Multiple Input Sensing and Signal Integration Using a Split Cas12a System

The ability to integrate biological signals and execute a functional response when appropriate is critical for sophisticated cell engineering using synthetic biology. Although the CRISPR-Cas system has been harnessed for synthetic manipulation of the genome, it has not been fully utilized for complex environmental signal sensing, integration, and actuation. Here, we develop a split dCas12a platform and show that it allows for the construction of multi-input, multi-output logic circuits in mammalian cells. The system is highly programmable and can generate expandable AND gates with two, three, and four inputs. It can also incorporate NOT logic by using anti-CRISPR proteins as an OFF switch. By coupling the split dCas12a design to multiple tumor-relevant promoters, we provide a proof of concept that the system can implement logic gating to specifically detect breast cancer cells and execute therapeutic immunomodulatory responses. [Display omitted] •Spontaneously dimerizing split Cas12a for construction of complex genetic circuits•Implementation of robust 2-, 3-, and 4-input AND gates to control endogenous genes•Split Cas12a, inducible guides, and anti-CRISPR allow higher order logic computation•Circuits can detect tumor-relevant signals for therapeutic gene expression Kempton et al. develop a split CRISPR-Cas12a toolbox for building multi-input, multi-output genetic circuits in mammalian cells. By splitting the Cas12a protein and effector into multiple components that can be individually controlled, they rationally engineer cells that can integrate information about multiple cues (e.g., tumor relevant) and execute programmed responses.