CRISPR/Cas9-mediated targeted T-DNA integration in rice
Combining with a CRISPR/Cas9 system, Agrobacterium-mediated transformation can lead to precise targeted T-DNA integration in the rice genome.
Agrobacterium-mediated T-DNA integration into the plant genomes is random, which often causes variable transgene expression and insertional mutagenesis. Because T-DNA preferentially integrates into double-strand DNA breaks, we adapted a CRISPR/Cas9 system to demonstrate that targeted T-DNA integration can be achieved in the rice genome. Using a standard Agrobacterium binary vector, we constructed a T-DNA that contains a CRISPR/Cas9 system using SpCas9 and a gRNA targeting the exon of the rice AP2 domain-containing protein gene Os01g04020. The T-DNA also carried a red fluorescent protein and a hygromycin resistance (hptII) gene. One version of the vector had hptII expression driven by an OsAct2 promoter. In an effort to detect targeted T-DNA insertion events, we built another T-DNA with a promoterless hptII gene adjacent to the T-DNA right border such that integration of T-DNA into the targeted exon sequence in-frame with the hptII gene would allow hptII expression. Our results showed that these constructs could produce targeted T-DNA insertions with frequencies ranging between 4 and 5.3% of transgenic callus events, in addition to generating a high frequency (50−80%) of targeted indel mutations. Sequencing analyses showed that four out of five sequenced T-DNA/gDNA junctions carry a single copy of full-length T-DNA at the target site. Our results indicate that Agrobacterium-mediated transformation combined with a CRISPR/Cas9 system can efficiently generate targeted T-DNA insertions.
KeywordsAgrobacterium-mediated transformation T-DNA integration Oryza sativa CRISPR/Cas9
We thank David Wright for providing the binary vector pDW3586. This project was partially supported by the Agriculture and Food Research Initiative Competitive Grant no. 2016–06247 from the USDA National Institute of Food and Agriculture (NIFA) to K.W., National Science Foundation Plant Genome Research Program Grant no IOS 1725122 to S.B.G. and K.W., by the USDA NIFA Hatch project # IOW04341, by State of Iowa funds, and by Crop Bioengineering Center of Iowa State University.
KW, ALE and KL designed the experiments; HZ and MM performed the Agrobacterium-mediated rice transformation; KL, ALE, RB, MEM, and MK analyzed the transgenic plants; KL, ALE, HZ, SBG and KW analyzed the data, and wrote and edited the manuscript. All authors contributed to discussion and revision of the manuscript
Compliance with ethical standards
Conflict of interest
The authors declare no conflict of interests.
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