March 2016

CRISPR has only been around a short while but is already revolutionising the way we do biological research - allowing scientists to study gene function in more robust or even previously unimagined ways.

If you'd like to understand CRISPR-Cas9 better, and bring this technology into your lab, then we've created the following three webinars for you. In them you'll be introduced to CRISPR-Cas9, where it's from and how it works. You'll learn how to plan a genome editing experiment and the potential pitfalls to avoid. And finally we'll discuss the types of genomic modifications that can be made with CRISPR

Watch one of our recorded webinars:

Ras mutations are amongst the most commonly occurring mutations in human cancer, present in approximately 49% of colorectal and 20% of lung cancers. Of these, mutations in K-Ras G12 and G13 are the most common. Understanding the role of mutant K-Ras in modulating drug response is critical to the successful development of novel therapeutics, and has been hampered by the lack of suitable in vitro tools.

We have generated suites of SW48 and LIM1215 colorectal cancer cells which harbor one of 7 different K-Ras G12 or G13 mutations. This system uses endogenous promoters and enables panels of cell lines to be studied which differ only by the point mutation of interest, providing patient relevant in vitro model systems.

Our data highlight the advantages of using this system in profiling molecularly targeted agents in order to identify potential mechanisms of resistance, and in rationally identifying combinations.

The discovery of the CRISPR‐Cas system in bacteria has initiated an impressive array of innovations that have enabled the use of the RNA‐guided Cas9 nuclease in functional genomic screens.

At Horizon, we have embraced these developments, as they provide new opportunities for drug target identification and validation. The case studies presented in this article highlight how we use this technology to successfully conduct genome wide and focused sgRNA library screens and to verify whether specific genes are required for the survival and/or proliferation of cancer cell lines.

We were interested in exploring novel ways to target tumour cells bearing mutant IDH1 alleles that were distinct from the obvious opportunity available to identify mutant-specific IDH1 inhibitors. The potential metabolic vulnerabilities of mutant IDH1 cancers raised the possibility that wild-type IDH1 might be essential for tumourigenesis or tumour maintenance in this context.

We therefore employed Horizon’s rAAV-mediated homologous recombination gene engineering technology to generate conditional knockouts of the IDH1+ or IDH1R132C alleles in the fibrosarcoma cell line, HT1080.

Isogenic panels of colorectal cancer cell lines can effectively model KRAS-mediated resistance to inhibitors of EGFR and MEK, and can be used to stratify patient-relevant genotypes into drug responsive and non-responsive populations.

Next generation screens that exploit both isogenic cell lines and cancer cell panels, and use a combination of knockdown (si/shRNA) and knock-out (CRISPR-Cas9-sgRNA) methodologies might be more effective at identifying novel targets that withstand validation. However, if we are to detect co-dependence as well as synthetic lethal interactions, screens must be performed under conditions where mutant KRAS alleles are essential for growth.

At Horizon Discovery we are combining our expertise in cancer cell biology and functional genomic screening to provide a more definitive target validation cascade in KRAS mutant cell lines. Our initial results indicate that RAF1 is an important mediator of mutant KRAS biology.

Optimised CRAF inhibitors might provide a well-tolerated therapy for lung cancer as long as established lung cancers remain dependent on CRAF for viability. To address this, we carried out a large shRNA validation studyassess the dependence of a panel of human non-small cell lung cancer (NSCLC) cells on KRAS and CRAF.

Plasmid DNA, PCR products, and single stranded oligonucleotides are routinely used as donors to introduce specific changes at the DSB site. The efficiency of introducing a desired change is dependent on many factors including

• the type of donor
• the length of homology
• the complexity of the desired change
• characteristics specific to the cell line

We have looked at whether rAAV vectors can be used as donors for DNA modification to obtain higher efficiencies than seen with other donor approaches.

While rAAV incorporates targeted changes without the requirement of a double strand DNA-break (DSB) it has been demonstrated that introduction of a DSB by nucleases such as CRISPR further increases the rates of targeted incorporation of the rAAV donor.

Below we detail the approaches we've taking to improving rAAV editing efficiency still futher, including:

• DNA mismatch repair pathway inhibition by RNA interference
• incorporation of negative selection into rAAV donors
• combination of rAAV and CRISPR with the above approaches

There exist now a range of techniques to perform genome editing, such as ZFN, CRISPR, TALENS and AAV, each with their own strengths and weaknesses. However, one consistent element that has a significant impact on the success of that editing event when generating an isogenic cell line is the choice of parental cell line to be engineered.

Below we detail Horizon's characterisation approach to select cell lines which not only improves the chance of gene editing success, but can also drive the selection of which engineering technique to use.

In particular, we highlight the benefits of assessing

• conditions required to generate clonal populations of a cell line
• copy number of the genetic locus of interest
• ability of the cell line to tolerate delivery of the targeting reagents by a variety of methods.