New Human Chromosomal Sites with “Safe Harbor” Potential for Targeted Transgene Insertion

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 AAVS1, hROSA26, and CCR5 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 in vitro and in vivo cleavage-sensitivity tests, and analyzed for populationlevel 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 PAX3/FOXO1 fusion oncogene in human rhabdomyosarcoma cells and as a Cas9-VPR fusion protein to upregulate expression of the muscle-specific transcription factor MYF5 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.

Safe harbor-targeted CRISPR-Cas9 homology-independent targeted integration for multimodality reporter gene-based cell tracking

“…Our final step for improving safety was to design our system to target a “safe harbor” locus in genomic DNA. Several of these loci have now been reported in the literature ( 49 ) and are described as sites where inserted genetic elements can function as intended, without causing alterations that would pose a risk to the host cell or organism ( 25 ). For this study, we targeted the AAVS1 site found within the human protein phosphatase 1 regulatory subunit 12C ( PP1R12C ) gene, as this has been one of the best characterized, to date.…”

CRISPR/dCas9‐mediated transposition with specificity and efficiency of site‐directed genomic insertions

“…53 However, a few human genomic targets have been used for site-directed integrations: the adeno-associated virus site 1 (AAVS1) in chromosome 19, 54 CCR5 locus in chromosome 3, 55 HPRT gene locus in the X-chromosome, 56,57 and the human ortholog of the mouse ROSA26 locus. 58 ROSA26 region in human cells has been identified based on homology with murine ROSA26 locus. 58 The same principle has been used to identify the sheep, 59 and the pig ROSA26 safe harbor regions.…”

“…58 ROSA26 region in human cells has been identified based on homology with murine ROSA26 locus. 58 The same principle has been used to identify the sheep, 59 and the pig ROSA26 safe harbor regions. 60 This region shows high recombination efficiency and stable expression of foreign genes, with no detectable effects on the growth of host cell.…”

Process analysis of pluripotent stem cell differentiation to megakaryocytes to make platelets applying European GMP

“…1b ). Both these safe harbour loci have been used extensively in mouse and human cells and are consistently and robustly expressed without compromising survival 66 . Dual antibiotic selection enables the quick and easy selection of dually targeted clones 65 .…”