Group II introns designed to insert into therapeutically relevant DNA target sites in human cells

Mobile group II intron RNAs insert directly into DNA target sites and are t hen reverse-transcribed into genomic DNA by the associated intron-encoded p rotein. Target site recognition involves modifiable base-pairing interactio ns between the intron RNA and a >14-nucleotide region of the DNA target sit e, as well as fixed interactions between the protein and flanking regions. Here, we developed a highly efficient Escherichia coli genetic assay to det ermine detailed target site recognition rules for the Lactococcus lactis gr oup II intron LL.LtrB and to select introns that insert into desired target sites. Using human immunodeficiency virus-type 1 (HIV-1) proviral DNA and the human CCR5 gene as examples, we show that group II introns can be retar geted to insert efficiently into virtually any target DNA and that the reta rgeted introns retain activity in human cells. This work provides the pract ical basis for potential applications of targeted group II introns in genet ic engineering, functional genomics, and gene therapy.