Patent ID: 7592161

Claim:
A method of increasing insertion of modified group II introns into a DNA target site in a host cell in vitro, comprising: a) introducing a nucleic acid construct into the host cell, said construct comprising: i.) a modified group II intron sequence encoding a modified group II intron RNA, said modified group II intron RNA comprising an EBS1 sequence, an EBS2 sequence, a delta sequence, and a deletion of subdomains IVB1, IVB2, IVB3, wherein one or more of said EBS1, EBS2 or delta sequences is modified to provide a modified EBS1, EBS2, and delta sequence that base pairs with an IBS1, IBS2, and delta prime sequence, respectively, in the DNA target site; ii) a promoter for regulating transcription of said modified group II intron sequence, said promoter being operably linked to the modified group II intron sequence; iii) a first flanking sequence upstream of the modified group II intron sequence, said flanking sequence encoding a first hybridizing sequence which is complementary to the EBS1 or the modified EBS1 sequence contained in said modified group II intron RNA, and a second flanking sequence downstream of the modified group II intron sequence encoding a second hybridizing sequence which is complementary to the EBS2 or the modified EBS2 sequence contained in said modified group II intron RNA; and iv) an open reading frame sequence encoding a wild-type or modified group II intron encoded protein, wherein said open reading frame sequence is located upstream, or downstream of the modified group II intron sequence, and wherein expression of said group II intron encoded protein is regulated by the promoter which is operably linked to the modified group II intron sequence or by a second promoter which is operably linked to the open reading frame sequence; and b) maintaining the cell under in vitro conditions which allow for expression of the modified group II intron sequence and the open reading frame sequence, formation of RNP particles comprising a modified excised group II intron RNA which is encoded by the modified group II intron sequence and a group II intron encoded protein which is encoded by the open reading frame sequence, and insertion of the modified group II intron into the DNA target site; wherein the method results in increased insertion of said modified group II introns into the DNA target site in the host cell in vitro as compared to a method where the group II intron RNA lacks deletion of subdomains IVB1, IVB2, and IVB3.