Generation of RNAi libraries for high-throughput screens

Abstract

The completion of the genome sequencing for several organisms has created a great demand for genomic tools that can systematically analyze the growing wealth of data. In contrast to the classical reverse genetics approach of creating specific knockout cell lines or animals that is time-consuming and expensive, RNA-mediated interference (RNAi) has emerged as a fast, simple, and cost-effective technique for gene knockdown in large scale. Since its discovery as a gene silencing response to double-stranded RNA (dsRNA) with homology to endogenous genes in Caenorhabditis elegans (C elegans), RNAi technology has been adapted to various high-throughput screens (HTS) for genome-wide loss-of-function (LOF) analysis. Biochemical insights into the endogenous mechanism of RNAi have led to advances in RNAi methodology including RNAi molecule synthesis, delivery, and sequence design. In this article, we will briefly review these various RNAi library designs and discuss the benefits and drawbacks of each library strategy.

Figure 1

Extracellular generation of siRNA molecules. (a) Sense and antisense strands of RNA are chemically synthesized and annealed to form 21–23 nt or 25–30 nt dsRNA molecules. 21–23 nt siRNA molecules can directly interact with RISC and guide degradation of the corresponding mRNA. 25–30 nt dsRNA molecules must first be cleaved by Dicer to generate the 21–23 nt siRNA molecule which can be loaded into RISC. (b) Recombinant Dicer or RNAse III enzymes can also be used to generate siRNA molecules with silencing capabilities. 21–23 nt siRNA molecules are cleaved from dsRNA and associate with the RISC.

Figure 2

Intracellular siRNA molecule generation via plasmids. Dual promoter systems can direct the production of siRNA sense and antisense strands which anneal and load into RISC. (a) Tandem sense and antisense strands are driven by individual U6 promoters. (b) A single template transcribes for both the sense and antisense strand via opposing promoter design. (c) The addition of a loop structure between the sense and antisense template driven by U6 promoter generates shRNA molecules. The shRNA is cleaved by Dicer producing the functional siRNA molecule. (d) Second-generation shRNA-mir construct is based on miR-30 primary transcript driven by a single U6 promoter. The shRNA-mir molecule is first cleaved by Drosha creating an shRNA molecule recognized by Dicer thereby entering the RNAi mechanism.

Figure 3

The generation of shRNA libraries from cDNA via enzymatic cleavage.