uk/biostudies/studies/S-EPMC9841515biostudies-literatureUnknown127(1)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 The journal of physical chemistry. BSingle Molecule FRET Analysis of CRISPR Cas9 Single Guide RNA Folding Dynamics.PMC9841515F31 GM134675GM 122569PHY-1430124R35 GM122569F31 GM 134675Okafor ICHa TfalseSingle 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 2023-01-01T00:00:00Z2023 Jan2026-05-28T11:30:31.731Z2025-04-07T03:17:05.345ZS-EPMC98415153656331410.1021/acs.jpcb.2c05428