<p>CRISPR/Cas9-mediated gene editing in insects conventionally relies on embryonic microinjection, which is challenging for non-model species. Therefore, adult injection-based methods have been developed to overcome this obstacle. One such simple and accessible method—direct parental CRISPR (DIPA-CRISPR)—enables gene editing by injecting the Cas9 ribonucleoprotein (RNP) into adult females. Subsequently, a modified approach called SYNCAS was developed by supplementing Cas9 RNPs with branched amphipathic peptide capsules and saponins as delivery enhancers. SYNCAS has been reported to yield higher gene editing efficiency (GEF) than DIPA-CRISPR in the spider mite. However, a direct experimental comparison of these methods under consistent conditions has not been conducted in insects. In the present study, we compared DIPA-CRISPR and SYNCAS methods in the predatory flower bug <i>Orius strigicollis</i> (Poppius) (Hemiptera: Anthocoridae), an important biocontrol agent. While peak GEF values were comparably high for both methods, SYNCAS-treated females continued to lay eggs with higher editing efficiency over a longer period than DIPA-CRISPR-treated females. It resulted in up to 4.8-fold higher yield of gene-edited progeny in the former group than in the latter group. These findings suggested that SYNCAS offers greater flexibility in terms of overall experimental design and gene editing outcomes than DIPA-CRISPR.</p>

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Comparison of DIPA-CRISPR and SYNCAS gene editing methods in the predatory bug Orius strigicollis (Hemiptera: Anthocoridae)

  • Naoki Matsuda,
  • Norihide Hinomoto,
  • Takaaki Daimon

摘要

CRISPR/Cas9-mediated gene editing in insects conventionally relies on embryonic microinjection, which is challenging for non-model species. Therefore, adult injection-based methods have been developed to overcome this obstacle. One such simple and accessible method—direct parental CRISPR (DIPA-CRISPR)—enables gene editing by injecting the Cas9 ribonucleoprotein (RNP) into adult females. Subsequently, a modified approach called SYNCAS was developed by supplementing Cas9 RNPs with branched amphipathic peptide capsules and saponins as delivery enhancers. SYNCAS has been reported to yield higher gene editing efficiency (GEF) than DIPA-CRISPR in the spider mite. However, a direct experimental comparison of these methods under consistent conditions has not been conducted in insects. In the present study, we compared DIPA-CRISPR and SYNCAS methods in the predatory flower bug Orius strigicollis (Poppius) (Hemiptera: Anthocoridae), an important biocontrol agent. While peak GEF values were comparably high for both methods, SYNCAS-treated females continued to lay eggs with higher editing efficiency over a longer period than DIPA-CRISPR-treated females. It resulted in up to 4.8-fold higher yield of gene-edited progeny in the former group than in the latter group. These findings suggested that SYNCAS offers greater flexibility in terms of overall experimental design and gene editing outcomes than DIPA-CRISPR.