CRISPR/Cas9-mediated homologous recombination in tobacco
Co-transformation of multiple T-DNA in a binary vector enabled CRISPR/Cas9-mediated HR in tobacco. HR occurred in a limited region around the gRNA target site.
In this study, CRISPR/Cas9-mediated homologous recombination (HR) in tobacco (Nicotiana tabacum L. ‘SR-1’) was achieved using binary vectors comprising two (T1–T2) or three (T1–T2–T3) independent T-DNA regions. For HR donor with the tobacco acetolactate synthase gene, SuRB, T-DNA1 contained ΔSuRBW568L, which lacked the N-terminus region of SuRB and was created by three nucleotide substitutions (ATG to GCT; W568L), leading to herbicide chlorsulfuron (Cs) resistance, flanked by the hygromycin (Hm)-resistant gene. T-DNA2 consisted of the hSpCas9 gene and two gRNA inserts targeting SuRB and An2. For the 2nd HR donor with the tobacco An2 gene encoding a MYB transcription factor involved in anthocyanin biosynthesis, T-DNA3 had a 35S promoter-driven An2 gene lacking the 3rd exon resulting in anthocyanin accumulation after successful HR. After selecting for Hm and Cs resistance from among the 7462 Agrobacterium-inoculated explants, 77 independent lines were obtained. Among them, the ATG to GCT substitution of endogenous SuRB was detected in eight T1–T2-derived lines and two T1–T2–T3-derived lines. Of these mutations, four T1–T2-derived lines were bi-allelic. All the HR events occurred across the endogenous SuRB and 5′ homology arm of the randomly integrated T-DNA1. HR of the SuRB paralog, SuRA, was also found in one of the T1–T2-derived lines. Sequence analysis of its SuRA-targeted region indicated that the HR occurred in a limited (< 153 bp) region around the gRNA target site. Even though some T1–T2–T3-derived lines introduced three different T-DNAs and modified the An2 gRNA target site, no signs of HR in the endogenous An2 could be observed.
KeywordsHomologous recombination CRISPR/Cas9 T-DNA Tobacco
Clustered regularly interspaced short palindromic repeats
Double-strand breaks. HMA, heteroduplex mobility assay
The authors would like to thank Dr. Roger Hellens (Queensland University of Technology), Dr. Philip Mullineaux (University of Essex) and Dr. Mark Smedley (John Innes Centre) for the pGreen vector system, Dr. Feng Zhang (Massachusetts Institute of Technology) for the pX330-U6-Chimeric_BB-CBh-hSpCas9 plasmid, Dr. Nam-Hai Chua (Rockefeller University) for the pER8 vector, and Dr. Hiroaki Ichikawa (NIAS, Japan) for the pSMAB704 vector. We also thank Editage (http://www.editage.jp) for English language editing.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
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