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CRISPR/Cas9-mediated targeted T-DNA integration in rice.

Abstract：

KEY MESSAGE: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.

journal_name

Plant Mol Biol

journal_title

Plant molecular biology

authors

Lee K,Eggenberger AL,Banakar R,McCaw ME,Zhu H,Main M,Kang M,Gelvin SB,Wang K

doi

10.1007/s11103-018-00819-1

subject

Has Abstract

pub_date

2019-03-01 00:00:00

pages

317-328

issue

4-5

eissn

0167-4412

issn

1573-5028

pii

10.1007/s11103-018-00819-1

journal_volume

99

pub_type

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