Understanding off-target effects through hybridization kinetics and thermodynamics.

In modern biotechnological and medical research, RNA-guided nucleases (RGNs) continue to be highly effective in targeted modification of genomes and the manipulation of gene expression (Sander and Joung [23]; Wang and Wang [30]). Improvement in specificity, on-target cleavage activity, and reduction of off-target-cleavage can be achieved through changes in CRISPR-derived nuclease, engineering of sgRNA, and/or Cas-sgRNA delivery modifications. Cas12a tightly binds DNA in two distinct kinetic stages, whereas PAM recognition is followed by a rate-limiting R-loop (a hybrid structure of Cas-RNA and target DNA) propagation. 4 Bisaria N, Jarmoskaite I, Herschlag D. Lessons from enzyme kinetics reveal specificity principles for RNA-guided nucleases in RNA interference and CRISPR-based genome editing. [Extracted from the article]