Loss-of-function screening using shRNA libraries is a powerful way to identify genes involved in almost any biological process. Over the past decade, shRNA screens, both in vivo and in cell culture systems, have generated many important discoveries. Knockdown screening has become a go-to ‘workhorse’ method for lead identification and gene network studies across many fields of biology. Whole-genome knockdown libraries are frequently used, as well as smaller libraries targeting subsets of genes, such as specific pathways or key biological regulators. Here are a few examples of major shRNA screening discoveries from the past few years:
Peroxisomes have a role in cholesterol transport
It has long been known that cholesterol undergoes intracellular transport, but the mechanism underlying this process remained unknown. Chu et al. developed an amphotericin B-based assay for impaired LDL-transport, and used it in a genome-wide, lentivirus-based shRNA screen1. They found that knockdown of important peroxisome genes led to an accumulation of cholesterol in lysosomes. Previously, peroxisomes had not been known to play a role in cholesterol transport. Furthermore, the group investigated mouse models and human patients with peroxisomal disorders, and found massive cellular cholesterol accumulation, suggesting a role for aberrant cholesterol transport in these diseases.
Many cancers are vulnerable to PRMT5 depletion
In many human cancers, the 5-methylthioadenosine phosphorylase (MTAP) gene is deleted due to its proximity to the tumor suppressor gene CDKN2A. This leads to alterations in methionine metabolism in these cancers2. Mavrakis et al. examined a large data set from an shRNA screen spanning 390 cancer cell lines, and found that the survival of MTAP-deficient cancer cells is impaired by depletion of the protein arginine methyltransferase PRMT5. Thus, inhibitors of PRMT5 have the potential to be highly effective treatments targeting MTAP/CDKN2A-deleted tumors.
CD55 is essential for malaria infection
Red blood cells do not have nuclei. This prevents the use of many types of genetic studies to identify host factors involved in Plasmodium falciparum infection. Egan et al. worked around this problem by performing an shRNA screen in red blood cells derived in culture from hematopoietic stem cells3. They screened a small, focused, lentivirus-based library targeting a subset of genes which could be knocked down without preventing erythroid development, and found that CD55 is an essential host factor for P. falciparum invasion. This finding makes CD55 a promising target for anti-malaria therapeutics.
Identification of a key regulator of liver regeneration
Among mammalian organs, the liver is unique in its ability to naturally regenerate. However, human liver diseases often prevent adequate liver regeneration, making the organ unable to compensate for hepatocyte loss, which leads to organ failure. Wuestefeld et al. performed an in vivo shRNA screen in mice to identify genes which modulate liver regeneration4. They stably delivered a focused, pooled shRNA library into hepatocytes of mouse livers undergoing regeneration, and identified the kinase MKK4 as a negative regulator of regeneration. They found that silencing MKK4 robustly increased liver regenerative capacity in mouse models of chronic liver failure and regeneration after liver damage.
If you would like to perform your own shRNA screen, VectorBuilder can help. We offer pooled, lentivirus-based whole-genome shRNA libraries and ‘elite’ shRNA libraries targeting the 2000 most frequently cited genes, with 5-6 shRNAs per gene. These are highly efficient and cost effective for conducting loss-of-function screens.
If you are interested in a custom, focused shRNA library, please contact us about your research plan.
- Chu BB, Liao YC, Qi W, Xie C, Du X, Wang J, Yang H, Miao HH, Li BL, Song BL. Cholesterol transport through lysosome-peroxisome membrane contacts. Cell. 2015 Apr 9;161(2):291-306.
- Mavrakis KJ, et al. Disordered methionine metabolism in MTAP/CDKN2A-deleted cancers leads to dependence on PRMT5. Science. 2016 Mar 11;351(6278):1208-13.
- Egan ES, et al. A forward genetic screen identifies erythrocyte CD55 as essential for Plasmodium falciparum invasion. Science. 2015 May 8;348(6235):711-4.
- Wuestefeld T, et al. A Direct in vivo RNAi screen identifies MKK4 as a key regulator of liver regeneration. Cell. 2013 Apr 11;153(2):389-401.