Massively parallel CRISPRi assays reveal concealed thermodynamic determinants of dCas12a binding

Significance Cas12a is a type V CRISPR-Cas endonuclease that relies on a programmable guide RNA to bind specific DNA sequences, and like most CRISPR-Cas systems, it suffers from spurious off-target binding. Improving the specificity of Cas12a is an important step toward its broader use as a tool for biomedical research, but a fundamental understanding of the biophysical determinants of nonspecific Cas12a binding is still lacking. Here, we developed a massively parallel CRISPR interference assay to reveal the thermodynamic factors involved in Cas12a off-target binding. Since our approach uses a parameter-free thermodynamic model that is broadly applicable to any RNA-guided endonucleases, our work should enhance our understanding of on- and off-target binding when applied to other CRISPR-Cas systems.