Directing RNase P-mediated cleavage of target mRNAs by engineered external guide sequences in cultured cells

Ribonuclease P (RNase P) complexed with external guide sequence (termed as EGS) represents a novel nucleic acid-based gene interference approach to modulate gene expression. In previous studies, by using an in vitro selection procedure, we have successfully generated EGS variants that are complementary to target mRNAs, and these variants exhibit higher efficiency in directing human RNase P to cleave the target mRNAs than those derived from nature RNAs in vitro. This chapter describes the procedure of using engineered EGSs for in vitro trans-cleavage of target viral mRNAs in cultured cells. Detailed information is focused on (1) generation and in vitro cleavage assay of the customized EGS variants and (2) stable expression of EGS and evaluation of its activity in inhibition of viral gene expression and growth in cultured cells. These methods should provide general guidelines for using engineered EGS to direct RNase P-mediated cleavage of target mRNAs in cultured cells.