A genome-wide map of adeno-associated virus–mediated human gene targeting

Abstract

To determine which genomic features promote homologous recombination, we created a genome-wide map of gene targeting sites. An adeno-associated virus vector was used to target identical loci introduced as transcriptionally active retroviral vector proviruses. A comparison of ~2,000 targeted and untargeted sites showed that targeting occurred throughout the human genome and was not influenced by the presence of nearby CpG islands, sequence repeats, or DNase I hypersensitive sites. Targeted sites were preferentially found within transcription units, especially when the target loci were transcribed in the opposite orientation to their surrounding chromosomal genes. The impact of DNA replication was determined by mapping replication forks, which revealed a preference for recombination at target loci transcribed towards an incoming fork. Our results constitute the first genome-wide screen of gene targeting in mammalian cells, and they demonstrate a strong recombinogenic effect of colliding polymerases.

Structural basis for retroviral integration into nucleosomes

“…The genomic coordinates were converted to the standard browser extensible data (BED) format, with each interval reporting the center dinucleotide of a PFV integration site. Distributions of the integration sites with respect to genomic features ( http://genome.ucsc.edu/cgi-bin/hgTables ), HT1080-specific gene expression activity (gene expression omnibus accession code GSE58968) 46 and lamina-associated domains (GSE22428) 47 , were analyzed using BEDtools software suite 48 . Nucleotide sequence logos were generated using WebLogo software ( http://weblogo.threeplusone.com/ ) 49 .…”

Structural basis for spumavirus GAG tethering to chromatin

“…HIV-1 and MLV integration sites in HEK293T cells alongside the matched random control simulated data were from published work (11). HT1080, HEK293T, MRC5, or HepG2 cell line-specific gene-expression activity was reported previously (GEO accession codes GSE58968, GSE11892, GSE63577, and GSE87958) (54)(55)(56)(57). All observable differences between WT and the corresponding mutant data were statistically significant (P < 0.05) (Dataset S1).…”

Investigation of the molecular biology underlying the pronounced high gene targeting frequency at the Myh9 gene locus in mouse embryonic stem cells

“…Interestingly, the trends of decreased percentage of SSR and SINEs in the homologous arms of these constructs were completely consistent with that of the GT efficiency for them. Importantly, the potential effects of these elements on gene targeting have been suggested [52][53][54][55][56], though direct evidence remain to be substantiated. Therefore, this finding presented here not only supports the concept, but also provide an excellent locus for in-depth investigating this issue in the future.…”