A Study of CRISPR Ribonucleoprotein Displacement in Cell-Free Systems

CRISPR/Cas-based transcription factors are a powerful
tool for
controlling gene expression in living cells and cell-free systems,
as their programmable DNA-binding activity makes them a powerful tool
for building and scaling up engineered genetic networks. The use of
guide RNAs for targeting Cas proteins to desired binding sites opens
up the possibility of using RNA engineering techniques to achieve
programmable and dynamic control of CRISPR/Cas-based transcription
factor activity and hence of gene expression. In this work, we investigate
the use of RNA strand displacement systems to remove bound CRISPR/Cas
ribonucleoprotein complexes from target DNA in cell-free systems.
The binding of catalytically inactive dCas9 is monitored by using
CRISPR interference to repress the expression of a reporter protein.
We express an antisense RNA complementary to an extended toehold on
an engineered guide RNA in an E. coli-based cell-free expression system with the goal of rapidly removing
bound CRISPR/Cas ribonucleoproteins via strand displacement. We find
that dCas9 appears to be surprisingly resistant to removal via this
mechanism, which indicates that other strategies for dynamic removal
of bound Cas proteins may prove to be more effective.