Engineering RNA endonucleases with customized sequence specificities

Rajarshi Choudhury, Yihsuan S. Tsai, Daniel Dominguez, Yang Wang and Zefeng Wang ()
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Rajarshi Choudhury: University of North Carolina at Chapel Hill
Yihsuan S. Tsai: University of North Carolina at Chapel Hill
Daniel Dominguez: University of North Carolina at Chapel Hill
Yang Wang: University of North Carolina at Chapel Hill
Zefeng Wang: University of North Carolina at Chapel Hill

Nature Communications, 2012, vol. 3, issue 1, 1-8

Abstract: Abstract Specific cleavage of RNAs is critical for in vitro manipulation of RNA and for in vivo gene silencing. Here we engineer artificial site-specific RNA endonucleases to function analogously to DNA restriction enzymes. We combine a general RNA cleavage domain with a series of Pumilio/fem-3-binding factor domains that specifically recognize different 8-nucleotide RNA sequences. The resulting artificial site-specific RNA endonucleases specifically recognize RNA substrates and efficiently cleave near their binding sites. The artificial site-specific RNA endonucleases can be devised to recognize and cleave various RNA target sequences, providing a useful tool to manipulate RNAs in vitro. In addition, we generate designer artificial site-specific RNA endonucleases to specifically silence an endogenous gene in Escherichia coli, as well as a mitochondrial-encoded gene in human cells, suggesting that artificial site-specific RNA endonucleases can serve as a gene-silencing tool with designed specificity.

Date: 2012
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DOI: 10.1038/ncomms2154

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