An In Vivo mutation Prevention System

¹Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, Massachusetts, USA.

²Department of Pathology, Massachusetts General Hospital, Boston, Massachusetts, USA.

³Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.

⁴Institute for Medical Engineering & Science, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

⁵Synthetic Biology Center, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

⁶Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

⁷Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.

⁸First two authors contributed equally

Point mutations have the remarkable ability to drastically alter organismal phenotype, conferring antibiotic resistance to bacterial pathogens and driving oncogenesis in healthy human cells. Despite a wealth of research into the mechanisms and implications of point mutations, there is no method to directly prevent their occurrence. To date we can only document when mutations occur and utilize this information to predict their effect on disease progression and chemotherapeutic outcome.

Through careful tuning of the RNA-guided endonuclease Cas9, we developed an in vivo “mutation prevention” system that can prevent the occurrence of targeted point mutations with little to no latent toxicity to the host organism.  We demonstrate the efficacy of the mutation prevention system by simultaneously inhibiting up to four targeted mutations that confer antibiotic resistance within the model organism E. coli.

Our results provide a general framework upon which to develop highly specific Cas9-gRNA complexes and make possible entirely new avenues of discovery in fields as diverse as molecular ecology and infection control.