Photo credits: JBS authors; top center image: Dan Cojocari [CC BY-SA 3.0 (http://creativecommons.org/licenses/by-sa/3.0)]; top right image: Opabinia regalis [CC-BY-SA-3.0 (http://creativecommons.org/licenses/by-sa/3.0/)].
RNAi is undergoing a "renaissance," according to the guest editors of the September 2015 JBS Special Issue on Screening by RNAi and Precise Genome Editing Technologies. The proof is in the issue's carefully selected papers, which reveal novel approaches and bioinformatics tools that are reducing the off-target effects and other problems that have caused researchers to question the value of the technology in the past.
A number of recent advances have helped "rehabilitate" RNAi, write Marc Bickle, Ph.D., of the Max Planck Institute of Molecular Cell Biology and Genetics in Dresden, Germany; Hakim Djaballah, Ph.D., of the Institut Pasteur Korea in Seongnam, South Korea; and Lorenz Martin Mayr, Ph.D. of AstraZeneca in Cambridge, UK, in their introduction to the special issue. These advances convinced the guest editors that it was time to highlight the ways in which some potential RNAi pitfalls are being addressed.
"We now have tools that allow us to use RNAi strategies in primary cells, minimize off-target effects and rapidly obtain high quality screening data," says Bickle. Djaballah adds that "the technology is on the rebound, but in a more conservative way than in previous years. Researchers are careful about the assays they're developing and about interpreting the data their work yields. We know RNAi is helpful when we go after genes of interest. While it hasn't been as useful yet for finding the needle in the haystack (identifying a gene based on a specific effect), the new tools will help."
Discovered in 1998 by Andrew Fire and Craig Mello, who shared a Nobel prize for the finding, RNAi is a natural biological process involving the production of small double-stranded RNA molecules, usually 20-25 base pairs in length. Those small interfering RNAs (siRNAs) bind to messenger RNA (mRNA) molecules with complementary nucleotide sequences, inhibiting the expression of specific genes.
According to the guest editors, "It was quickly recognized that this natural process could be harnessed using synthetic siRNAs or plasmid-encoded short hairpin RNAs (shRNAs) to down-regulate any gene. Whole genome siRNA libraries were synthesized and commercialized in the early 2000s leading to the development of automated RNAi transfection protocols on high-throughput liquid handling robots in high-density microtiter plates."
The pharmaceutical industry began using RNAi technologies to investigate specific genes as potential targets for small and large molecules, and to support decision making about whether or not to move forward on a drug-development project. But as problems such as off-target effects emerged, "people went from celebrating the new technology to deciding it was absolutely worthless," Bickle says. "That's because the expectations were too high, and the technology wasn't mature enough to deliver what was expected of it."
Those expectations were driven in part by the fact that RNAi was developed in academic centers and the results were published in high-profile publications. "There was lots of publicity and few questions asked about what was really happening," Djaballah observes. "Ultimately, reproducibility became an issue. In 2008, a comparison of the hit lists of three RNAi genome-wide screens against HIV replication showed an overlap of only three genes from a total of 834 identified as hits in various papers" (see references 3-6 in the From the Guest Editors column in the special issue). But since that low point, technologies have emerged that are making it easier to manage RNAi, validate targets and maximize RNAi's potential both for screening applications and therapeutic use.
These tools and approaches are among those helping to re-establish RNAi and are featured in the September 2015 special issue of JBS:
Power Decoder. Researchers using pooled shRNA screens to determine the biological relevance of genes for a specific phenotype will be especially interested in the Power Decoder Simulator developed by Jesse Stombaugh of Annaleen Vermeulen, Dharmacon (GE Healthcare) and colleagues, according to Bickle. "This open-source software tool, which is freely available for download, can tell you the power of your screen, whether you need to repeat it and whether it's robust enough to answer the questions you're asking," he says.
"Assessing an shRNA pooled screen's performance is difficult in practice; one can estimate the performance only by using reproducibility as a proxy for power or by employing a large number of validated positive and negative controls," the authors report. With the power decoder, a screen's statistical power, or sensitivity, can be estimated in silico by modeling the relative abundance of multiple shRNAs in a single screening replicate, as well as the biological noise between replicates for the individual shRNAs. "The ability to do fast, easy, accurate power analyses before screening will enable researchers to perform adequately powered experiments, thereby delivering reliable answers to crucial biological questions," they conclude.
Electroporation. A major hurdle for the use of RNAi in high-throughput screening is delivery to cells and tissues. "Even with virus-based delivery systems, we're still not very good at transfecting non-adherent cells, especially blood cells," Djaballah says. "The review article, Electroporation Knows No Boundaries: The Use of Electrostimulation for siRNA, by Robin Ketteler and Christin Luft of University College, London, UK, demonstrates that many companies are using the approach to develop new instrumentation, methodologies and reagents that enable cells to take in siRNA oligonucleotides."
"Electroporation is a powerful and versatile method for delivery of RNA, DNA, peptides and small molecules into cell lines and primary cells, as well as whole tissues and organisms," write Ketteler and Luft. Applying brief, high-intensity electrical pulses to cells leads to the formation of pores in the membranes that facilitate the uptake of macromolecules; turning the electrical pulses off allows the pores to reseal. Various parameters, including the intensity, duration and spacing of the pulses, determine the efficiency of the process, they explain. Their review includes a look at selected instruments for high-throughput electroporation in multi-well formats, barriers to widespread implementation of the method and devices currently in development.
