Barcoding CRISPR/Cas9 improves the search for novel drug targets

Development of a novel barcoded CRISPR screening library for next generation target discovery

Scientists in the IMED Biotech Unit at AstraZeneca working with colleagues at the IMBA, Vienna BioCenter, have developed a new approach, termed CRISPR-UMI (Unique Molecular Identifier). In the recent issue of Nature Methods, the authors describe how they have added thousands of short unique sequences, or barcodes, to each guide RNA present in a gene library to improve the efficacy of how we can identify novel gene targets for drug discovery.

 

This has been a really great collaboration with Dr. Elling at IMBA that we started through our Ideas Incubator programme in IMED – think a friendly version of Dragon’s Den where blue-skies ideas get pitched to a panel of experts for funding opportunities. We were successful in our pitch and together we developed a novel way to tag thousands of guide-RNA’s - the warheads that take the CRISPR/Cas9 machinery to the gene, with unique signatures. The addition of these unique signatures enables us to home-in on gene targets of interest

said Roberto Nitsch, Discovery Sciences Senior Scientist, IMED Biotech Unit.

Since the advent of CRISPR/Cas9 technology, one of its widest applications has been in ‘recessive genetic screening’. In these large cellular screens, CRISPR/Cas9 is used to knock-out thousands of specific individual genes so researchers can discover novel links to any observed cellular effects associated with disease. By adding these unique barcodes to each gene knockout means it is now possible to interrogate the effects of gene-loss on the single cell level. This development has a huge advantage over the current ‘pooled’ CRISPR screening approaches as they only report effect on a population-basis. Even more, the specificity of the barcodes also means we can look at cell-to-cell genetic variation to better represent disease heterogeneity.

 

One drawback of the great technology CRISPR/Cas9 is that after the precise cut of the gene, the genetic outcome of the repair mechanism cant be controlled and generates a mixture of knockout and wild-type cells. However, on the single cell level, each cell is either wild-type or knockout. By adding unique fingerprints to each cell, we can now overcome this drawback in CRISPR-Cas9 screens and exploit it’s full potential for gene-discovery.” says Ulrich Elling, group leader at IMBA (Institute of Molecular Biotechnology Austria) at the Vienna BioCenter.

says Ulrich Elling, group leader at IMBA (Institute of Molecular Biotechnology Austria) at the Vienna BioCenter.

In order to demonstrate the efficiency of this approach, two proof of principle screens were performed. The first aimed to identify genes that sensitised cells to the DNA-damaging agent, etoposide. As expected, genes involved in the repair of DNA, notably in the Non-Homologous End Joining repair pathway were identified but in addition, several unanticipated genes such as Abcc1, Zfp451, Rad9a and Erbb4 were also identified. “These genes represent potential new drug targets for combination therapy in cancer.” says Georg Michlits, first author of the study, “and we even showcase the power of CRISPR-UMI in a second screen, where we identified the gas pedal for reprogramming skin cells back to pluripotent stem cells.”

Whilst in its infancy, this novel screen is already having impact in the drug discovery of novel targets across AstraZeneca’s therapy areas and in particular helping to identify mechanisms of resistance to our DNA Damaging Response portfolio in Oncology.

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