Gene targeting by homologous recombination can be variable and researchers are itching to understand what key factors underlie variable success in editing experiments. Could it be an unfavorable chromatin environment, repetitive sequences, or maybe just bad luck?
An unrelenting group of researchers at the University of Washington recently set out to shed some light on the genomic elements that influence homologous recombination.
To avoid sequence specific biases, David Deyle and colleagues first created a human fibrosarcoma cell line library introducing identical target sites at thousands of chromosomal positions with retroviral vectors.
These target sites harbored a truncated Neomycin resistant gene, which they used to select for clones that underwent homologous recombination using a homology donor delivered by adeno-associated virus (AAV) vectors.
They then applied massive parallel sequencing to identify the genomic sequences that contained successful target events and compared them to the untargeted retroviral insertion sites.
With this genome-wide approach they were able to nail down several characteristics of the genomic elements that promote homologous recombination and came up with some surprising results.
Genomic elements that do not influence targeting frequencies:
- Repetitive sequences
- CpG islands
- DNase I-hypersensitive sites
- Recombination occurred on all chromosomes at an equal rate
Factors that favor homologous recombination:
- Active transcription of the targeted locus slightly increased targeting frequencies
- Overlapping, convergent transcription at target sites significantly stimulates homologous recombination
- Target sites transcribed in the opposite direction to replication fork movement had a 2-fold higher likelihood to undergo homologous recombination
The researchers hypothesize that the reason for the higher recombination frequencies of target sites transcribed in the opposite direction of the genomic locus or the replication fork movement could be colliding polymerases that expose single-stranded DNA regions.
Check out the details at Nature Structural & Molecular Biology, October 2014