Integrating Omics and CRISPR Technology for Identification and Verification of Genomic Safe Harbor Loci in the Chicken Genome

Nima Dehdilani; Lena Goshayeshi; Sara Yousefi Taemeh; Ahmad Reza Bahrami; Sylvie Rival Gervier; Bertrand Pain; Hesam Dehghani

doi:10.1186/s12575-023-00210-5

Integrating Omics and CRISPR Technology for Identification and Verification of Genomic Safe Harbor Loci in the Chicken Genome

Biol Proced Online. 2023 Jun 24;25(1):18. doi: 10.1186/s12575-023-00210-5.

Authors

Nima Dehdilani¹, Lena Goshayeshi^{1

2}, Sara Yousefi Taemeh^{1

2}, Ahmad Reza Bahrami^{3

4}, Sylvie Rival Gervier⁵, Bertrand Pain⁵, Hesam Dehghani^{6

7

8}

Affiliations

¹ Stem Cell Biology and Regenerative Medicine Research Group, Research Institute of Biotechnology, Ferdowsi University of Mashhad, Azadi Square, Mashhad, Iran.
² Division of Biotechnology, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran.
³ Department of Biology, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran.
⁴ Industrial Biotechnology Research Group, Institute of Biotechnology, Ferdowsi University of Mashhad, Mashhad, Iran.
⁵ Stem Cell and Brain Research Institute, University of Lyon, Université Lyon 1, INSERM, INRAE, U1208, USC1361, 69500, Bron, France.
⁶ Stem Cell Biology and Regenerative Medicine Research Group, Research Institute of Biotechnology, Ferdowsi University of Mashhad, Azadi Square, Mashhad, Iran. dehghani@um.ac.ir.
⁷ Division of Biotechnology, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran. dehghani@um.ac.ir.
⁸ Department of Basic Sciences, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran. dehghani@um.ac.ir.

Abstract

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.

Keywords: CRISPR/Cas9 Technology; Chicken Genome; Genomic Safe Harbor Loci; Stably-Expressed Transgene; Transgenesis; Weakened Promoter.