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Background <p>Limitations in genome editing of avian species, including chicken, due to the inaccessibility of one-cell zygotes, have led to the manipulation of primordial germ cells (PGCs) as the primary approach for generating genetically engineered birds. Although plasmid-mediated delivery of clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) has been widely used, it has several limitations, including delayed nuclease activation, increased off-target effects, cytotoxicity, and the inability to apply in vivo selection strategies. Collectively, these limitations highlight the need to develop strategies that achieve high on-target activity at the initial editing step.</p> Results <p>In this study, we compared plasmid- and ribonucleoprotein (RNP)-mediated CRISPR/Cas9 delivery in Leghorn male hepatoma (LMH) cells and in PGCs targeting three loci: deleted in azoospermia like (<i>DAZL</i>), chicken vasa homologue (<i>CVH</i>), and stimulated by retinoic acid 8 (<i>STRA8</i>). RNP delivery showed comparable or higher cell viability and editing efficiency than plasmid delivery in both cell types. Insertion/deletion (indel) profiling revealed broader and more diverse mutation patterns with RNPs, consistent with a shift in repair pathway engagement toward microhomology-mediated end joining (MMEJ), which favors larger deletions. Off-target analysis further showed substantially reduced off-target editing and high specificity with RNP delivery.</p> Conclusions <p>Together, these findings demonstrate that RNP delivery improves efficiency, reduces cytotoxicity, and improves precision, providing a more reliable platform for chicken genetic engineering.</p>

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Improvement of chicken genome editing efficiency in vitro using ribonucleoprotein-mediated CRISPR/Cas9 delivery

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年5月4日

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  • Yujin Han,
  • Chan Young Kwon,
  • Jae Yong Han

摘要

Background

Limitations in genome editing of avian species, including chicken, due to the inaccessibility of one-cell zygotes, have led to the manipulation of primordial germ cells (PGCs) as the primary approach for generating genetically engineered birds. Although plasmid-mediated delivery of clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) has been widely used, it has several limitations, including delayed nuclease activation, increased off-target effects, cytotoxicity, and the inability to apply in vivo selection strategies. Collectively, these limitations highlight the need to develop strategies that achieve high on-target activity at the initial editing step.

Results

In this study, we compared plasmid- and ribonucleoprotein (RNP)-mediated CRISPR/Cas9 delivery in Leghorn male hepatoma (LMH) cells and in PGCs targeting three loci: deleted in azoospermia like (DAZL), chicken vasa homologue (CVH), and stimulated by retinoic acid 8 (STRA8). RNP delivery showed comparable or higher cell viability and editing efficiency than plasmid delivery in both cell types. Insertion/deletion (indel) profiling revealed broader and more diverse mutation patterns with RNPs, consistent with a shift in repair pathway engagement toward microhomology-mediated end joining (MMEJ), which favors larger deletions. Off-target analysis further showed substantially reduced off-target editing and high specificity with RNP delivery.

Conclusions

Together, these findings demonstrate that RNP delivery improves efficiency, reduces cytotoxicity, and improves precision, providing a more reliable platform for chicken genetic engineering.