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Loss-of-Function Genetic Screening with Pooled shRNA or sgRNA Libraries

Loss-of-function genetic screening using pooled libraries of effector molecules, such as RNAi hairpins (shRNA) or CRISPR guide molecules (sgRNA) provides a powerful tool to identify the genetic elements functionally needed to:

  • manifest specific cell phenotypes
  • activate signaling pathways
  • produce responses to drugs or other factors
  • and/or maintain viability

Genetic screens with pooled shRNA or sgRNA libraries can be utilized to investigate any aspect of biology that can be recapitulated in a cell culture model and provide an unbiased approach to identify genes that act to produce various biological responses that enable cell development, differentiation, and disease progression.

Historically, pooled shRNA libraries have been used for genetic screening in mammalian cells–specifically loss-of-function genetic screening–since shRNA interferes with gene expression. More recently, CRISPR sgRNA libraries, which induce knockouts of target genes on the genomic level, have been used in similar sorts of screens. Both of these approaches require only RNA sequences of ca. 20 bases to target and disrupt a specific gene with very high efficiency. As a result, it is possible to easily design large numbers of shRNA- or sgRNA-encoding oligonucleotides that, when cloned into expression constructs, can efficiently target and disrupt thousands of genes. It is this feature that makes these technologies suitable for large-scale cell screening.

Loss-of-Function Genetic Screening Experimental Workflow

For pooled screening, a heterogeneous mix of constructs expressing a collection of different sgRNAs, shRNAs, or other effector molecules is typically packaged as viral particles and transduced into a population of cells. Typically, lentiviral systems or similar systems are used that ensure a high-efficiency introduction of mostly single constructs into each cell’s genomic DNA, where they are stably expressed and maintained in progeny cells.

Following introduction of the library, the cells are now essentially a mixed collection, where a different gene is disrupted or perturbed in each of the cells. A selection of the cell population can be done by any number of means, including some of the following more common approaches:

  • Treatment with a factor that kills most of the cells
  • Culturing the cells under optimal conditions for multiple doublings
  • Treatment with a compound at sub-lethal levels to kill off hypersensitive cells
  • FACS selection of cells having activated or lost a fluorescent reporter

As a result of the selection, then, cells containing some of the effector molecules that are more fit, will increase relative to the whole population, and those less fit, will decrease. For example, some cells may die out, or some may be more likely to be selected by FACS in the positive gated fraction. Regardless of how the selection is done, cells that become more or less prevalent will carry their respective effector, and so, the frequency the effector they contain (the sgRNA or shRNA) appears in the population is directly proportional to the number of reads that correspond to it in the next-generation sequencing (NGS) data.

As a result of this straightforward assay, it is easy to determine which effectors help promote the phenotype of interest (e.g., activation of a report, viability of the cells, etc.) and, by implication, which genes or genetic elements are functionally involved in the underlying biology for that phenotype.

For more details on how RNAi genetic screening with pooled shRNA libraries works, please see the Pooled Lentiviral shRNA Library User Manual. Also, please review Cellecta’s CRISPR and RNAi Genetic Screening Services.

For CRISPR screening, you may see the CRISPR sgRNA Library User Manual and see the CRISPR and RNAi Screening Services web page.

For additional information on Cellecta Genetic Screening services, please contact us.