Eric Rhodes Chief Technology Officer Horizon Discovery

Over the last 10 years, genome-scale RNA-interference screens have been widely established as important tools to investigate mammalian functional genomics.

Large-scale pooled-format screens employing libraries of shRNAs in retroviral/lentiviral vectors have identified genes whose silencing conferred resistance to insults, or whose silencing led to inviability or reduced proliferation. Initially these screens employed shRNA barcoding coupled with transcriptional arrays for analysis; more recently, next-generation sequencing has amplified the power and flexibility of this approach. RNA-interference screens, however, have two major limitations: incomplete knockdown and off-target effects.

RNA-guided Cas9 nuclease is revolutionizing gene editing in mammalian cells. This key component of an evolutionally ancient bacterial adaptive immunity system has been engineered to be active in the nucleus, where engineered single-guide RNAs (sgRNAs) can target the nuclease to any 17-20 nucleotide sequence where it generates a double stranded break that when repaired by non-homologous end joining creates small insertions and deletions that inactivate gene function.

sgRNAs are close in size (70–80 nt) to the short-hairpin RNAs that are used in RNA interference screens. So can the Cas9/sgRNA system be readily adapted to the existing shRNA lentiviral screening infrastructure to enable genome-wide knockout screening? And would this technology overcome some of the disadvantages of RNA interference?

This year both these questions have been answered with a definitive “Yes.” Four reports have been published presenting the results of whole genome screens using lentiviruses to deliver Cas9 and sgRNA libraries into human or murine cells and next-generation sequencing to quantify the depletion or accumulation of the integrated lentiviral DNA sequences specifying each sgRNA [1-4].


CRISPR gene-targeting

All four papers demonstrate the superlative performance of sgRNA in positive selection screens (where editing of the sgRNA target confers a proliferation advantage). For example, Feng Zhang’s group found that the frequency of all the sgRNAs targeting tumor suppressors whose loss is associated with resistance to the BRAF inhibitor vemurafenib were dramatically elevated in a treated population. Results with shRNAs were far less clear. Similarly, all 6 sgRNAs vs. enhanced green-fluorescent protein tested proved able to eliminate GFP expression in >95% of stably transduced cells, whereas shRNAs only reduced expression 7–30 fold.  

sgRNA screening is set to become an important tool in oncology for identifying synthetic lethal/co-dependence targets and can also be applied to other therapeutic areas where pathway function can be engineered to regulate survival or expression of fluorescent markers.


Cells deleted for tumor suppressors accumulated under vemurafenib selection

Eric Rhodes (e.rhodes@horizondiscovery.com) is SVP, R&D and CTO of Horizon Discovery.

References:
1. Wang T, Wei JJ, Sabatini DM, Lander ES. Genetic screens in human cells using the CRISPR-Cas9 system. Science, 343: 80-84 (2014)
2. Shalem O, Sanjana NE, Hartenian E, Shi X, Scott DA, Mikkelsen TS, Heckl D, Ebert BL, Root DE, Doench JG, Zhang F. Genome-scale CRISPR-Cas9 knockout screening in human cells. Science, 343: 84-87 (2014).
3. Koike-Yusa H, Li Y, Tan EP, Velasco-Herrera Mdel C, Yusa K. Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library.  Nat Biotechnol. 32: 267-273 (2014).
4. Zhou Y, Zhu S, Cai C, Yuan P, Li C, Huang Y, Wei W. High-throughput screening of a CRISPR/Cas9 library for functional genomics in human cells. Nature, 509: 487-491 (2014).