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PERSPECTIVELessons from Nature: microRNA-based
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6Kenneth Chang1, Stephen J Elledge2 & Gregory J Hannon1Loss-of-function genetics has proven essential for interrogating the functions of genes
and for probing their roles within the complex circuitry of biological pathways. In
many systems, technologies allowing the use of such approaches were lacking before
the discovery of RNA interference (RNAi). We have constructed first-generation short
hairpin RNA (shRNA) libraries modeled after precursor microRNAs (miRNAs) and secondgeneration libraries modeled after primary miRNA transcripts (the Hannon-Elledge
libraries). These libraries were arrayed, sequence-verified, and cover a substantial portion
of all known and predicted genes in the human and mouse genomes. Comparison of firstand second-generation libraries indicates that RNAi triggers that enter the RNAi pathway
through a more natural route yield more effective silencing. These large-scale resources
are functionally versatile, as they can be used in transient and stable studies, and for
constitutive or inducible silencing. Library cassettes can be easily shuttled into vectors
that contain different promoters and/or that provide different modes of viral delivery.RNAi is an evolutionarily conserved, sequence-specific
gene-silencing mechanism that is induced by dsRNA.
Each dsRNA silencing trigger is processed into 2125 bp
dsRNAs called small interfering RNAs (siRNAs)13 by
Dicer, a ribonuclease III family (RNase III) enzyme4. The
resulting small RNAs enter the RNA-induced silencing
complex (RISC), which uses a single-stranded version
of the small RNA as a guide to substrate selection1,3,5,6.
Perfect complementarity between the substrate and the
small RNA leads to target-RNA cleavage by an RNAse H
like active site within an Argonaute protein that forms
the core of RISC7,8. For each small RNA, the two strands
of the Dicer product are treated differently. The strand
with the less stable duplex at its 5 end is incorporated
preferentially into RISC9,10. This realization has led to
rational designs of effective siRNA sequences and yielded substantial improvements in both the efficiency and
reliability of RNAi.miRNAs are a class of endogenous dsRNAs that
exert their effects through the RNAi pathway. More than a decade elapsed between the discovery of the first
miRNA, Caenorhabditis elegans lin-4 (refs. 11,12) and
the achievement of at least a superficial understanding
of the biosynthesis, processing and mode of action of
this class of noncoding regulatory RNAs13,14. miRNAs