DNA-CLEAVING RNASE P RNA

摘要

Disclosed are modified, or variant, RNase P RNA molecules that specifically cleave DNA with a catalytic efficiency greater than wild-type RNase P RNA. Sequence-specific targeting of the variant RNase P RNA is provided by guide sequences that specifically hybridize to the target DNA and cause the variant RNase P RNA to recognize the DNA molecule as a substrate. Thus, the disclosed variant RNase P RNA molecules are universal DNA cleavage enzymes. The disclosed variant RNase P RNA molecules have a wide variety of uses. For example, the disclosed variant RNase P RNA molecules can be used to cleave any desired DNA sequence in vitro or in vivo. These uses specifically include diagnostic methods for detection, quantitation, or cataloging of DNA sequences, forensic methods, genome dissection methods, biostatistical methods, and population genetics methods, cleaving genomic DNA at particular sequences, creating gene knockouts by gene cleavage, killing specific cells by specific cleavage of DNA, cleaving pathogen DNA in a host cell, and killing mutant cells by specific cleavage of mutant DNA in the cell. Also disclosed is a method of producing variant RNase P RNA molecules that catalyze efficient cleavage of DNA substrates. The disclosed methods allow efficient and predictable generation of a variety of RNase P RNA variants having significant DNA-cleaving activity. The disclosed method thus provides a ready means of producing numerous variant RNase P RNA molecules for use in any of the disclosed methods for use of such variant RNase P RNA molecules. The disclosed variant RNase P RNA molecules can be used with a separate guide sequence molecule or with a guide sequence covalently coupled to the variant RNase P RNA.