Discovery of cancer drug targets by CRISPR-Cas9 screening of protein domains

Developers

Junwei Shi, Eric Wang, Joseph P Milazzo, Justin B Kinney, Christopher R Vakoc, etc.

Description of the technology

CRISPR-Cas9 genome editing technology holds great promise for discovering therapeutic targets in cancer and other diseases. Current screening strategies target CRISPR-Cas9-induced mutations to the 5′ exons of candidate genes, but this approach often produces in-frame variants that retain functionality, which can obscure even strong genetic dependencies.

The proposed technology overcomes this limitation by targeting CRISPR-Cas9 mutagenesis to exons encoding functional protein domains. This generates a higher proportion of null mutations and substantially increases the potency of negative selection. It was also showed that the magnitude of negative selection could be used to infer the functional importance of individual protein domains of interest. The RNA-guided endonuclease Cas9, a component of the type II CRISPR (clustered regularly interspaced short palindromic repeats) system of bacterial host defense is used a tool for genome editing in this technology. Ectopic expression of Cas9 and a single guide RNA (sgRNA) is sufficient to direct the formation of a DNA double-strand break (DSB) at a specific region of interest. In the absence of a homology-directed repair DNA template, these DSBs are repaired via the non-homologous end joining pathway to generate an assortment of short deletion and insertion mutations (indels) in the vicinity of the sgRNA recognition site. The capabilities of CRISPR-based genetic screens are particularly evident in the performing of positive selection, such as identifying mutations that confer drug resistance. In negative selection screens, it has been shown that sgRNA hits are statistically enriched for essential gene classes (ribosomal, RNA processing, and DNA replication factors).

A screen of 192 chromatin regulatory domains in murine acute myeloid leukemia cells identified six known drug targets and 19 additional dependencies. A broader application of this approach may allow comprehensive identification of protein domains that sustain cancer cells and are suitable for drug targeting.

Practical application

This technology is a potentially valuable approach to identify cancer dependencies suitable for pharmacological inhibition; herewith sgRNA libraries should be designed to target exons that encode protein domains.

Thus, the technology exploits utility of domain-focused CRISPR screening as a tool for anti-cancer drug target discovery.

Laboratories

• Cold Spring Harbor Laboratory, Cold Spring Harbor (USA)
• Molecular and Cellular Biology Program, Stony Brook University, Stony Brook (USA)

Links

Publications

• Shi, J. et al. «Discovery of cancer drug targets by CRISPR-Cas9 screening of protein domains." 33.6 Nat Biotechnol. (2015): 661−667.
• Shi, J. & Vakoc, C.R. «The mechanisms behind the therapeutic activity of BET bromodomain inhibition." 54 Mol. Cell (2014): 728–736.
• Shi, J. et al. «Role of SWI/SNF in acute leukemia maintenance and enhancer-mediated Myc regulation." 27 Genes Dev. (2013): 2648–2662.