  CRISPR systems experience off-target effects that interfere with the ability
to accurately perform genetic edits. While empirical models predict off-target
effects in specific platforms, there is a gap for a wide-ranging mechanistic
model of CRISPR systems based on evidence from the essential processes of
target recognition. In this work, we present a first principles model supported
by many experiments performed in vivo and in vitro. First, we observe and model
the conformational changes and discrete stepped DNA unwinding events of
SpCas9-CRISPR target recognition process in a cell-free
transcription-translation system using truncated guide RNAs, confirming
structural and FRET microscopy experiments. Second, we implement an energy
landscape to describe single mismatch effects observed in vivo for spCas9 and
Cas12a CRISPR systems. From the mismatch analysis results we uncover that
CRISPR class 2 systems maintain their kinetic proofreading mechanism by
utilizing intermittent energetic barriers and we show how to leverage the
landscape to improve target specificity.
