Abstract Viral vector systems are very useful for delivering genes to cells. Commonly used systems include vectors derived from retroviruses, lentiviruses, adenoviruses, adeno‐associated viruses and herpesviruses.

We use an Epstein-Barr virus (EBV) plasmid model chromosome system to study how different donor plasmid constructs affect recombination stimulated by an I-SceI-induced double-strand break in the target sequence in human cells. The entire 3.5 kb lacZ gene was efficiently recombined into a target EBV vector lacking lacZ sequences, but having limited homology to the donor plasmid. A donor plasmid with lacZ flanked by sequence homologous to the target consistently generated gene conversion events and was more effective than a donor carrying lacZ outside the same sequence homology. Reducing the length of homology between the target and donor from 5.5 kb to 1 kb caused only a 3-fold drop in recombination frequency, contrasting with the exponential dependence on homology length seen when no DSB is present in the target. These results document a DSB-induced 175-fold increase in recombination of a heterologous gene into a target, requiring only limited flanking homology.

Homologous recombination stimulated by a double-strand break at a desired target site offers a method to achieve site-specific integration useful for gene therapy and other genetic engineering. To test parameters needed for this strategy, we developed an Epstein-Barr virus shuttle vector model system as a genetic tool. This extrachromosomal plasmid assay system has several advantages over a chromosomal assay. The system detects all classes of recombination events without selection and allows rapid analysis of the frequency and nature of recombination events. We found that a double-strand break at the target site stimulated a large increase in recombination frequency. The resulting recombinants included one-sided insertion events, as well as two-sided or gene conversion events. A circular donor substrate was more effective in recombination than linearized donor DNA.