New from my lab in @NatureRevGenet - we take a deep dive into the tools and platforms that can be used to discover and characterize cancer drug targets. nature.com/articles/s4157…
Work from my lab and from several others has demonstrated that many genes previously reported to be cancer-essential are in fact dispensable for cancer growth. 
In many cases, these incorrect results were due to experiments done with off-target RNAi reagents and/or promiscuous small molecules.
How can we solve this problem? On one level, head-to-head comparisons clearly show that CRISPR is less prone to off-target artefacts when compared to RNAi - but CRISPR is absolutely not a perfect system.
CRISPR causes DNA damage - potentially resulting in false-positives - and mRNA misregulation - potentially resulting in false-negatives. No single approach is flawless!
Every genetic perturbation system has its benefits and limitations. CRISPR-interference gives you the specificity of CRISPR without DNA damage, but you need to know the gene’s TSS. New systems like Cas12 are easy to multiplex but may suffer from lower on-target activity.
We suggest that the key is orthogonal genetic validation: hit a target with CRISPR and RNAi, or Cas9 and Cas12. The more ways you prove a point, the less likely it is to represent a false-positive.
The same goes for small molecules. The gold-standard for on-target drug activity is if you can rescue the effect with a point mutation that blocks drug binding. Crystal structures, in vitro activity assays, and CETSA are great but are not definitive.
At the same time, resistance mutations aren’t possible for every gene in the genome, and sometimes resistance mutations occur in genes that aren’t a drug’s direct target (e.g., BRAFi resistance mutations in NRAS or MEK1). So resistance mutations aren’t actually definitive either!
For drugs, orthogonal validation using multiple independent modalities - both genetic and biochemical - is the best path forward.
Finally - how do we even know if something is a good cancer drug? Is it something that kills MDA-MB-231 breast cancer cells in a petri dish? Sunlight and bleach will do that too - but I promise you that neither will actually cure cancer.
Cell lines are a mainstay of cancer research and provide immense experimental flexibility - but many have spent decades evolving to optimize their growth on plastic when fed sugar water with cow’s blood. Not very physiological!
Newer approaches, like patient-derived organoids and xenografts, may better reflect the genetics and environments of real tumors. Showing that something is or isn’t essential in MDA-MB-231 is a good start - but it’s far from definitive. Validate targets in different systems!
I hope that this paper can serve as a useful reference to the cancer research community. I’m happy to do my best to answer any questions that people have!
