Comparative Analysis of Sequence‐Specific DNA Recombination Systems in Human Embryonic Stem Cells

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Abstract
The great potential of human embryonic stem cells (hESCs) in basic research, regenerative medicine, and gene therapy is widely recognized. Controlled manipulation of hESC genomes through sequence-specific DNA recombination (SSR) may play a significant role in future hESC applications. However, very little is known about the functionality of SSR systems in hESCs. We demonstrate here that mutant phage lambda integrase, phage P1 Cre recombinase, and mutant gammadelta resolvase displayed distinct activities on episomal recombination substrates. Interestingly, cofactor-independent lambda integrase catalyzed the integrative pathway five times more efficiently than the excisive pathway. Such a degree of directionality in hESCs could be explored for sequential gene insertions into predetermined genomic sequences. We also report an improved, easy-to-use plasmid transfection system that employs silica microspheres and, in combination with SSR, could be applied to hESC genome engineering.