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.
Highlights
Genome editing via CRISPR/Cas[9] is a powerful technology, which has been widely applied to improve traits in cereals, vegetables and even fruit trees
A case of inverted repeats may be ascribed to line GT110.5, where upstream to a partial left border (LB) site we found a sequence containing U6At, gRNA, cleavage target sites (CTS) and right border (RB). (v) Microhomology In the lines free of filler unknown DNA, we analyzed the extent of microhomology between T-DNA processed ends and the genomic sequence by comparing the pre-insertion genomic DNA sequence from the point of integration in the forward direction, with the exogenous sequence at the border
We focused on seeking the LB sequence that in theory would be present in all the organisms modified via Agrobacterium, like a specific footprint proving the biotechnological origin of the product
Summary
Genome editing via CRISPR/Cas[9] is a powerful technology, which has been widely applied to improve traits in cereals, vegetables and even fruit trees. Editing technology, based predominantly on CRISPR systems, has been applied to various crop species offering tremendous opportunities for trait improvement in vegetatively propagated crops, which can conserve the overall genetic make-up[1,2]. This huge biotechnological potential is sometimes hampered by legislative boundaries which regulate agricultural production at a national level. USA, Argentina, Brazil and Chile have established that if no foreign genes or genetic material are present in the edited plant variety, the additional regulatory oversight and risk assessment for GMO products will not apply[3] Other countries, such as Australia and New Zealand, are still examining the regulation of new breeding technologies[4]. Agrobacterium tumefaciens (A.t.) where stable integration of exogenous DNA is followed by backcrossing (only feasible for sexually propagated annual plants), transient expression of plasmids carrying CRISPR c omponents[8] or methods relying on the delivery of CRISPR/Cas[9] protein-RNA complex (RNP) to p rotoplasts[2] are being developed
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