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

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

doi:10.1186/s12575-023-00210-5

Cited by 3 publications

(1 citation statement)

References 73 publications

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“…However, the initial targeting rate was very low (1.8%). Currently, two novel genomic safe harbor loci, cHIPP and cROSA, are found in the genome of DF-1 cells [41]. Nonetheless, further in vivo investigation is necessary for genetically engineered chickens.…”

Section: Introductionmentioning

confidence: 99%

Comparison of Multiple Strategies for Precision Transgene Knock-In in Gallus gallus Genome via Microhomology-Mediated End Joining

Wang,

Sun,

Liu

et al. 2023

IJMS

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Precision exogenous gene knock-in is an attractive field for transgenic Gallus gallus (chicken) generation. In this article, we constructed multiple Precise Integration into Target Chromosome (PITCh) plasmid systems mediated by microhomology-mediated end-joining (MMEJ) for large-fragment integration in DF-1 cells and further assess the possibility of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) as a genomic safe harbor for chickens. We designed three targeted sgRNAs for the all-in-one plasmid at the 3′UTR of GAPDH near the stop codon. The donor-plasmid-carrying microhomology arms correspond to sgRNA and EGFP fragments in the forward and reverse directions. MMEJ-mediated EGFP insertion can be efficiently expressed in DF-1 cells. Moreover, the differences between the forward and reverse fragments indicated that promoter interference does affect the transfection efficiency of plasmids and cell proliferation. The comparison of the 20 bp and 40 bp microhomology arms declared that the short one has higher knock-in efficiency. Even though all three different transgene insertion sites in GAPDH could be used to integrate the foreign gene, we noticed that the G2-20R-EGFP cell reduced the expression of GAPDH, and the G3-20R-EGFP cell exhibited significant growth retardation. Taken together, G1, located at the 3′UTR of GAPDH on the outer side of the last base of the terminator, can be a candidate genomic safe harbor (GSH) loci for the chicken genome. In addition, deleted-in-azoospermia-like (DAZL) and actin beta (ACTB) site-specific gene knock-in indicated that MMEJ has broad applicability and high-precision knock-in efficiency for genetically engineered chickens.

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Section: Introductionmentioning

confidence: 99%

Comparison of Multiple Strategies for Precision Transgene Knock-In in Gallus gallus Genome via Microhomology-Mediated End Joining

Wang,

Sun,

Liu

et al. 2023

IJMS

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Exploring the Function of Gene Promoter Regulatory Elements Using CRISPR Tools

Yousefi Taemeh,

Dehdilani,

Goshayeshi

et al. 2024

Methods in Molecular Biology

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Advancements in mammalian display technology for therapeutic antibody development and beyond: current landscape, challenges, and future prospects

Slavny,

Hegde,

Doerner

et al. 2024

Front. Immunol.

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The evolving development landscape of biotherapeutics and their growing complexity from simple antibodies into bi- and multi-specific molecules necessitates sophisticated discovery and engineering platforms. This review focuses on mammalian display technology as a potential solution to the pressing challenges in biotherapeutic development. We provide a comparative analysis with established methodologies, highlighting key aspects of mammalian display technology, including genetic engineering, construction of display libraries, and its pivotal role in hit selection and/or developability engineering. The review delves into the mechanisms underpinning developability-driven selection via mammalian display and their broader implications. Applications beyond antibody discovery are also explored, alongside advancements towards function-first screening technologies, precision genome engineering and AI/ML-enhanced libraries, situating them in the context of mammalian display. Overall, the review provides a comprehensive overview of the current mammalian display technology landscape, underscores the expansive potential of the technology for biotherapeutic development, addresses the critical challenges for the full realisation of this potential, and examines advances in related disciplines that might impact the future application of mammalian display technologies.

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

Cited by 3 publications

References 73 publications

Comparison of Multiple Strategies for Precision Transgene Knock-In in Gallus gallus Genome via Microhomology-Mediated End Joining

Comparison of Multiple Strategies for Precision Transgene Knock-In in Gallus gallus Genome via Microhomology-Mediated End Joining

Exploring the Function of Gene Promoter Regulatory Elements Using CRISPR Tools

Advancements in mammalian display technology for therapeutic antibody development and beyond: current landscape, challenges, and future prospects

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