New human chromosomal safe harbor sites for genome engineering with CRISPR/Cas9, TAL effector and homing endonucleases

Abstract

292 words) Safe Harbor Sites (SHS) are genomic locations where new genes or genetic elements can be introduced without disrupting the expression or regulation of adjacent genes. We have identified 35 potential new human SHS in order to substantially expand SHS options beyond the three widely used canonical human SHS, AAVS1 , CCR5 and hROSA26 . All 35 potential new human SHS and the three canonical sites were assessed for SHS potential using 9 different criteria weighted to emphasize safety that were broader and more genomics-based than previous efforts to assess SHS potential. We then systematically compared and rank-ordered our 35 new sites and the widely used human AAVS1 , hROSA26 and CCR5 sites, then experimentally validated a subset of the highly ranked new SHS together versus the canonical AAVS1 site. These characterizations included in vitro and in vivo cleavage-sensitivity tests; the assessment of population-level sequence variants that might confound SHS targeting or use for genome engineering; homology-dependent and -independent, SHS-targeted transgene integration in different human cell lines; and comparative transgene integration efficiencies at two new SHS versus the canonical AAVS1 site. Stable expression and function of new SHS-integrated transgenes were demonstrated for transgene-encoded fluorescent proteins, selection cassettes andCas9 variants including a transcription transactivator protein that were shown to drive large deletions in a PAX3/FOXO1 fusion oncogene and induce expression of the MYF5 gene that is normally silent in human rhabdomyosarcoma cells. We also developed a SHS genome engineering 'toolkit' to enable facile use of the most extensively characterized of our new human SHS located on chromosome 4p.We anticipate our newly identified human SHS, located on 16 chromosomes including both arms of the human X chromosome, will be useful in enabling a wide range of basic and more clinically-oriented human gene editing and engineering.

Multimodal CRISPR perturbations of GWAS loci associated with coronary artery disease in vascular endothelial cells

“…For each experiment, we measured the mean fluorescent intensity (MFI) obtained for sgRNA of interest and compared it with the MFI for control sgRNA (safe-harbor and/or scrambled sgRNA). Safe-harbor sgRNA sequences were based on Pellenz et al [ 64 ]. We performed each experiment at least three times.…”

Gulo gene locus, a new gene editing locus for mammalian cells

“…New safe harbor loci were reported, including CCR5 [22,23] and hROSA26. [24] The CCR5 gene locus encodes the chemokine receptor, the major receptor for HIV-1. The spontaneous null mutation in the CCR5 locus was observed in the resistance against HIV-1 infection.…”

An intergenic “safe haven” region in Cryptococcus neoformans serotype D genomes

“…Thus, integration in a designated gene free region in the genome is preferred. Several gene free regions were named as "Safe Harbor" sites for alien gene integration in human cellular studies (Pellenz et al, 2018;Sadelain et al, 2011). Similarly, "safe haven" sites have recently been identified in the fungus Aspergillus fumigatus reference strains CEA10/Af293 and C. neoformans serotype A reference strain H99 (Arras et al, 2015;Pham et al, 2020;Upadhya et al, 2017).…”