Abstract
Current breeding relies mostly on random mutagenesis and recombination to generate novel genetic variation. However, targeted genome editing is becoming an increasingly important tool for precise plant breeding. Using the CRISPR-Cas system combined with the bean yellow dwarf virus rolling circle replicon, we optimized a method for targeted mutagenesis and gene replacement in tomato. The carotenoid isomerase (CRTISO) and phytoene synthase 1 (PSY1) genes from the carotenoid biosynthesis pathway were chosen as targets due to their easily detectable change of phenotype. We took advantage of the geminiviral replicon amplification as a means to provide a large amount of donor template for the repair of a CRISPR-Cas-induced DNA double-strand break (DSB) in the target gene, via homologous recombination (HR). Mutagenesis experiments, performed in the Micro-Tom variety, achieved precise modification of the CRTISO and PSY1 loci at an efficiency of up to 90%. In the gene targeting (GT) experiments, our target was a fast-neutron-induced crtiso allele that contained a 281-bp deletion. This deletion was repaired with the wild-type sequence through HR between the CRISPR-Cas-induced DSB in the crtiso target and the amplified donor in 25% of the plants transformed. This shows that efficient GT can be achieved in the absence of selection markers or reporters using a single and modular construct that is adaptable to other tomato targets and other crops.
| Original language | English |
|---|---|
| Pages (from-to) | 5-16 |
| Number of pages | 12 |
| Journal | Plant Journal |
| Volume | 95 |
| Issue number | 1 |
| Early online date | 18 Apr 2018 |
| DOIs | |
| Publication status | Published - Jul 2018 |
Funding
The authors would like to thank Prof. Daniel Voytas (University of Minnesota) for the BeYDV geminiviral replicon system; Prof. Antonio Granell and Dr Diego Orzàez (Università Politecnica Valencia) for the Golden‐Braid cloning system; Prof. Holger Puchta (Karlsruhe Institute of Technology) for the pDE‐PcUbi4:Sp_Cas9 plasmid; Tamar Azulay and Timor Grinevich for excellent technical assistance; and Ziva Amsellem and Muriel Chemla for assistance with the Micro‐Tom transformation protocol and help in tissue culture. The authors would like to thank all the Levy lab members for fruitful discussions during the course of this study. This work was supported by a grant from the Israeli Ministry of Agriculture (IMOAG) to AAL. T.D.‐M. planned and performed most of the experiments, and wrote the manuscript; S.F.‐H. contributed to the planning of some of the experiments; C.M.‐B. prepared the high‐throughput sequencing libraries and the S. blot analysis, and contributed to the planning of some of the experiments; S.B. and A.A. provided the SlUbiquitin10 promoter; H.C. provided supervision and expertise with the use of the geminiviral replicon; A.A.L. planned most of the experiments, wrote the manuscript and supervised the work.