November 1, 2015 (Vol. 35, No. 19)
Gene-Silencing and Gene-Disabling Techniques Are Moving To the Heart of Drug Discovery
Genes can be knocked down with RNA interference (RNAi) or knocked out with CRISPR-Cas9. RNAi, the screening workhorse, knocks down the translation of genes by inducing rapid degradation of a gene target’s transcript.
CRISPR-Cas9, the new but already celebrated genome-editing technology, cleaves specific DNA sequences to render genes inoperative. Although mechanistically different, the two techniques complement one another, and when used together facilitate discovery and validation of scientific findings.
RNAi technologies along with other developments in functional genomics screening were discussed by industry leaders at the recent Discovery on Target conference. The conference, which emphasized the identification and validation of novel drug targets and the exploration of unknown cellular pathways, included a symposium on the development of CRISPR-based therapies.
RNAi screening can be performed in either pooled or arrayed formats. Pooled screening provides an affordable benchtop option, but requires back-end deconvolution and deep sequencing to identify the shRNA causing the specific phenotype. Targets are much easier to identify using the arrayed format since each shRNA clone is in an individual well.
“CRISPR complements RNAi screens,” commented Ryan Raver, Ph.D., global product manager of functional genomics at Sigma-Aldrich. “You can do a whole genome screen with either small interfering RNA (siRNA) or small hairpin RNA (shRNA), then validate with individual CRISPRs to ensure it is a true result.”
A powerful and useful validation method for knockdown or knockout studies is to use lentiviral open reading frames (ORFs) for gene re-expression, for rescue experiments, or to detect whether the wild-type phenotype is restored. However, the ORF randomly integrates into the genome. Also, with this validation technique, gene expression is not acting under the endogenous promoter. Accordingly, physiologically relevant levels of the gene may not be expressed unless controlled for via an inducible system.
In the future, CRISPR activators may provide more efficient ways to express not only wild-type but also mutant forms of genes under the endogenous promoter.
Choice of screening technique depends on the researcher and the research question. Whole gene knockout may be necessary to observe a phenotype, while partial knockdown might be required to investigate functions of essential or lethal genes. Use of both techniques is recommended to identify all potential targets.
For example, recently, a whole genome siRNA screen on a human glioblastoma cell line identified a gene, known as FAT1, as a negative regulator of apoptosis. A CRISPR-mediated knockout of the gene also conferred sensitivity to death receptor–induced apoptosis with an even stronger phenotype, thereby validating FAT1’s new role and link to extrinsic apoptosis, a new model system.
Dr. Raver indicated that next-generation RNAi libraries that are microRNA-adapted might have a more robust knockdown of gene expression, up to 90–95% in some cases. Ultracomplex shRNA libraries help to minimize both false-negative and false-positive rates by targeting each gene with ~25 independent shRNAs and by including thousands of negative-control shRNAs.
Recently, a relevant paper emerged from the laboratory of Jonathan Weissman, Ph.D., a professor of cellular and molecular pharmacology at the University of California, San Francisco. The paper described how a new ultracomplex pooled shRNA library was optimized by means of a microRNA-adapted system. This system, which was able to achieve high specificity in the detection of hit genes, produced robust results. In fact, they were comparable to results obtained with a CRISPR pooled screen. Members of the Weissman group systematically optimized the promoter and microRNA contexts for shRNA expression along with a selection of guide strands.
Using a sublibrary of proteostasis genes (targeting 2,933 genes), the investigators compared CRISPR and RNAi pooled screens. Data showed 48 hits unique to RNAi, 40 unique to CRISPR, and an overlap of 21 hits (with a 5% false discovery rate cut-off). Together, the technologies provided a more complete research story.
RNA interference (RNAi) silences, or knocks down, the translation of a gene by inducing degradation of a gene target’s transcript. To advance RNAi applications, Thermo Fisher Scientific has developed two types of small RNA molecules: short interfering RNAs and microRNAs. The company also offers products for RNAi analysis in vitro and in vivo, including libraries for high-throughput applications.
