High efficiency gene editing and phenotype generation in avian embryos using lipid nanoparticles as a vector delivery platform
- 1Colossal Bioscience
Abstract
Birds are a longstanding developmental biology model because easy access to their fertilized eggs allows for direct observation and experimental manipulation during any stage of embryonic growth prior to hatching. However, functional genetics studies in avian embryos continue to rely primarily on in ovo electroporation and viral transduction, each of which is limited by variable efficiency, procedure-induced embryonic defects, substantial embryo mortality, and ineffective delivery of gene editing reagents such as CRISPR/Cas9. Here we establish lipid nanoparticle (LNP)-mediated delivery as a reproducible alternative for manipulating gene expression in avian embryos with high viability. Using the chicken embryo (Gallus gallus), we targeted migrating cranial NCCs, which contribute extensively to the craniofacial skeleton. We optimized LNP formulation and injection timing and frequency to build a robust and adaptable pipeline that offers efficient delivery of plasmid DNA and RNA cargos. We show that LNP delivery can support the piggyBac transposon system, enabling persistent reporter expression and long-term labeling of cranial NCC derivatives. To test if LNP-mediated delivery of RNA-based CRISPR/Cas9 components can achieve efficient somatic genome editing in ovo, we targeted Runt-related transcription factor 2 (RUNX2), which is required for osteogenesis. Embryos with RUNX2 edits in their cranial NCCs show a severe loss and disruption of craniofacial ossification on the targeted side of their craniofacial skeletons, demonstrating the potential of this approach for rapid functional interrogation of gene function in vivo. We also show that LNPs can mediate transfection in domestic pigeon (Columba livia) and Pekin duck (Anas platyrhynchos) embryos, indicating this method can be deployed across avians as a vector delivery platform for studying development, disease, and evolution.
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BibTeX
@misc{keyte2026high,
title = {{High efficiency gene editing and phenotype generation in avian embryos using lipid nanoparticles as a vector delivery platform}},
author = {A. L. Keyte and J. P. Zanin and B. M. Collerton and S. K. Nguyen and W. N. Feist and M. L. Coquelin and J. N. Stout and B. B. Blair and J. B. Papizan and R. A. Schneider and X. Zhan},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.13.753869},
url = {https://www.biorxiv.org/content/10.64898/2026.10.13.753869v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - High efficiency gene editing and phenotype generation in avian embryos using lipid nanoparticles as a vector delivery platform
AU - A. L. Keyte
AU - J. P. Zanin
AU - B. M. Collerton
AU - S. K. Nguyen
AU - W. N. Feist
AU - M. L. Coquelin
AU - J. N. Stout
AU - B. B. Blair
AU - J. B. Papizan
AU - R. A. Schneider
AU - X. Zhan
PY - 2026
DA - 2026/10/13
PB - bioRxiv
DO - 10.64898/2026.10.13.753869
UR - https://www.biorxiv.org/content/10.64898/2026.10.13.753869v1
AB - Birds are a longstanding developmental biology model because easy access to their fertilized eggs allows for direct observation and experimental manipulation during any stage of embryonic growth prior to hatching. However, functional genetics studies in avian embryos continue to rely primarily on in ovo electroporation and viral transduction, each of which is limited by variable efficiency, procedure-induced embryonic defects, substantial embryo mortality, and ineffective delivery of gene editing reagents such as CRISPR/Cas9. Here we establish lipid nanoparticle (LNP)-mediated delivery as a reproducible alternative for manipulating gene expression in avian embryos with high viability. Using the chicken embryo (Gallus gallus), we targeted migrating cranial NCCs, which contribute extensively to the craniofacial skeleton. We optimized LNP formulation and injection timing and frequency to build a robust and adaptable pipeline that offers efficient delivery of plasmid DNA and RNA cargos. We show that LNP delivery can support the piggyBac transposon system, enabling persistent reporter expression and long-term labeling of cranial NCC derivatives. To test if LNP-mediated delivery of RNA-based CRISPR/Cas9 components can achieve efficient somatic genome editing in ovo, we targeted Runt-related transcription factor 2 (RUNX2), which is required for osteogenesis. Embryos with RUNX2 edits in their cranial NCCs show a severe loss and disruption of craniofacial ossification on the targeted side of their craniofacial skeletons, demonstrating the potential of this approach for rapid functional interrogation of gene function in vivo. We also show that LNPs can mediate transfection in domestic pigeon (Columba livia) and Pekin duck (Anas platyrhynchos) embryos, indicating this method can be deployed across avians as a vector delivery platform for studying development, disease, and evolution.
ER -