Group II Introns Designed to Insert into Therapeutically Relevant DNA Target Sites in Human Cells
Huatao Guo,1
Michael Karberg,1
Meredith Long,2
J.
P. Jones III,2
Bruce Sullenger,2
Alan M. Lambowitz1*
Mobile group II intron RNAs insert directly into DNA target sites
and are then reverse-transcribed into genomic DNA by the associated
intron-encoded protein. Target site recognition involves modifiable
base-pairing interactions between the intron RNA and a >14-nucleotide
region of the DNA target site, as well as fixed interactions between
the protein and flanking regions. Here, we developed a highly efficient
Escherichia coli genetic assay to determine detailed target
site recognition rules for the Lactococcus lactis group 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 retargeted to insert efficiently into virtually any
target DNA and that the retargeted introns retain activity in human
cells. This work provides the practical basis for potential
applications of targeted group II introns in genetic engineering,
functional genomics, and gene therapy.
1 Institute for Cellular and Molecular Biology,
Department of Chemistry and Biochemistry, and Section of Molecular
Genetics and Microbiology, School of Biological Sciences, University of
Texas, Austin, TX 78712, USA.
2 Center for Genetic
and Cellular Therapies, Department of Surgery, Duke University Medical
Center, Box 2601, Durham, NC 27710, USA.
*
To whom correspondence should be addressed. E-mail:
lambowitz{at}mail.utexas.edu
