Adenoviral vector DNA for accurate genome editing with engineered nucleases

摘要

The exchange of genetic information between native acceptor loci and exogenous donor DNA through error-free HR is an established strategy to manipulate prokaryote and eukaryote genomes with nucleotide-level precision. However, in mammalian somatic cells, typical frequencies of spontaneous HR-mediated gene-targeting range from 10~(-8) to 10~(-6) events per transfected cell, with most exogenous DNA being found randomly integrated throughout host-cell chromosomes. Importantly, the deployment of sequence-specific nucleases greatly increases the odds of retrieving cells with specific allelic alterations. Generation of double-stranded DNA breaks (DSBs) at predefined chromosomal positions together with the introduction of donor DNA containing sequences identical to those bracketing the genomic lesion can increase gene targeting by several orders of magnitude. In gene therapy, for instance, inserting transcriptional units into specific genomic positions (i.e., so-called safe harbors) or directly repairing faulty genes within their native chromosomal context is a highly desirable goal.