Optimising CRISPR-CAS systems for targeted gene editing in aphids and whiteflies

James, Reuben Philip (2024) Optimising CRISPR-CAS systems for targeted gene editing in aphids and whiteflies. Doctoral thesis, University of East Anglia.

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Abstract

The green peach aphid (Myzus persicae) and the silverleaf whitefly (Bemisia tabaci) are global, polyphagous hemipteran pests. Defined by piercing-sucking mouthparts, these insects feed on phloem sap, causing plant damage and transmitting viruses. They also secrete effector proteins to suppress plant defences. Gene editing technologies could be used to knock out effector, and viral transmission associated genes, enabling a deeper understanding of these damaging processes, with the potential of identifying an ‘Achilles heel’ to target for insect control.

This thesis investigated Receptor Mediated Ovary Transduction of Cargo (ReMOT Control) for gene editing in M. persicae. This involved injecting females with a Cas9 protein fused to a vitellogenin (Vg)-derived peptide, enabling the Cas9-sgRNA complex to reach developing preblastodermal embryos for germline editing. This technique has been successful in insects and one arachnid, indicating its potential in aphids. Often, CRISPR in insects occurs via egg injection; because aphids only lay eggs under autumn-winter conditions, ReMOT offers a more feasible alternative to direct egg injection.

To optimize ReMOT Control in M. persicae, Vgs were characterized, and an embryo-targeting peptide (MpRV) was identified. Its targeting efficiency was tested alongside the Drosophila melanogaster P2C peptide, which has been used for embryo-targeting in other arthropods. Cas9 fusions with MpRV and P2C were successfully expressed, purified, and shown to cleave the aphid white gene in vitro, a gene predicted to alter eye colour. Upon optimizing injection protocols, evidence of low-efficiency somatic gene editing in aphids was obtained.

For B. tabaci, a smaller CRISPR enzyme, CasΦ, was tested due to its compact size and AT-rich targeting preference. A CasΦ chimera fused with the whitefly Vg-derived peptide BtKV was developed, and ReMOT experiments showed preliminary evidence of white gene editing.

In summary, ReMOT Control technologies represent a promising advancement for achieving more efficient gene editing in aphids and whiteflies.

Item Type:	Thesis (Doctoral)
Faculty \ School:	Faculty of Science > School of Biological Sciences
Depositing User:	Chris White
Date Deposited:	10 Nov 2025 09:37
Last Modified:	10 Nov 2025 09:37
URI:	https://ueaeprints.uea.ac.uk/id/eprint/100932
DOI:

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