Rapid delivery of Cas9 gene into the tomato cv.‘Heinz 1706’through an optimized Agrobacterium-mediated transformation procedure

Solanum lycopersicum‘Heinz 1706’is a pioneer model cultivar for tomato research,whose whole genome sequence valuable for genomics studies is available.Nevertheless,a genetic transformation procedure for this cultivar has not yet been reported.Meanwhile,various genome editing technologies such as transfection of clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated(Cas)ribonucleoprotein complexes into cells are in the limelight.Utilizing the Cas9-expressing genotype possessing a reference genome can simplify the verification of an off-target effect,resolve the economic cost of Cas9 endonuclease preparation,and avoid the complex assembly process together with single-guide RNA(sgRNA)in the transfection approach.Thus,this study was designed to generate Cas9-expressing‘Heinz 1706’lines by establishing an Agrobacterium tumefaciens-mediated transformation(ATMT)procedure.Here,we report a rapid and reproducible transformation procedure for‘Heinz 1706’by finetuning various factors:A.tumefaciens strain,pre-culture and co-culture durations,a proper combination of phytohormones at each step,supplementation of acetosyringone,and shooting/rooting method.Particularly,through eluding subculture and simultaneously inducing shoot elongation and rooting from leaf cluster,we achieved a short duration of three months for recovering the transgenic plants expressing Cas9.The presence of the Cas9 gene and its stable expression were confirmed by PCR and qRT-PCR analyses,and the Cas9 gene integrated into the T_(0) plant genome was stably transmitted to T_(1) progeny.Therefore,we anticipate that our procedure appears to ease the conventional ATMT in‘Heinz 1706’,and the created Cas9-expressing‘Heinz 1706’lines are ultimately useful in gene editing via unilateral transfection of sgRNA into the protoplasts.

A simple, flexible and high-throughput cloning system for plant genome editing via CRISPR-Cas system

CRISPR-Cas9 system is now widely used to edit a target genome in animals and plants. Cas9 protein derived from Streptococcus pyogenes（Sp Cas9） cleaves double-stranded DNA targeted by a chimeric single-guide RNA（sg RNA）. For plant genome editing, Agrobacterium-mediated T-DNA transformation has been broadly used to express Cas9 proteins and sg RNAs under the control of Ca MV 35 S and U6/U3 promoter, respectively. We here developed a simple and high-throughput binary vector system to clone a 19 20 bp of sg RNA, which binds to the reverse complement of a target locus, in a large T-DNA binary vector containing an Sp Cas9 expressing cassette. Twostep cloning procedures：（1） annealing two target-specific oligonucleotides with overhangs specific to the Aar I restriction enzyme site of the binary vector; and（2） ligating the annealed oligonucleotides into the two Aar I sites of the vector, facilitate the high-throughput production of the positive clones. In addition, Cas9-coding sequence and U6/U3 promoter can be easily exchanged via the GatewayTMsystem and unique Eco RI/Xho I sites on the vector, respectively. We examined the mutation ratio and patterns when we transformed these constructs into Arabidopsis thaliana and a wild tobacco, Nicotiana attenuata. Our vector system will be useful to generate targeted large-scale knock-out lines of model as well as non-model plant.