Efficacy of In Vivo Electroporation on the Delivery of Molecular Agents into Aphid (Hemiptera: Aphididae) Ovarioles

Abstract

While the wealth of genomic data presently available is increasing rapidly, the advancement of functional genomics technologies for the large majority of these organisms has lagged behind. The Clustered Regularly Interspaced Palindromic Repeats (CRISPR)/Cas9 system is an emerging gene-editing technology derived from a bacterial adaptive immune system that has proven highly effective in multiple model systems. Here, the CRISPR/Cas9 system was delivered into the ovarioles of the pea aphid, Acyrthosiphon pisum (Harris) (Hemiptera, Aphididae), with a new delivery method utilizing in vivo electroporation. To validate gene-editing, a target sequence within the marker tor pigment gene was chosen, and gene-editing was predicted to result in white pigmentation in the offspring of treated adult aphids. Adult aphids (10-d old) were injected with the tor single guide RNA and Cas9 complex and subsequently subjected to electroporation. Adult aphids were given 4 d to produce viviparous offspring. After offspring developed for 6 d, DNA was extracted and sequenced to validate if CRISPR/Cas9-directed gene editing occurred. A survival rate over 70% was found in treated adult aphids. A distinct white pigmentation was found in 2.5% of aphids; however, gene-editing within the target sequence was not found in any of the individuals screened. Presence of white aphids without gene-editing suggests other mechanisms may have influenced pigmentation. High survival rates in experimental treatments demonstrate the robustness of this new technique, and further refinement of this technique may prove it as an effective functional genomics tool for viviparous insects and/or gene editing at a somatic level.

In-vivo egfp expression in the honeybee Apis mellifera induced by electroporation and viral expression vector

“…The feasibility of the application of in-vivo electroporation and viral transduction in insect species where a genetic tool kit is not available has been demonstrated in several recent studies. For example, to deliver CRISPR/Cas9 [ 36 ] or RNAi [ 37 ] vectors by in vivo electroporation or for the optogenetic manipulation of neurons by viral transduction [ 38 ]. Species specific differences, like the cellular density of a tissue or the expression of surface receptors, are one of the factors influencing the outcome of a physiological manipulation.…”

Host and symbiont genetic determinants of nutritional phenotype in a natural population of the pea aphid

“…An important topic for future research is the contribution of the various candidate aphid genes identified in this study to the regulatory circuits that influence the EAA nutritional phenotype of the insect. Such future studies will be facilitated by ongoing innovations in methods to manipulate aphid genomes (Jamison, Thairu, & Hansen, ; Le Trionnaire et al, ), as well as expanded effort to identify candidate genes. In particular, full genome sequencing is predicted to yield further SNPs of interest, beyond those obtained in this study by the reduced‐representation genome sequencing by GBS.…”

In vivo direct cell-penetrating peptide mediated protein transduction system in Acyrthosiphon pisum

“…The Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-based genome editing system can be applied to various species, including non-model insects, which is crucial to develop novel pest control strategies [ 1 ]. To date, single guide ribonucleic acid (sgRNA)–Cas9 (CRISPR associated protein 9) ribonucleoprotein (RNP) complexes have been attempted to be delivered using DNA electroporation targeting the Target of Rapamycin ( Tor ) gene in viviparous aphid ovaries, with no indel mutations detected in the target gene in the offspring of treated individuals [ 2 ]. Although the CRISPR/Cas9 system has been tested in aphids, its deployment is still challenging due to the difficulty of injecting into aphid embryos because oogenesis occurs without apparent vitellogenesis.…”