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 below 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.
Have you been overwhelmed by the number of CRISPR articles published in 2017? PubMed alone has cited over 3,000 CRISPR publications in 2017! We wanted to save you from having to sieve through the databases by asking our experts to select what they thought were the most important CRISPR publications from last year.
A revolution is under way in functional genomics which is spearheaded by the CRISPR-Cas9 system and its application to pooled genetic screening. Remarkable new tools, made possible by dCas9, are coming to fruition that will allow for a new kind of interrogation of gene function, allowing us to ask more sophisticated questions about the biology of drug targets.
January 2013 was marked by a major breakthrough in genome engineering. Four labs simultaneously engineered the bacterial and archaeal CRISPR-Cas9 system to induce precise cleavage at mammalian genomic loci1–4. Within a year’s time, two back-to-back papers documented the application of CRISPR-Cas9 knockout technology to forward genetic screening5,6. These studies showed not just proof of concept of a new technology, but a spectacular jump in what is possible within functional genomics. Many studies have since capitalised on these discoveries and several publications, including from Horizon7, have demonstrated screening platforms with even greater precision and performance.
Recently published in a paper on Nature.com in Scientific Reports, Horizon Discovery have conducted a detailed analysis of CRISPR-Cas9 sensitivity (drop-out) screening to come up with a highly improved and optimised platform. In our analysis, we used a custom ultra-complex sgRNA library and capitalised on Horizon’s streamlined screening pipeline to evaluate fundamental aspects of functional genomic screening, including:
- Side-by-side comparison of the impact of a novel tracrRNA sequence on screen performance
- Direct analysis of the efficacy of two different sgRNA design algorithms
- Evaluation of the effect of cell line ploidy on KO rate and screen quality
- Time-resolved gene drop-out analysis to evaluate the kinetics of CRISPR-Cas9 driven gene knockout
Thanks to next generation sequencing (NGS), we are starting to understand the mutational changes that occur across the board in the cancer genome. With this knowledge comes potential – novel mutated genes and the proteins that they encode are candidates for prognostic markers and/or new drug targets.