| 摘要 |
Methods for intracellularly generating single stranded DNA molecules that are active in mediating triplex-dependent or independent chromosomal events are provided herein. The method is based on the discovery that one can introduce viral vectors or plasmids into the cells, where they generate within the cells to be engineered the single stranded DNA molecules that bind to the target chromosomal DNA to form a triplex, which may induce a desired mutation, and/or be recombinagenic and induce a change to the chromosomal DNA by incorporation of a donor DNA molecule. The vectors or plasmid not only encode the TFO and optionally the donor DNA, but also a reverse transcriptase, and optionally, a restriction enzyme, which is present in the preferred embodiment as a fusion protein which reverse transcribes the RNA encoded by the vector or plasmid, then cleaves it at a restriction enzyme site to yield a single stranded DNA. The single stranded DNA may be produced directly, or initially as a double stranded stem-single stranded loop structure, which is then cleaved to yield the single stranded DNA.
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