Integrated approach for the molecular characterization of edited plants obtained via Agrobacterium tumefaciens-mediated gene transfer

AbstractAgrobacterium tumefaciens-mediated gene transfer—actually the most used method to engineer plants—may lead to integration of multiple copies of T-DNA in the plant genome, as well as to chimeric tissues composed of modified cells and wild type cells. A molecular characterization of the transformed lines is thus a good practice to select the best ones for further investigation. Nowadays, several quantitative and semi-quantitative techniques are available to estimate the copy number (CN) of the T-DNA in genetically modified plants. In this study, we compared three methods based on (1) real-time polymerase chain reaction (qPCR), (2) droplet digital PCR (ddPCR), and (3) next generation sequencing (NGS), to carry out a molecular characterization of grapevine edited lines. These lines contain a knock-out mutation, obtained via CRISPR/Cas9 technology, in genes involved in plant susceptibility to two important mildew diseases of grapevine. According to our results, qPCR and ddPCR outputs are largely in agreement in terms of accuracy, especially for low CN values, while ddPCR resulted more precise than qPCR. With regard to the NGS analysis, the CNs detected with this method were often not consistent with those calculated by qPCR and ddPCR, and NGS was not able to discriminate the integration points in three out of ten lines. Nevertheless, the NGS method can positively identify T-DNA truncations or the presence of tandem/inverted repeats, providing distinct and relevant information about the transgene integration asset. Moreover, the expression analysis of Cas9 and single guide RNA (sgRNA), and the sequencing of the target site added new information to be related to CN data. This work, by reporting a practical case-study on grapevine edited lines, explores pros and cons of the most advanced diagnostic techniques available for the precocious selection of the proper transgenic material. The results may be of interest both to scientists developing new transgenic lines, and to laboratories in charge of GMO control.

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Strategies to produce T-DNA free CRISPRed fruit trees via Agrobacterium tumefaciens stable gene transfer

Scientific Reports ◽

10.1038/s41598-020-77110-1 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Lorenza Dalla Costa ◽

Stefano Piazza ◽

Valerio Pompili ◽

Umberto Salvagnin ◽

Alessandro Cestaro ◽

...

Keyword(s):

Agrobacterium Tumefaciens ◽

Gene Transfer ◽

Genomic Dna ◽

Fruit Trees ◽

Plant Genome ◽

Stable Gene ◽

Binary Vectors ◽

Exogenous Dna ◽

Left Behind ◽

Dicotyledonous Plants

AbstractGenome editing via CRISPR/Cas9 is a powerful technology, which has been widely applied to improve traits in cereals, vegetables and even fruit trees. For the delivery of CRISPR/Cas9 components into dicotyledonous plants, Agrobacterium tumefaciens mediated gene transfer is still the prevalent method, although editing is often accompanied by the integration of the bacterial T-DNA into the host genome. We assessed two approaches in order to achieve T-DNA excision from the plant genome, minimizing the extent of foreign DNA left behind. The first is based on the Flp/FRT system and the second on Cas9 and synthetic cleavage target sites (CTS) close to T-DNA borders, which are recognized by the sgRNA. Several grapevine and apple lines, transformed with a panel of CRISPR/SpCas9 binary vectors, were regenerated and characterized for T-DNA copy number and for the rate of targeted editing. As detected by an optimized NGS-based sequencing method, trimming at T-DNA borders occurred in 100% of the lines, impairing in most cases the excision. Another observation was the leakage activity of Cas9 which produced pierced and therefore non-functional CTS. Deletions of genomic DNA and presence of filler DNA were also noticed at the junctions between T-DNA and genomic DNA. This study proved that many factors must be considered for designing efficient binary vectors capable of minimizing the presence of exogenous DNA in CRISPRed fruit trees.

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Gene transfer and molecular characterization of introgression from wildOryzaspecies into rice

Rice Genetics Collection - Rice Genetics II ◽

10.1142/9789812814289_0051 ◽

2008 ◽

pp. 477-486 ◽

Cited By ~ 4

Author(s):

D. S. Brar ◽

R. Dalmacio ◽

R. Elloran ◽

R. Aggarwal ◽

R. Angeles ◽

...