Arrayed CRISPR Screens
“RNA screens are well accepted and will continue to be used, but it is important biologically that researchers step away from the RNA mechanism to further study and validate their hits to eliminate potential bias,” explained Louise Baskin, senior product manager, Dharmacon, part of GE Healthcare. “The natural progression is to adopt CRISPR in the later stages.”
RNAi uses the cell’s endogenous mechanism. All of the components required for gene knockdown are already within the cell, and the delivery of the siRNA starts the process. With the CRISPR gene-editing system, which is derived from a bacterial immune defense system, delivery of both the guide RNA and the Cas9 nuclease, often the rate limiter in terms of knockout efficiency, are required.
In pooled approaches, the cell has to either drop out or overexpress so that it is sortable, limiting the types of addressable biological questions. A CRISPR-arrayed approach opens up the door for use of other analytical tools, such as high-content imaging, to identify hits of interest.
To facilitate use of CRISPR, GE recently introduced Dharmacon Edit-R synthetic CRISPR RNA (crRNA) libraries that can be used to carry out high-throughput arrayed analysis of multiple genes. Rather than a vector- or plasmid-based gRNA to guide the targeting of the Cas9 cleavage, a synthetic crRNA and tracrRNA are used. These algorithm-designed crRNA reagents can be delivered into the cells very much like siRNA, opening up the capability to screen multiple target regions for many different genes simultaneously.
GE showed a very strong overlap between CRISPR and RNAi using this arrayed approach to validate RNAi screen hits with synthetic crRNA. The data concluded that CRISPR can be used for medium- or high-throughput validation of knockdown studies.
“We will continue to see a lot of cooperation between RNAi and gene editing,” declared Baskin. “Using the CRISPR mechanism to knockin could introduce mutations to help understand gene function at a much deeper level, including a more thorough functional analysis of noncoding genes.
“These regulatory RNAs often act in the nucleus to control translation and transcription, so to knockdown these genes with RNAi would require export to the cytoplasm. Precision gene editing, which takes place in the nucleus, will help us understand the noncoding transcriptome and dive deeper into how those genes regulate disease progression, cellular development and other aspects of human health and biology.”
Synthetic crRNA:tracrRNA reagents can be used in a similar manner to siRNA reagents for assessment of phenotypes in a cell population. Top row: A reporter cell line stably expressing Cas9 nuclease was transfected with GE Dharmacon’s Edit-R synthetic crRNA:tracrRNA system, which was used to target three positive control genes (PSMD7, PSMD14, and VCP) and a negative control gene (PPIB). Green cells indicate EGFP signaling occuring as a result of proteasome pathway disruption. Bottom row: A siGENOME siRNA pool targeting the same genes was used in the same reporter cell line.
Tool Selection
The functional genomics tool should fit the specific biology; the biology should not be forced to fit the tool. Points to consider include the regulation of expression, the cell line or model system, as well as assay scale and design. For example, there may be a need for regulatable expression. There may be limitations around the cell line or model system. And assay scale and design may include delivery conditions and timing to optimally complete perturbation and reporting.
“Both RNAi- and CRISPR-based gene modulation strategies have pros and cons that should be considered based on the biology of the genes being studied,” commented Gwen Fewell, Ph.D., chief commercial officer, Transomic Technologies.
RNAi reagents, which can produce hypomorphic or transient gene-suppression states, are well known for their use in probing drug targets. In addition, these reagents are enriching studies of gene function. CRISPR-Cas9 reagents, which produce clean heterozygous and null mutations, are important for studying tumor suppressors and other genes where complete loss of function is desired.
Timing to readout the effects of gene perturbation must be considered to ensure that the biological assay is feasible. RNAi gene knockdown effects can be seen in as little as 24–72 hours, and inducible and reversible gene knockdown can be realized. CRISPR-based gene knockout effects may become complete and permanent only after 10 days.
Both RNAi and CRISPR reagents work well for pooled positive selection screens; however, for negative selection screens, RNAi is the more mature tool. Current versions of CRISPR pooled reagents can produce mixed populations containing a fraction of non-null mutations, which can reduce the overall accuracy of the readout.
