Unveiling Cas8 dynamics and regulation of a transposon-encoded cascade-TniQ complex.

Abstract

Csy (CRISPR-System Yersinia, or ‘Cascade’) is a class 1, type 1F CRISPR-Cas system which utilizes RNA-guided strand invasion to target viral DNA and designate it for cleavage by an external nuclease (Cas2/3). It has been recently shown, in Vibrio Cholerae, that there exists a variant of Cascade encoded in a transposon (Tn6677). This variant binds to a TniQ homodimer, resulting in a ‘Cascade-TniQ’ complex which, unlike the original Cascade that targets viral DNA for cleavage, recruits a heteromeric transposase responsible for integration of Tn6677. This integration process starts with binding of Cascade's crRNA to the DNA target sequence located in the bacterial genome, resulting in the formation of the ‘R Loop,’ this step is the focus of the current research. Both classical molecular dynamics (cMD) and enhanced sampling methods (GaMD) have been employed to better understand the dynamical state of the system in the RNA-only and the DNA-bound configurations. We have found evidence for a multi-state like behavior, with drastic differences in the state population and transition barriers upon DNA binding. Furthermore, it appears that the TniQ homodimer undergoes changes in large-scale conformations dependent on its level of intrinsic stability. This allowed us to make a tentative claim on a role of Zinc-coordination within TniQ's Zinc-finger binding domains. Tying our findings together with data from literature allowed us to make general comments on how the RNA-bound state and R Loop formation may be regulated. Hopefully, these findings will eventually allow for a more streamlined effort in re-engineering Cascade-TniQ for use in mammalian cells.