Engineering RNA endonucleases with customized sequence specificities.

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.