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

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Activation and cutting of a transcriptional reporter using gRNAs with progressively shorter 5’ end length. (a) Deletion analysis of truncated gRNAs on a synthetic transcriptional reporter. Samples were transfected with the indicated Cas9 construct and gRNA length. Data indicates the mean +/- s.e.m (n = 2 independent transfections).transfections). (b) Quantification of activation for truncated gRNAs via a fluorescent transcriptional reporter. Data indicates the mean +/- s.e.m (n = 2 independent transfections).
Professor Ron Weiss
Department of Biological Engineering, MIT
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Samira Kiani
Department of Biological Engineering, MIT
Mohammad Ebrahimkhani
Department of Biological Engineering, MIT
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Jon Gilbert
MIT Technology Licensing Officer
Patent Protection

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

PCT Patent Application WO 2017-040786
Cas9 gRNA engineering for genome editing, activation and repression
Nature Methods, September 7, 2015
Highly efficient Cas9-mediated transcriptional programming
Nature Methods, March 2, 2015


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