To meet the needs of varied and complex biological questions, Transomic Technologies has developed both RNAi and CRISPR tools with options for optimal expression, selection, and assay scale. For example, the company’s shERWOOD-UltramiR shRNA reagents incorporate advances in design and small RNA processing to produce increased potency and specificity of knockdown, particularly important for pooled screens.
Sequence-verified pooled shRNA screening libraries provide flexibility in promoter choice, in vitro formats, in vivo formats, and a choice of viral vectors for optimal delivery and expression in biologically relevant cell lines, primary cells or in vivo.
The company’s line of lentiviral-based CRISPR-Cas9 reagents has variable selectable markers such that guide RNA- and Cas9-expressing vectors, including inducible Cas9, can be co-delivered and selected for in the same cell to increase editing efficiency. Promoter options are available to ensure expression across a range of cell types.
“Researchers are using RNAi and CRISPR reagents individually and in combination as cross-validation tools, or to engineer CRISPR-based models to perform RNAi-based assays,” informs Dr. Fewell. “Most exciting are parallel CRISPR and RNAi screens that have tremendous potential to uncover novel biology.”
Schematic of a pooled shRNA screening workflow developed by Transomic Technologies. Cells are transduced, and positive or negative selection screens are performed. PCR amplification and sequencing of the shRNA integrated into the target cell genome allows the determination of shRNA representation in the population.
Converging Technologies
The convergence of RNAi technology with genome-editing tools, such as CRISPR-Cas9 and transcription activator-like effector nucleases, combined with next-generation sequencing will allow researchers to dissect biological systems in a way not previously possible.
“From a purely technical standpoint, the challenges for traditional RNAi screens come down to efficient delivery of the RNAi reagents and having those reagents provide significant, consistent, and lasting knockdown of the target mRNAs,” states Ross Whittaker, Ph.D., a product manager for genome editing products at Thermo Fisher Scientific. “We have approached these challenges with a series of reagents and siRNA libraries designed to increase the success of RNAi screens.”
Thermo Fisher’ provides lipid-transfection RNAiMax reagents, which effectively deliver siRNA. In addition, the company’s Silencer and Silencer Select siRNA libraries provide consistent and significant knockdown of the target mRNAs. These siRNA libraries utilize highly stringent bioinformatic designs that ensure accurate and potent targeting for gene-silencing studies. The Silencer Select technology adds a higher level of efficacy and specificity due to chemical modifications with locked nucleic acid (LNA) chemistry.
The libraries alleviate concerns for false-positive or false-negative data. The high potency allows less reagent use; thus, more screens or validations can be conducted per library.
Dr. Whittaker believes that researchers will migrate regularly between RNAi and CRISPR-Cas9 technology in the future. CRISPR-Cas9 will be used to create engineered cell lines not only to validate RNAi hits but also to follow up on the underlying mechanisms. Cell lines engineered with CRISPR-Cas9 will be utilized in RNAi screens. In the long term, CRISPR-Cas9 screening will likely replace RNAi screening in many cases, especially with the introduction of arrayed CRISPR libraries.
Validating Antibodies with RNAi
Unreliable antibody specificity is a widespread problem for researchers, but RNAi is assuaging scientists’ concerns as a validation method.
The procedure introduces short hairpin RNAs (shRNAs) to reduce expression levels of a targeted protein. The associated antibody follows. With its protein knocked down, a truly specific antibody shows dramatically reduced or no signal on a Western blot. Short of knockout animal models, RNAi is arguably the most effective method of validating research antibodies.
The method is not common among antibody suppliers—time and cost being the chief barriers to its adoption, although some companies are beginning to embrace RNAi validation.
“In the interest of fostering better science, Proteintech felt it was necessary to implement this practice,” said Jason Li, Ph.D., founder and CEO of Proteintech Group, which made RNAi standard protocol in February 2015. “When researchers can depend on reproducibility, they execute more thorough experiments and advance the treatment of human diseases and conditions.”
