CRISPR-based drug target discovery : implications for CRISPR-based therapies

The CRISPR system was discovered in prokaryotic cells, and it is now one of the most efficient molecular tools to modify genes in eukaryotic cells. It is widely used to inactivate or modify genes in cell lines, animal models, and is also used in several clinical gene therapy trials. Additionally, CRISPR-based screens have been developed as a high throughput methodology to identify drug targets and mechanisms contributing to diseases. However, safety concerns related to the CRISPR system have been emphasized, which challenges the clinical applications of this molecular tool. The consequence of CRISPR-induced DNA damage is one of the challenges and has so far been less studied.

In Paper I, we showed that CRISPR-induced DNA damage enriches for cells with mutations in genes of a core CRISPR-p53 tumor suppressor interactome. Such enrichment may contribute to cancer development and is a potential challenge for clinical CRISPR use. We also discovered that such enrichment could be suppressed by transient p53 inhibition. In addition, we discovered factors affecting the enrichment of p53 mutated cells from a database of >800 human cancer cell lines. In Paper II, we presented the Rapid CRISPR Competitive (RCC) assay, which is a rapid and universal experimental approach to discover potential drug targets. In this, we leverage the genetic heterogeneity induced by CRISPR and use Sanger sequencing to discover how different genes are involved in the studied cellular behavior or phenotype based on the enrichment or depletion of mutations. In Paper III, we identified that IL-4 can suppress B16-F10 melanoma tumor model growth by inducing a Gcn1l1 regulated amino acid deprivation response. We used gene expression analysis, mass spectrometry, and an in vivo CRISPR screen to link the potent therapeutic activity of IL-4 to ARG1-mediated arginine depletion and identify Gcn1l1 as a potential synergistic treatment target.

In conclusion, we extensively studied p53 biology in the context of DNA damage induced by CRISPR, and identified strategies for safer CRISPR use. We also developed a rapid and universal CRISPR-based experimental approach to discover potential drug targets. Finally, we use an in vivo CRISPR-based screen approach to discover a novel combinatorial cancer therapy.

List of scientific papers

I. Jiang L, Ingelshed K, Shen Y, Boddul SV, Iyer VS, Kasza Z, Sedimbi S, Lane DP, Wermeling F. CRISPR/Cas9-induced DNA damage enriches for mutations in a p53-linked interactome: implications for CRISPR-based therapies. Cancer Res. 2021 Nov 8:canres.1692.2021.
https://doi.org/10.1158/0008-5472.CAN-21-1692

II. Shen Y, Jiang L, Iyer VS, Raposo B, Dubnovitsky A, Boddul SV, Kasza Z, Wermeling F. A rapid CRISPR competitive assay for in vitro and in vivo discovery of potential drug targets affecting the hematopoietic system. Comput Struct Biotechnol J. 2021 Sep 20;19:5360-5370.
https://doi.org/10.1016/j.csbj.2021.09.020

III. Kasza Z, Jiménez-Andrade Y, Jiang L, Shen Y, Iyer VS, Boddul SV, Malin S, Jain M, Nilsson R, and Wermeling F. IL-4 can potently suppress tumor growth by inducing a Gcn1l1 regulated amino acid deprivation response. [Manuscript]