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Updated: Jul 25, 2025

HOX Loci Focused CRISPR/sgRNA Library Screening Identifying Critical CTCF Boundaries
Published on: March 31, 2019
Integrating Omics and CRISPR Technology for Identification and Verification of Genomic Safe Harbor Loci in the
Nima Dehdilani1, Lena Goshayeshi1,2, Sara Yousefi Taemeh1,2
1Stem Cell Biology and Regenerative Medicine Research Group, Research Institute of Biotechnology, Ferdowsi University of Mashhad, Azadi Square, Mashhad, Iran.
Background:
One of the most prominent questions in the field of transgenesis is 'Where in the genome to integrate a transgene?'. Escape from epigenetic silencing and promoter shutdown of the transgene needs reliable genomic safe harbor (GSH) loci. Advances in genome engineering technologies combined with multi-omics bioinformatics data have enabled rational evaluation of GSH loci in the host genome. Currently, no validated GSH loci have been evaluated in the chicken genome.
Results:
Here, we analyzed and experimentally examined two GSH loci in the genome of chicken cells. To this end, putative GSH loci including chicken HIPP-like (cHIPP; between DRG1 and EIF4ENIF1 genes) and chicken ROSA-like (cROSA; upstream of the THUMPD3 gene) were predicted using multi-omics bioinformatics data. Then, the durable expression of the transgene was validated by experimental characterization of continuously-cultured isogenous cell clones harboring DsRed2-ΔCMV-EGFP cassette in the predicted loci. The weakened form of the CMV promoter (ΔCMV) allowed the precise evaluation of GSH loci in a locus-dependent manner compared to the full-length CMV promoter.
Conclusions:
cHIPP and cROSA loci introduced in this study can be reliably exploited for consistent bio-manufacturing of recombinant proteins in the genetically-engineered chickens. Also, results showed that the genomic context dictates the expression of transgene controlled by ΔCMV in GSH loci.
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