How to use siRNA, shRNA, microRNA and CRISPR libraries

Considerations for Functional Genomics Studies by High-throughput Library Screening

Gene function(s) can be determined in a dynamic living system through systematic knockdown, knockout or over-expression of genes. This experimental approach is known as functional genomics, and has become a key discovery tool in many areas of biological research such as drug target identification, drug resistance, host‑pathogen interactions, and biological pathway analysis.

In 2005, Dharmacon, Inc produced the first siRNA reagent library to target each gene in the human genome. Whole-genome libraries enabled individual down-regulation of thousands of genes in parallel, and provided another strategy for functional genomics studies beyond gene expression microarrays.

Today, high-throughput genetic screening approaches combine libraries of small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA or CRISPR-Cas9 guide RNA (gRNA) with sophisticated automation, biological assays, high-content data capture and powerful bio-statistical analysis. These approaches are routinely employed in academic, pharmaceutical and biotechnology laboratories. Genome-scale RNAi libraries are used with chemical compound screening and classical genetic tools to understand the roles of genes, explore a diverse range of biological functions, and characterize pathways and networks. (1, 2)

Types and formats of screening libraries

Reagent libraries of siRNA, shRNA, microRNA and CRISPR-Cas9 gRNA for targeting mammalian genomes for knockdown or knockout are available from a range of sources, both academic and commercial. Scientists must consider the goals of the intended experiments to choose which type of screening library will be most appropriate. Additionally, reagent design, production quality control and technical support are important considerations when choosing a screening library source. Each type of library has advantages over the other in specific aspects, and for this reason, many advanced functional genomics screening laboratories employ combinations of siRNA, shRNA, microRNA and CRISPR-Cas9 gRNA libraries.

In most cell types, siRNAs can be delivered effectively using transfection reagents, and only require resuspension in water or buffer before use. Some siRNAs have been chemically modified for delivery into difficult-to-transfect cells without the use of lipid transfection reagents of electroporation. Generally, gene silencing peaks within 48-72 hours and disappears entirely by 5-7 days in actively dividing cells. Depending on the assay or phenotype being measured, this duration of knockdown may not be sufficient.

Plasmid Libraries

shRNA libraries can be supplied in microtiter plates as plasmid-transformed E. coli glycerol stocks which provide a renewable source of RNAi material. These libraries are labor-intensive to use as each clone must be grown and maintained for quality and viability. Further, time-consuming plasmid preparation is required prior to use. Silencing is transient, and is a function of the cellular expression level of the shRNA. For this reason, the choice of a promoter that will have high activity in the cell type under study is an important consideration.

Lentiviral Libraries

The potential of shRNA is most fully realized when viral methods are used for delivery to cells. Many cell types, including primary and non-dividing cells, can be efficiently transduced with shRNA vector constructs packaged into non-replicating lentiviral particles. However, production of lentiviral particles arrayed in 96-well plates is both difficult and costly. It technically difficult to control for well-to-well and plate-to-plate titer variations. Further, achieving high-titer usually requires individual packaging of each construct and further manipulation to array the viruses into a useful screening format. For these reasons, while lentiviral shRNA approaches are ideal for observing long-term, integrated silencing effects, the technical limitations of an arrayed lentiviral format make this high-throughput genome-wide screening strategy costly and labor intensive.

Pooled Lentiviral Libraries

Pooled lentiviral shRNA methods have the distinct advantage of combining lentiviral delivery with high-throughput screening. With this strategy, hundreds or thousands of unique shRNAs or sgRNAs are added together into one pool, and then packaged into lentiviral particles. With this method, cells can be transduced in a larger group, selection pressure can be applied, and cells expressing individual hairpins can be isolated and identified using several selection strategies. This innovative approach does not require liquid handling robotics and is therefore accessible to more research groups. Pooled lentiviral shRNA screening has produced a number of published functional genomic screens both in vitro and in vivo (3-7).

