According to PubMed, over the last four years the number of scientific papers with CRISPR-Cas in the title has soared from 45 to 501. But why the sudden burst of interest in a relatively obscure immune response found only in prokaryotes? Nicola McCarthy, Oncology Program Manager for Horizon Discovery, uses some recent publications to explain their growing obsession.
Clustered regularly interspaced short palindromic repeats (CRISPRs) and the CRISPR-associated (Cas) genes were first described in Bacteria and Archaea by Ruud Jansen and colleagues1 in 2002. This rudimentary prokaryotic ‘immune response’ has been thrust into the limelight recently because the nuclease activity of the Cas proteins is being harnessed to engineer the mammalian genome in radically new ways.
Cas nucleases cleave DNA and can be guided to specific DNA sequences through the use of single guide RNAs (sgRNAs). The DNA strand breaks that Cas nucleases induce are repaired by a variety of DNA repair pathways which can often lead to the generation of insertions or deletions (indels). These indels can result in missense mutations, thereby preventing the expression of a targeted gene, for example. Hence, sgRNA-Cas has been used for genome wide knockout screens and is a useful alternative to current techniques such as siRNA and shRNA screens, which can have substantial off target effects. If, in addition to the sgRNA-Cas construct, a template for DNA repair is included, specific mutations can be introduced through homologous directed DNA repair. Thus, at Horizon Discovery we are using sgRNA–Cas along with rAAV vectors as templates to generate isogenic cell lines with specific point mutations in addition to carrying-out genome wide and gene specific screens.
However, this is not the only reason we have an invested interest in this technology. The uses of sgRNA–Cas are expanding rapidly and include the generation of relevant tumour models for the identification and validation of new and efficacious treatments for cancer. Two recent papers published in Nature nicely illustrate this.
In the first paper, Andrea Ventura and colleagues2 used sgRNAs to direct Cas9-mediated DNA cutting to the Eml4 and Alk genes in the lung. This resulted in some of the lung cells having an Eml4–Alk inversion, mimicking the EML4–ALK inversion that occurs in a subset of patients with non-small-cell lung cancer. Indeed, mice with the engineered Eml4–Alk inversion developed lung lesions followed by lung tumours. Importantly, treatment of these mice with the ALK inhibitor crizotinib resulted in complete disease regression in the majority of mice, verifying that this mouse model mimics patients with ALK positive non-small cell lung cancer.
Taking a slightly different approach, Tyler Jacks and colleagues3 used sgRNA–Cas9 to examine how the deletion of different tumour suppressor genes (Nkx2-1, Pten and Apc) impacts lung cancer development driven by Kras mutations in mice. Their results support the evidence that the underlying genetic background of the tumour effects its development and how aggressive it is likely to be.
Use of these types of models during preclinical drug development might better support target identification and validation, as well as informing patient selection and stratification. Importantly, these kinds of approaches might also help us to determine why some mutations are selected for during tumour development and thereby identify new targets for drug development.
- Jansen, R., Embden J.D., Gaastra, W., & Schouls, L.M. Identification of genes that are associated with DNA repeats in prokaryotes. Mol Microbiol. 43, 1565-1575 (2002).
- Maddalo, D., et al. In vivo engineering of oncogenic chromosomal rearrangements with the CRISPR/Cas9 system. Nature. doi 10.1038/nature13902
- Sanchez-Rivera, F.J, et al. Rapid modelling of genetic events in cancer through somatic editing. Nature. doi 10.1038/nature13906
If you’d like more information on CRISPR genome editing, Horizon Discovery have created a video which can be watched here