Payacán Ortiz, Claudia Verónica (2022) A non-integrative CRISPR/Cas9 genome-editing approach for use in vegetable crop breeding. PhD thesis, University of Warwick.

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Abstract

The Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/CRISPRassociated nuclease 9 (Cas9) system has become the most widespread method to produce edited plants with improved traits. A DNA-free genome editing approach was developed using the plant virus vectors Potato virus X (PVX) and Tobacco rattle virus (TRV) to deliver and express Cas9 and sgRNAs. Splitting two Cas9 orthologs was explored to avoid viral instability due to the delivery of large transgenes. Manipulating flowering time in crops is a long-term goal in plant biotechnology. To achieve this, targeted genome edits were introduced in the SD flowering Nicotiana tabacum var. Maryland Mammoth FLOWERING LOCUS T 4 (NtFT4) gene.

The expression of the split Cas9 was corroborated in N. benthamiana leaves and in vivo activity was validated in N. tabacum protoplasts by edition of the PDS gene. A single transcription unit was constructed encoding the Cas9 and the sgRNA flanked by ribozymes. Mature sgRNAs were detected after self-cleavage of the ribozymes, enabling them to guide the Cas9 to the NtFT4 target sites. Genome editing was found in N. tabacum leaves inoculated with TRV full-length or split Cas9, with frequencies between 3.03% to 0.29%, depending on the enzyme and target site. A delay in flowering time was observed in some plants, however other factors, such as strong viral symptoms, cannot be discarded. Cas9 protein was detected only in inoculated leaves with TRV-full-length SpCas9, and TRV or PVX vectors expressing the SaCas9 C-terminal end, while Cas9 mRNA was found in all inoculated leaves. Even though systemic symptoms of TRV viral infection were seen, full-length or split SpCas9 mRNA was not found, while weak expression of full-length and N-terminal SaCas9 mRNA was observed. In contrast, SaCas9 C-terminal mRNA was detected strongly in TRV systemic leaves, whilst weak expression was found in one PVX systemic leaf sample. Thus, a cargo below 1.3 kb is suggested for viral stability. To obtain fully edited plants, shoots from inoculated tobacco leaves with the different viral constructs were regenerated, but only non-edited plants were found.

In summary, this work demonstrates that viral-mediated genome editing is feasible. Improvements to this system, such as the use of newly developed Cas enzymes, are also discussed and may prove useful in generating crops with new desirable agronomical traits.

Item Type:	Thesis (PhD)
Subjects:	Q Science > QH Natural history > QH426 Genetics Q Science > QK Botany S Agriculture > S Agriculture (General) S Agriculture > SB Plant culture
Library of Congress Subject Headings (LCSH):	Gene editing, Potato virus X, Tobacco rattle virus, Field crops -- Breeding, Plants, Flowering of -- Flowering time
Official Date:	July 2022
Dates:	Date Event July 2022 UNSPECIFIED
Institution:	University of Warwick
Theses Department:	School of Life Sciences
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Jackson, Stephen D.
Sponsors:	Chile. Comisión Nacional de Investigación Científica y Tecnológica
Format of File:	pdf
Extent:	ix, 204 leaves : illustrations (mostly colour), photographs
Language:	eng

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