High-throughput functional genomics screening workflow - general considerations

Today, high-throughput functional genomic screening is routinely used to determine molecular mechanisms in both normal and disease physiology. These loss-of-function, or gain-of-function in the case of microRNA, screens are based on measurable phenotypes that result from using libraries of siRNA, shRNA, microRNA and CRISPR-Cas9 guide RNA (crRNA or sgRNA).

For arrayed screening, instrumentation is required for multiple functions within the workflow including liquid handling, plate stacking, lidding and de-lidding, incubator capacity sufficient for a large number of plates, assay read-out equipment, and data management and analysis systems. Robotic systems are often employed to increase accuracy and reproducibility for large-scale screens (e.g. whole genome, druggable targets, etc.). Techniques such as reverse transfection are also employed to reduce the number of liquid handling steps in an arrayed screening workflow, thereby reducing variability.

Collecting phenotypic screening data

While single-gene experiments share the same general experimental workflow and factors critical for success, functional genomic screens generally need more stringent assay validation and optimization before beginning. Phenotypic read-outs from RNAi and CRISPR-Cas9 screening can be performed on equipment that is standard in many laboratories such as plate readers. There are many end-point assays that employ colorimetric, fluorescent or luminescent measurement to assess cell proliferation, protein secretion, reporter gene activity, and apoptotic induction.

Recently, high-throughput RNAi screening workflows have included automated fluorescent microscopy platforms and sophisticated high-content imaging instrumentation to record spatial, temporal and kinetic multi-parametric data sets. High-content analysis (HCA) enables the measurement of detailed loss-of-function phenotypes in a broader biological context and may result in increased screening sensitivity due to the ability to characterize several parameters in each well.

General screening workflow

General Workflow

Figure 1. Success of RNAi and CRISPR-Cas9 screening depends on rigorous testing, planning and implementation of each critical phase. Before the screen can be started, conditions for cell culture, RNAi reagent delivery, and assay read-out must be determined. These conditions must also be validated on the automated systems and data collection instruments that will be used during screening. This will provide information on the variability of the assay, and therefore the number of technical and biological replicates that will be needed to achieve statistically significant results. To obtain hits from the primary screen, statistical analysis that is appropriate to the type of data being collected is applied. Initial positive hits will need to be confirmed by at least one secondary orthologous method.

  See the  for additional screening information on:
  • assay development and validation
  • siRNA screening
  • pooled lentiviral shRNA library screening
  • hit stratification
  • confirmation of results
  • tips for successful screening

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Featured Resources


  1. Lord, C.J., S.A. Martin, and A. Ashworth, RNA interference screening demystified. J Clin Pathol, 2009. 62(3): p. 195-200.
  2. Falschlehner, C., et al., High-throughput RNAi screening to dissect cellular pathways: a how-to guide. Biotechnol J. 5(4): p. 368-76.
  3. Hurov, K.E., C. Cotta-Ramusino, and S.J. Elledge, A genetic screen identifies the Triple T complex required for DNA damage signaling and ATM and ATR stability. Genes Dev, 2010. 24(17): p. 1939-50.
  4. Smogorzewska, A., et al., A genetic screen identifies FAN1, a Fanconi anemia-associated nuclease necessary for DNA interstrand crosslink repair. Mol Cell, 2010. 39(1): p. 36-47.
  5. Boettcher, M., et al., Decoding pooled RNAi screens by means of barcode tiling arrays. BMC Genomics, 2010. 11: p. 7.
  6. Burgess, D.J., et al., Topoisomerase levels determine chemotherapy response in vitro and in vivo. Proc Natl Acad Sci U S A, 2008. 105(26): p. 9053-8.
  7. Mendes-Pereira, A.M., et al., Breast Cancer Special Feature: Genome-wide functional screen identifies a compendium of genes affecting sensitivity to tamoxifen. Proc Natl Acad Sci U S A, 2011.