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

Scientific Reports
Lorenza Dalla CostaMickael Malnoy

Abstract

Genome 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...Continue Reading

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Citations

May 2, 2021·Horticulture Research·Xianhang WangXiping Wang
May 5, 2021·Plant Biotechnology Journal·Gaetano GiudiceWalter Chitarra

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Methods Mentioned

BETA
transgenic
phosphotransferase
PCR
Illumina sequencing
transfection
genotyping
electrophoresis

Software Mentioned

Blast
fastq
VSEARCH
GenomeWalker
SAM

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