Plant organelles including mitochondria and chloroplasts contain their own genomes, which encode many genes essential for respiration and photosynthesis, respectively. Gene editing in plant organelles, an unmet need for plant genetics and biotechnology, has been hampered by the lack of appropriate tools for targeting DNA in these organelles. In this study, we developed a Golden Gate cloning system1, composed of 16 expression plasmids (8 for the delivery of the resulting protein to mitochondria and the other 8 for delivery to chloroplasts) and 424 transcription activator-like effector subarray plasmids, to assemble DddA-derived cytosine base editor (DdCBE)2 plasmids and used the resulting DdCBEs to efficiently promote point mutagenesis in mitochondria and chloroplasts. Our DdCBEs induced base editing in lettuce or rapeseed calli at frequencies of up to 25% (mitochondria) and 38% (chloroplasts). We also showed DNA-free base editing in chloroplasts by delivering DdCBE mRNA to lettuce protoplasts to avoid off-target mutations caused by DdCBE-encoding plasmids. Furthermore, we generated lettuce calli and plantlets with edit frequencies of up to 99%, which were resistant to streptomycin or spectinomycin, by introducing a point mutation in the chloroplast 16S rRNA gene.

CRISPR-Cas9 system is now widely used to edit a target genome in animals and plants. Cas9 protein derived from Streptococcus pyogenes (SpCas9) cleaves double-stranded DNA targeted by a chimeric single-guide RNA (sgRNA). For plant genome editing, Agrobacterium-mediated T-DNA transformation has been broadly used to express Cas9 proteins and sgRNAs under the control of CaMV 35S and U6/U3 promoter, respectively. We here developed a simple and high-throughput binary vector system to clone a 19-20 bp of sgRNA, which binds to the reverse complement of a target locus, in a large T-DNA binary vector containing an SpCas9 expressing cassette. Two-step cloning procedures: (1) annealing two target-specific oligonucleotides with overhangs specific to the AarI restriction enzyme site of the binary vector; and (2) ligating the annealed oligonucleotides into the two AarI 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 Gateway(TM) system and unique EcoRI/XhoI 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.

SummaryThe genes that encode the ethylene biosynthesis enzyme 1‐aminocyclopropane‐1‐carboxylate oxidase (ACO) are thought to be involved in flower senescence. Hence, we investigated whether the transcript levels of PhACO genes (PhACO1, PhACO3 and PhACO4) in Petunia cv. Mirage Rose are associated with ethylene production at different flowering stages. High transcript levels were detected in the late flowering stage and linked to high ethylene levels. PhACO1 was subsequently edited using the CRISPR/Cas9 system, and its role in ethylene production was investigated. PhACO1‐edited T0 mutant lines, regardless of mutant type (homozygous or monoallelic), exhibited significantly reduced ethylene production and enhanced flower longevity compared with wild‐type. Flower longevity and the reduction in ethylene production were observed to be stronger in homozygous plants than in their monoallelic counterparts. Additionally, the transmission of the edited gene to the T1 (lines 6 and 36) generation was also confirmed, with the results for flower longevity and ethylene production proving to be identical to those of the T0 mutant lines. Overall, this study increases the understanding of the role of PhACO1 in petunia flower longevity and also points to the CRISPR/Cas9 system being a powerful tool in the improvement of floricultural quality.

Chloroplast DNA (cpDNA) encodes up to 315 (typically, 120–130) genes1, including those for essential components in photosystems I and II and the large subunit of RuBisCo, which catalyses CO2 fixation in plants. Targeted mutagenesis in cpDNA will be broadly useful for studying the functions of these genes in molecular detail and for developing crops and other plants with desired traits. Unfortunately, CRISPR–Cas9 and CRISPR-derived base editors, which enable targeted genetic modifications in nuclear DNA, are not suitable for organellar DNA editing2, owing to the difficulty of delivering guide RNA into organelles. CRISPR-free, protein-only base editors (including DddA-derived cytosine base editors3–8 and zinc finger deaminases9), originally developed for mitochondrial DNA editing in mammalian cells, can be used for C-to-T, rather than A-to-G, editing in cpDNA10–12. Here we show that heritable homoplasmic A-to-G edits can be induced in cpDNA, leading to phenotypic changes, using transcription activator-like effector-linked deaminases13.

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