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A robust and Efficient CRISPR-Cas9 mutagenesis protocol in the pea aphid

Abstract : CRISPR-Cas9 has emerged in the last few years as a very efficient functional analysis tool in model and non-model organisms. Aphids damages on crops are largely explained by their unusual life cycle, since they reproduce most of the time asexually (by parthenogenesis during spring and summer), sexual reproduction only occurring at the end of autumn, in response to photoperiod shortening. Sexual individuals thus mate and lay eggs that over-winter through an obligatory 3 months diapause. In most of insect species where sexuality is the only reproductive mode, mutagenesis protocols rely on the injection of fertilized eggs to generate stable and germline transmissible mutational events. Aphids thus represent a very challenging model since sexual reproductive mode switch must be triggered by carefully controlled photoperiod conditions. Here we developed a step-by-step CRISPR-Cas9 mutagenesis protocol in the pea aphid by targeting Stylin-1, a cuticular protein gene likely to be involved in phytovirus vection. To achieve that, we injected fertilized eggs with a mixture of single guide RNAs (targeting coding sequences of the gene) and Cas9 protein. The overall procedure takes almost 7 months from photoperiodic induction of sexual morphs (2 months), mating and eggs injection (2 weeks), obligatory diapause for surviving eggs (3 months), lineages hatching (3 weeks) and germline transmission evaluation in their offspring (3 weeks). We could first estimate directly after injection an in-ovo mutation rate of nearly 70%. Then after diapause hatching rate of melanized eggs was about 8% ended up in total with 16 established lineages producing offspring. Among those, 6 lineages showed stable mutations of one or both alleles. Germline transmission rate of mutations was thus around 37.5%. Despite the peculiarities of the biology of the aphid our study clearly demonstrates the efficiency of the CRISPR-Cas9 mutagenesis protocol in this insect. These stylin-1 edited aphids represent so far the first mutant lineages ever generated and open exciting opportunities to eventually demonstrate and validate gene function in this major crop pest.
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https://hal.inrae.fr/hal-02733649
Contributor : Migration Prodinra <>
Submitted on : Tuesday, June 2, 2020 - 12:52:08 PM
Last modification on : Thursday, April 15, 2021 - 12:26:05 PM

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  • HAL Id : hal-02733649, version 1
  • PRODINRA : 445125

Citation

Gaël Le Trionnaire, Denis Tagu, Amaury Herpin, Marilyne Uzest, Sylvie Tanguy, et al.. A robust and Efficient CRISPR-Cas9 mutagenesis protocol in the pea aphid. 11. European Congress of Entomology - ECE 2018, Jul 2018, Napoli, Italy. 334 p. ⟨hal-02733649⟩

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