Targeting Long Noncoding RNAs. Many transcripts identified through large sequencing efforts, such as ENCODE, are long noncoding RNAs (lncRNAs) that have no protein-coding potential. According to Djaballah, the paper by Frank Buchholz, formerly of the Max Planck Institute of Cell Biology and Genetics and now at Medical Systems Biology in Dresden, and colleagues "highlights a novel way to address the main questions everyone has about these transcripts: what is their function—especially those found in high frequency throughout a genome—and why are they made?"
In the report Targeting Human Long Noncoding Transcripts by Endoribonuclease-Prepared siRNAs, the authors explain that endoribonuclease-prepared siRNAs (esiRNAs) are pools of hundreds of individual siRNAs derived from a single target transcript, pooled together to reduce off-target effects. They note that an esiRNA library for targeting mouse lncRNAs has been used successfully in a loss-of-function screen, and they felt that, similarly, a library for screening human esiRNAs could be developed. Indeed, in this paper, they present the first generation of such a library, which targets 1779 different human lncRNAs. They also specify esiRNAs that can be used to target lncRNAs in commonly used cell lines and provide lncRNA expression data for 11 human cancer cell lines.
iScreen Technology. The application note by Guanghua Xiao of The University of Texas Southwestern Medical Center in Dallas, TX, and colleagues entitled iScreen: Image-Based High-Content RNAi Screening Analysis Tools, is one of two that describe ways to obtain multidimensional data from high-content RNAi screens—an important advance, according to Bickle. "Now that there are tools to help researchers get into primary cells, we still have to worry about off-target effects. These multivariate tools can help by analyzing multidimensional data while controlling for potential confounders."
iScreen is an R package—freely available software for the statistical modeling and visualization of image-based high-content screening. Xiao and colleagues describe two case studies that demonstrate the capabilities of the software, and state that in addition to using the default analysis functions, users have the option of integrating other software or their own analytic functions into the package to perform customized data analysis.
Multivariate Robust Analysis Method. In A Multivariate Computational Method to Analyze High-Content RNAi Screening Data, Michael Yaffe of the Massachusetts Institute of Technology in Cambridge, MA, and colleagues observe that most multivariate techniques applied to high-content RNAi screens are "complicated, multistep" procedures. Therefore, they write, "the majority of researchers pursuing HCS still rely on univariate methods to analyze multidimensional screening data. Analyzing multidimensional data with univariate methods is a self-imposed bottleneck that makes HC assays factually low content."
To address these and other challenges to RNAi screening, the team developed the multivariate robust analysis method (M-RAM)—described in detail in the paper and supplementary materials—applied it in the laboratory and attained meaningful results, including "transform[ing] shRNA-level data into gene-level data, capturing consistency and variability of differential shRNA effects and achiev[ing] a nearly sevenfold reduction in dimensionality."
Much has been said and written about whether a relative newcomer to gene editing, CRISPR (clustered regularly interspaced short palindromic repeats), will supplant RNAi. Therefore, Bickle, Djaballah and Mayr include two relevant review papers in the JBS special issue—one by Mark Wade of Instituto Italiano di Tecnologia in Milan, Italy and the other by Simon Woodcock and Jessica Taylor of AstraZeneca, Cheshire, UK. Both reviews explore the potential of CRISPR technologies and compare them to RNAi technologies, and both conclude that the technologies complement, rather than compete, with each other.
Djaballah and Bickle share that perspective. "Certainly with respect to screening, CRISPR is still in its infancy, with only a handful of published papers so far," Djaballah says. "In addition, there are things RNAi can do—for example, targeting non-coding RNA sequences—that can't be done with CRISPR." Rather than rushing into the CRISPR space, as happened earlier with RNAi, researchers are being more cautious. "They're developing small, focused libraries for CRISPR, rather than genome-wide libraries. In addition, some will use RNAi technologies to confirm CRISPR findings, whereas some journals are asking for CRISPR confirmation of RNAi results." Djaballah currently is working on a project to compare the results of functional screens using an siRNA library, an shRNA library and a CRISPR library.
"The overall take-home message from this JBS special issue is that all is not lost for RNAi," Djaballah affirms. "RNAi is still here; we just need to be a bit patient and look to the next two-to-five years to see whether the enhanced tools we've discussed will result in the discovery and validation of new targets, as well as increase the potential of RNAi for therapeutic uses." Bickle, who currently is working with RNAi in 3D cell cultures and as a potential therapeutic for liver conditions, agrees. "RNAi is still kicking and will be with us for years to come, as CRISPR and other gene editing technologies work out the challenges that they now face, and will continue to face in the future." [Ed. note: An SLAS2016 Short Course, Gene Editing for Drug Discovery, will be presented on Sunday, January 24, 2016; SLAS will host a webinar, Introduction to Gene Editing for Drug Discovery: Pooled Genetic Screens on Sept. 30; and an SLAS e-zine editorial feature is available.]
In addition to the contributions from Stombaugh, Ketteler and Luft, Buchholz, Xiao and Yaffe, the JBS Special Issue on Screening by RNAi and Precise Genome Editing Technologies features additional review articles and original reports on technological and methodological advances that have alleviated many of the challenges that hindered the progress of RNAi in recent years.
The special issue is part of the SLAS commitment to improving access to information for health professionals, scientists and policy makers around the world, according to SLAS President Dean Ho. SLAS participates in the World Health Organization's Research4Life initiative by providing free or low-cost access to JBS and JALA to more than 6,000 publicly funded non-profit institutions in over 100 countries and territories in the developing world. In addition, deeply discounted membership rates make it easier for life sciences R&D professionals in emerging economies to join SLAS and enjoy full access to its many programs, products, services and events.
August 31, 2015