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

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 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.

Whole-genome sequencing reveals rare off-target mutations in CRISPR/Cas9-edited grapevine

“…The risk of off-target mutations is increased in plants where the CRISPR/Cas9 construct is active for a long time 50 . Some researchers are committed to developing a way to obtain clean edited plants to reduce such off-target risks 50 , 51 . Six of the Cas9-edited grapevines of W52 used in this study were obtained in our previous studies 12 .…”

LIFE-Seq: A universal Large Integrated DNA Fragment Enrichment Sequencing strategy for transgene integration in genetically modified organisms

“…During the process of GM crop production, transgenes are introduced into the recipient genome randomly, and sometimes accompanied by host gene interruptions, multiple exogenous DNA insertions in one or more chromosomal locations, and host genome rearrangement around the insertion site, which may affect the functional expression and genetic stability of trait-determining genes (38). Therefore, comprehensively characterising the whole transgene integration in the recipient genome is a prerequisite not only for GM crop production, but also for commercialisation.…”

Induced Genetic Variations in Fruit Trees Using New Breeding Tools: Food Security and Climate Resilience

“…Furthermore, Sanger sequencing can be difficult, laborious, and time-consuming. The Sanger sequencing platform is the most widely opted for mechanism of mutation detection after executing the CRISPR-Cas and has been successfully executed in a variety of plants [ 30 , 230 , 231 , 232 , 233 , 234 ].…”