ABSTRACT
Efficient incorporation of novel DNA sequences into a specific site in the genome of living human cells remains a challenge despite its potential utility to genetic medicine, biotechnology, and basic research. We find that a precisely placed double-strand break induced by engineered zinc finger nucleases (ZFNs) can stimulate integration of long DNA stretches into a predetermined genomic location, resulting in high-efficiency site-specific gene addition. Using an extrachromosomal DNA donor carrying a 12-bp tag, a 900-bp ORF, or a 1.5-kb promoter-transcription unit flanked by locus-specific homology arms, we find targeted integration frequencies of 15%, 6%, and 5%, respectively, within 72 h of treatment, and with no selection for the desired event. Importantly, we find that the integration event occurs in a homology-directed manner and leads to the accurate reconstruction of the donor-specified genotype at the endogenous chromosomal locus, and hence presumably results from synthesis-dependent strand annealing repair of the break using the donor DNA as a template. This site-specific gene addition occurs with no measurable increase in the rate of random integration. Remarkably, we also find that ZFNs can drive the addition of an 8-kb sequence carrying three distinct promoter-transcription units into an endogenous locus at a frequency of 6%, also in the absence of any selection. These data reveal the surprising versatility of the specialized polymerase machinery involved in double-strand break repair, illuminate a powerful approach to mammalian cell engineering, and open the possibility of ZFN-driven gene addition therapy for human genetic disease.
Subject(s)
Deoxyribonucleases/genetics , Gene Targeting/methods , Gene Transfer Techniques , Genetic Engineering/methods , Genome, Human/genetics , Zinc Fingers/genetics , Base Sequence , Evaluation Studies as Topic , Humans , Molecular Sequence DataABSTRACT
Fifty-two case-hardened, severely self-abusive and aggressive autistic and retarded students, living in six different community residences, were trained to exit their residences, without the presence or assistance of staff, when a fire alarm sounded at night. On a pretest, prior to an 11-week period of intensive environmental and behavioral interventions, only 25% of the students exited within 2 1/2 minutes and independent of staff assistance. At posttest, 100% exited successfully, in a mean evacuation time of 60 seconds. The study was conducted at the Behavior Research Institute (BRI).
