Item request has been placed! ×
Item request cannot be made. ×
loading  Processing Request

An integrated protocol for targeted mutagenesis with CRISPR-Cas9 system in the pea aphid

Item request has been placed! ×
Item request cannot be made. ×
loading   Processing Request
  • معلومة اضافية
    • Contributors:
      Institut de Génétique, Environnement et Protection des Plantes (IGEPP); Institut National de la Recherche Agronomique (INRA)-Université de Rennes 1 (UR1); Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-AGROCAMPUS OUEST; Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro); Biologie et Génétique des Interactions Plante-Parasite (UMR BGPI); Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)-Institut National de la Recherche Agronomique (INRA)-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro); Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Centre international d'études supérieures en sciences agronomiques (Montpellier SupAgro); Virologie UMR1161 (VIRO); École nationale vétérinaire d'Alfort (ENVA)-Institut National de la Recherche Agronomique (INRA)-Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES); Laboratoire de Physiologie et Génomique des Poissons (LPGP); Institut National de la Recherche Agronomique (INRA)-Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique ); This work was funded by an INRA-SPE grant (2016-2017) lead by Gaël Le Trionnaire and Véronique Brault (INRA Colmar, UMR SVQV) and the ANR SexAphid (2018-2020) project (ANR-17-CE20-677 0010) lead by Denis Tagu.; ANR-17-CE20-0010,SexAphid,Alternance du mode de reproduction : parts de la génétique et de l'environnement(2017)
    • بيانات النشر:
      HAL CCSD
      Elsevier
    • الموضوع:
      2019
    • Collection:
      Archive ouverte HAL (Hyper Article en Ligne, CCSD - Centre pour la Communication Scientifique Directe)
    • نبذة مختصرة :
      International audience ; CRISPR-Cas9 technology is a very efficient functional analysis tool and has been developed in several insects to edit their genome through injection of eggs with guideRNAs targeting coding sequences of genes of interest. However, its implementation in aphids is more challenging. Aphids are major pests of crops worldwide thatalternate during their life cycle between clonality and sexual reproduction. The production of eggs after mating of sexual individuals is a single yearly event and isnecessarily triggered by a photoperiod decrease. Fertilized eggs then experience an obligate 3-month diapause period before hatching as new clonal colonies. Takinginto consideration these particularities, we developed in the pea aphid Acyrthosiphon pisum a step-by-step protocol of targeted mutagenesis based on the microinjectionwithin fertilized eggs of CRISPR-Cas9 components designed for the editing of a cuticular protein gene (stylin-01). This protocol includes the following steps:i) the photoperiod-triggered induction of sexual morphs (2 months), ii) the mating and egg collection step (2 weeks), iii) egg microinjection and melanization, iv) the3-month obligate diapause, v) the hatching of new lineages from injected eggs (2 weeks) and vi) the maintenance of stable lineages (2 weeks). Overall, this 7-monthlong procedure was applied to three different crosses in order to estimate the impact of the choice of the genetic combination on egg production dynamics by femalesas well as hatching rates after diapause. Mutation rates within eggs before diapause were estimated at 70–80%. The hatching rate of injected eggs following diapauseranged from 1 to 11% depending on the cross and finally a total of 17 stable lineages were obtained and maintained clonally. Out of these, 6 lineages were mutated atthe defined sgRNAs target sites within stylin-01 coding sequence, either at the two alleles (2 lineages) or at one allele (4 lineages). The final germline transmission rateof the mutations was thus around 35%. Our ...
    • Relation:
      info:eu-repo/semantics/altIdentifier/pmid/31015023; hal-02154486; https://hal.archives-ouvertes.fr/hal-02154486; https://hal.archives-ouvertes.fr/hal-02154486/document; https://hal.archives-ouvertes.fr/hal-02154486/file/2019_Le%20Trionnaire_IBMB_1.pdf; PRODINRA: 471158; PUBMED: 31015023; WOS: 000472127200004
    • الرقم المعرف:
      10.1016/j.ibmb.2019.04.016
    • Rights:
      info:eu-repo/semantics/OpenAccess
    • الرقم المعرف:
      edsbas.3260B4E5