High throughput methods for functionally determining RNA interference efficiency

摘要

Provided is a single construct combining a sequence encoding an RNAi molecule, a sequence encoding a reporter, and a target sequence specific for the RNAi molecule. The construct can be used to determine the potency of the encoded RNAi molecule in a direct and unbiased way. These results can be used to inform the design of potent RNAi molecules of various types and can be extended to several other applications, including: (1) generation of tiled libraries comprising every possible RNAi molecule-encoding sequence for a given gene target; (2) large-scale parallel validation of RNAi molecules targeting many genes to generate validated RNAi molecule-encoding libraries; (3) experimental comparison of design algorithms and strategies; and (4) investigation of RNAi biology in target site mutagenesis assays by screening pools containing single nucleotide changes in target sites and/or in the RNAi molecule to identify the most relevant sequence characteristics of potent RNAi-target site predictions.

主权项

1. A single construct comprising:
a first sequence comprising a first promoter and a sequence encoding an shRNA molecule, operably linked to the first promoter, wherein the shRNA molecule comprises a guide strand; and a second sequence comprising a second promoter and a target sensor, operably linked to the second promoter, the target sensor comprising: a sequence encoding a reporter and a target sequence that is co-transcribed with the sequence encoding the reporter, and the target sequence comprises from about 8 to about 29 contiguous nucleotides complementary to at least a portion of the guide strand of the shRNA molecule, such that the functional shRNA molecule is specific for the target sequence and is capable of suppressing reporter expression.