Keyword(s):

Gene Transfer ◽

Molecular Characterization

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Improvement of adventitious organogenesis for regeneration of transgenic plants in blackberry

Genetika ◽

10.2298/gensr1502599g ◽

2015 ◽

Vol 47 (2) ◽

pp. 599-608 ◽

Cited By ~ 1

Author(s):

Alena Gajdosová ◽

Tatjana Vujovic ◽

Miroslava Súkeníková ◽

Gabriela Libiaková

Keyword(s):

Agrobacterium Tumefaciens ◽

Plant Regeneration ◽

Gene Transfer ◽

Transgenic Plants ◽

Plant Genome ◽

Ms Medium ◽

Regeneration System ◽

Promising Technique ◽

Rubus Fruticosus ◽

Foreign Dna

The introduction of foreign DNA into the plant genome by Agrobacterium tumefaciens is a promising technique of targeted gene transfer which depends on good working regeneration system. The aim of the work was to elaborate the system for efficient adventitious organogenesis and transgenic plant regeneration in Rubus fruticosus L. using explants from mature plants. Regeneration of putative transgenic shoots took place from flag explants cultivated vertically on MS medium with 1 mg l-1 TDZ and 0.02 mg l-1 IBA followed by transfer on MS medium with 1 mg l-1 BAP, 0.02 mg l-1 IBA and 0.1 mg l-1 GA3 supplemented with 10-15 mg l-1 hygromycin after transformation by A. tumefaciens strain LBA 4404 carrying plasmid pCambia 1304. Four putative transgenic plants of cv. 'Cacanska Bestrna' were rooted and acclimatized.

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Comparison of circulating tumor cells and cell-free DNA in the molecular characterization of patients with head and neck cancer.

Journal of Clinical Oncology ◽

10.1200/jco.2020.38.15_suppl.e18521 ◽

2020 ◽

Vol 38 (15_suppl) ◽

pp. e18521-e18521

Author(s):

Santiago Cabezas-Camarero ◽

Vanesa García-Barberán ◽

Virginia De la Orden-García ◽

Beatriz Mediero-Valeros ◽

Isabel Díaz-Millán ◽

...

Keyword(s):

Head And Neck Cancer ◽

Parotid Gland ◽

Head And Neck ◽

Molecular Characterization ◽

Neck Cancer ◽

Liquid Biopsy ◽

Digital Pcr ◽

Cell Free Dna ◽

Free Dna

e18521 Background: The role of liquid biopsy in diagnosis and therapy monitoring in patients with head and neck cancer has been much less studied compared to other cancers. Our aim was to evaluate the perfomance in the isolation and recovery for molecular characterization of circulating tumour cells (CTC) of a new immunoafinity-based method and to compare it with the molecular diagnostic yield of plasma cell-free DNA. Methods: Patients with recurrent/metastatic (RM) head and neck cancer (HNC) were enrolled prospectively. Forty mililiters (ml) of plasma were collected at one or several time-points. First blood draw was always collected before starting a new therapeutic intervention or at the time of radiologic progression. For CTC detection and isolation, either anti-EpCAM or both anti-EpCAM + anti-EGFR antibodies were used. Digital PCR and castPCR were used to study KRAS and PI3KCA mutations in non-squamous HNC. A 15-gene customized NGS panel was used to characterized both CTC and cfDNA in patients with squamous HNC. Results: Between February 2016 and October 2018, 14 patients with R/M HNC were included (n = 1 local-only disease, n = 10 local and distant disease, n = 3 distant-only disease). Squamous histology (S): n = 9. Non-squamous (NS): n = 5 (1 naso-ethmoidal intestinal-type adenocarcinoma, 1 parotid gland exadenoma pleomorfic carcinoma, 2 parotid-gland salivary duct carcinomas (SDC), 1 parotid-gland high-grade neuroendocrine carcinoma). Twenty-five CTC determinations were performed. In 5 patients serial CTC determinations were performed. Median CTC was 4 (min-max: 0-49). Median CTC among 11 CTC determinations in S-HNC was 4 (min-max: 0-49). Median CTC was 3 CTC (min-max: 0-26) among the 14 determinations performed in NS-HNC. Digital PCR unveiled mutations in CTC and in cfDNA in 2 of 4 patients tested with NS histology (KRAS, PIK3CA), with one of them being concordant for the specific mutation. NGS unveiled mutations in CTC in 7/9 patients and in cfDNA in 6/9 patients, with only one loci-concordant case between CTC and plasma. Conclusions: IsoFlux detected CTC in the majority of patients with R/M HNC, regardless of the histologic type, and allowed for molecular characterization of CTC using different techniques for mutational analysis. Both NGS and digital PCR allowed for the detection in cell-free DNA of commonly mutated genes in HNC. Liquid biopsy should be more actively studied in this disease in order to better define its role in diagnosis and therapeutic monitoring.

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