Uncovering thermodynamic determinants of CRISPR-Cas gene circuit design.

The versatility of CRISPR-Cas endonucleases as a tool for synthetic biology has lead to diverse applications in gene editing, programmable transcriptional control, and nucleic acid detection. Most CRISPR-Cas systems, however, suffer from off-target effects and unpredictable non-specific binding that negatively impact their reliability and broader applicability. To better evaluate the impact of mismatches on DNA target recognition and binding, we develop a massively parallel CRISPR interference assay to measure the binding energy between tens of thousands of CRISPR RNA and target DNA sequences. By developing a general thermodynamic model of CRISPR-Cas binding dynamics, our results unravel a comprehensive map of the energetic landscape of CRISPR-Cas as it searches for its DNA target. Our generalizable approach provides a mechanistic understanding of target recognition and DNA binding by CRISPR-Cas variants, which should contribute to the advancement of recent synthetic biology efforts to repurpose dCas as gene circuit elements that behave orthogonally and operate independently without crosstalk.