Abstract IA27: Retooling CRISPR to turn genes on and off

Abstract

Abstract 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. These experiments establish CRISPRi and CRISPRa as powerful tools that provide rich and complementary information for mapping complex pathways. Recently, we have used our CRISPRi/a functional genomics platform to determine the genetics of cellular response to HSP70 inhibitors, which are a new class of anti-cancer drugs targeting a non-oncogene addiction. Together, our experiments demonstrate the utility of our CRISPRi/a functional genomics approach for studying principles of oncogene and non-oncogene addiction in cancer biology and cancer therapy. Citation Format: Luke A. Gilbert, Max A. Horlbeck, Jacqueline Villalta, Shao Hao, Jason E. Gestwicki, Jonathan S. Weissman. Retooling CRISPR to turn genes on and off. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr IA27.