Journal article
New Human Chromosomal Sites with "Safe Harbor" Potential for Targeted Transgene Insertion
Human gene therapy, Vol.30(7), pp.814-828
07/01/2019
PMID: 30793977
Abstract
This study identified 35 new sites for targeted transgene insertion that have the potential to serve as new human genomic "safe harbor" sites (SHS). SHS potential for these 35 sites, located on 16 chromosomes, including both arms of the human X chromosome, and for the existing human SHS
,
, and
was assessed using eight different desirable, widely accepted criteria for SHS verifiable with human genomic data. Three representative newly identified sites on human chromosomes 2 and 4 were then experimentally validated by
and
cleavage-sensitivity tests, and analyzed for population-level and cell line-specific sequence variants that might confound site targeting. The highly ranked site on chromosome 4 (SHS231) was further characterized by targeted homology-dependent and -independent transgene insertion and expression in different human cell lines. The structure and fidelity of transgene insertions at this site were confirmed, together with analyses that demonstrated stable expression and function of transgene-encoded proteins, including fluorescent protein markers, selectable marker cassettes, and Cas9 protein variants. SHS-integrated transgene-encoded Cas9 proteins were shown to be capable of introducing a large (17 kb) gRNA-specified deletion in the
fusion oncogene in human rhabdomyosarcoma cells and as a Cas9-VPR fusion protein to upregulate expression of the muscle-specific transcription factor
in human rhabdomyosarcoma cells. An engineering "toolkit" was developed to enable easy use of the most extensively characterized of these new human sites, SHS231, located on the proximal long arm of chromosome 4. The target sites identified here have the potential to serve as additional human SHS to enable basic and clinical gene editing and genome-engineering applications.
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Details
- Title
- New Human Chromosomal Sites with "Safe Harbor" Potential for Targeted Transgene Insertion
- Creators
- Stefan Pellenz - University of WashingtonMichael Phelps - University of WashingtonWeiliang Tang - University of WashingtonBlake T Hovde - University of WashingtonRyan B Sinit - University of WashingtonWenqing Fu - University of WashingtonHui Li - University of WashingtonEleanor Chen - University of WashingtonRaymond J Monnat, Jr - University of Washington
- Publication Details
- Human gene therapy, Vol.30(7), pp.814-828
- Academic Unit
- Department of Animal Sciences
- Grant note
- T32 HG000035 / NHGRI NIH HHS RL1 CA133831 / NCI NIH HHS P01 CA077852 / NCI NIH HHS
- Identifiers
- 99901397958101842
- Language
- English
- Resource Type
- Journal article