Multilayer Genetic Safety Kill Circuits Based on Single Cas9 Protein and Multiple Engineered gRNA in Mammalian Cells


This invention is a multifunctional CRISPR system to engineer genetic circuits (simple and multi-layers) that can generate safety off and kill switches with lesser genetic materials than current methods.

Problem Addressed

Gene and cell-based therapies have revolutionized cancer therapy and other hard to treat diseases, but additional regulatory mechanisms to control for specificity of these biological treatments or minimize off target effects are greatly needed. Accordingly, the development of synthetic genetic circuits in mammalian cells has become increasingly faster and efficient due to CRISPR. However, as genetic circuits become more complex they become more difficult to engineer due to the metabolic load they create on cells. This invention is a multi-layered and complex genetic kill switch in human cells which utilizes a single Cas9 protein with multiple functionality.


Multiple functionality (cleaving and transcriptional activation/repression) of the modified Cas9 protein, a Cas9 nuclease fused to an activation domain (VPR), is useful because it provides better flexibility to engineer complex and multilayer genetic switches since these functionalities are achieved by altering small and easy to engineer guide RNAs (gRNAs). This technology technology is based on the discovery by the Church lab that truncating gRNA from the 5’ end decreases nuclease activity of Cas9-VPR complex while retaining its DNA binding capacity. The inventors have developed several genetic kill switches (and off switches) with increasing complexity using shared and single Cas9-VPR and multiple gRNAs of different length. The lesser DNA footprint (sallows generation of circuits that can be better packaged into delivering vehicles such as viruses that contain load limit for therapeutic potentials.  Furthermore, this technology is easy to engineer and has great potential for therapeutic application (safety switches).


  • Easy to engineer
  • Safety switches for therapeutics
  • Allows more complex systems which may be better packaged