RT Journal Article
SR Electronic
T1 Specificity Principles in RNA-Guided Targeting
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 055954
DO 10.1101/055954
A1 Namita Bisaria
A1 Inga Jarmoskaite
A1 Daniel Herschlag
YR 2016
UL http://biorxiv.org/content/early/2016/05/28/055954.abstract
AB 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. Here we demonstrate that simple kinetic considerations of the targeting reaction can explain these and other literature observations. The kinetic models described provide a foundation for understanding RGN systems and a necessary physical and functional framework for their rational engineering.