Genome editing and gene expression engineering using CRISPR-Cas systems
in plants usually rely on labor-intensive tissue culture approaches to
generate stably transformed plants that express the components of the
reaction. Viral vectors have demonstrated to be a quick and effective
alternative to express multiple guide RNAs, DNA templates for homologous
recombination, and even Cas nucleases. Here we have developed an
improved vector system based on tobacco rattle virus (TRV) to simplify
logistics in genome editing and gene silencing approaches. The new
system consists in a single Agrobacterium tumefaciens clone
co-transformed with two compatible mini binary vectors from which TRV
RNA1 and an engineered version of TRV RNA2 are expressed. Sequences of
recombinant proteins, gene fragments for virus-induced gene silencing
(VIGS) or guide RNAs can be easily inserted by one-step
digestion-ligation and homology-based cloning methods in the RNA2
plasmid to produce vectors with a size substantially smaller than usual.
Using this new one-Agrobacterium TRV mini vector system, we show robust
VIGS of an endogenous host gene after infiltration of bacterial
suspensions at low optical densities, and efficient production of
recombinant proteins in Nicotiana benthamiana. Most importantly, we also
show highly efficient heritable genome editing in more than half of the
seedling originating from inoculated N. benthamiana plants that express
Cas9.
A new vector coupling ligation-independent cloning with sortase a fusion for efficient cloning and one-step purification of tag-free recombinant proteins
