CRISPR (clustered regularly interspaced short palindromic repeats) is an adaptive genome defense system in bacteria and archaea. CRISPR has been widely repurposed as an RNA-guided genome editing method. We have shown that the endonuclease domains of the Cas9 protein can be mutated to create a programmable RNA-guided DNA-binding protein. In human cells, we have shown we can control transcription of endogenous coding and non-coding genes over a wide range (up to 1000 fold) by using dCas9 chimeras to deliver protein domains that either repress (CRISPRi) or activate (CRISPRa) transcription. These experiments provide proof of principle that we can use dCas9 as an RNA-guided DNA binding platform to deliver in theory any protein to a specific genomic location, which may be useful for studying transcription, epigenetic regulation, DNA replication and DNA repair. We have developed a genome-scale CRISPRi/a functional genomics platform for loss- and gain-of-function screening in human cells. We have demonstrated that our CRISPRi/a screening platform can robustly identify pathways and proteins required for cancer cell growth and survival. Gain-of-function CRISPRa screens promise to reveal a layer of genetic information missing from previous efforts. We have shown in a cell growth screen that CRISPRa reveals latent tumor suppressors and transcription factors that regulate cancer cell state in chronic myeloid leukemia cells. In proof of principle, we have also used CRISPRi/a screens to determine the mechanism of cellular uptake, trafficking, retro-translocation, and toxicity for a chimeric bacterial toxin. We have now applied our CRISPRi/a functional genomics platform as a new tool in chemical biology. We have shown we can use functional genomics to determine mechanisms of cellular response to new drugs and to identify the direct molecular target of candidate anti-cancer drugs.

Retooling CRISPR to turn genes on and off

253rd National Meeting (2017)

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.