title: Lessons from Enzyme Kinetics Reveal Specificity Principles for RNA-Guided Nucleases in RNA Interference and CRISPR-Based Genome Editing.
abstract: RNA-guided nucleases (RGNs) provide sequence-specific gene regulation through base-pairing interactions between a small RNA guide and target RNA or DNA. RGN systems, which include CRISPR-Cas9 and RNA interference (RNAi), hold tremendous promise as programmable tools for engineering and therapeutic purposes. However, pervasive targeting of sequences that closely resemble the intended target has remained a major challenge, limiting the reliability and interpretation of RGN activity and the range of possible applications. Efforts to reduce off-target activity and enhance RGN specificity have led to a collection of empirically derived rules, which often paradoxically include decreased binding affinity of the RNA-guided nuclease to its target. We consider the kinetics of these reactions and show that basic kinetic properties can explain the specificities observed in the literature and the changes in these specificities in engineered systems. The kinetic models described provide a foundation for understanding RGN targeting and a necessary conceptual framework for their rational engineering.
authors.affiliation: Department of Biochemistry, Stanford University, Stanford, CA 94305, USA.
authors.forename: Namita
authors.initials: N
authors.lastname: Bisaria
authors.affiliation: Department of Biochemistry, Stanford University, Stanford, CA 94305, USA.
authors.forename: Inga
authors.initials: I
authors.lastname: Jarmoskaite
authors.affiliation: Department of Biochemistry, Stanford University, Stanford, CA 94305, USA; Department of Chemical Engineering, Stanford University, Stanford, CA 94305, USA; Department of Chemistry, Stanford University, Stanford, CA 94305, USA; Stanford ChEM-H, Stanford University, Stanford, CA 94305, USA. Electronic address: herschla@stanford.edu.
authors.forename: Daniel
authors.initials: D
authors.lastname: Herschlag
mesh_terms: D064130:CRISPR-Associated Proteins / Q000235:genetics; D064113:CRISPR-Cas Systems / Q000235:genetics / Q000502:physiology; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004720:Endonucleases / Q000235:genetics; D004798:Enzymes / Q000493:pharmacokinetics; D000072669:Gene Editing; D005818:Genetic Engineering; D006801:Humans; D007700:Kinetics; D012313:RNA / Q000737:chemistry; D034622:RNA Interference / Q000502:physiology*; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000737:chemistry*; D015203:Reproducibility of Results; D012260:Ribonucleases / Q000235:genetics / Q000493:pharmacokinetics*
chemical_list: D064130:CRISPR-Associated Proteins; D004798:Enzymes; D000094704:RNA, Guide, CRISPR-Cas Systems; D012313:RNA; D004720:Endonucleases; D012260:Ribonucleases