Improving CRISPR gene circuit design using Physics and Synthetic Biology

The versatility of CRISPR-Cas endonucleases as a tool for synthetic biology has lead to diverse applications beyond gene editing that include 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 for CRISPR-Cas variants, we develop a massively parallel CRISPR interference assay to measure the binding energy between tens of thousands of target sequences. Using a general thermodynamic model of CRISPR-Cas binding dynamics, we unravel the complex energetic landscape of several CRISPR-Cas systems. Our approach provides a mechanistic understanding of target recognition and DNA binding of 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.