Chloroplast and mitochondrial DNA editing in plants

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.

TALE array and a uracil glycosylase inhibitor (UGI) function as heterodimers to catalyse cytosine deamination, inducing C-to-T conversions, within a spacer region between the two TALE protein binding sites in target DNA. In this study, we present a rapid and convenient system to assemble DdCBE plasmids for expression in mitochondria and chloroplasts and use the resulting DdCBEs to demonstrate highly efficient organelle base editing in plants ( Supplementary Fig. 1).
To this end, we first developed a Golden Gate assembly system to construct chloroplast-targeting DdCBE (cp-DdCBE) plasmids or mitochondrial-targeting DdCBE (mt-DdCBE) plasmids ( Fig. 1). Our expression plasmids encode fusion proteins composed of a chloroplast transit peptide (CTP) or a mitochondrial targeting sequence (MTS), the TALE N-or C-terminal domains, split-DddA tox halves (G1333N, G1333C, G1397N and G1397C) and UGI, which are codon-optimized for expression in dicot plants, under the control of the parsley ubiquitin (PcUbi) promoter and pea3A terminator. DdCBE plasmids with custom-designed TALE DNA-binding arrays can be constructed in a single subcloning step by mixing an expression vector and six TALE subarray plasmids in an Eppendorf tube. A total of 424 (6 × 64 tripartite + 2 × 16 bipartite + 2 × 4 monopartite) modular TALE subarray plasmids 1 are available for making cp-DdCBEs or mt-DdCBEs that recognize DNA sequences of 16-20 base pairs in length, including a conserved T at the 5′ terminus. As a result, a functional DdCBE heterodimer recognizes 32-to 40-base-pair DNA sequences.
To assess whether our DdCBEs can promote base editing in chloroplasts, we constructed four pairs of cp-DdCBE plasmids specific to the chloroplast 16S rRNA gene encoding the RNA component of the 30S ribosomal subunit, co-transfected each pair into lettuce and rapeseed protoplasts, and measured base editing efficiencies using targeted deep sequencing at day 7 post-transfection (Fig. 2a,b). The best-performing cp-DdCBE pair (Left-G1397-N + Right-G1397-C) induced C•G-to-T•A conversions in the 15-base-pair spacer region between the two TALE array-binding sites at frequencies of 30% in lettuce protoplasts and 15% in rapeseed protoplasts (Fig. 2b). In line with the previous results in mammalian cells 2 and mice 14 , cytosines (C9 and C13) in a 5′-TC motif were converted to thymine preferentially by this cp-DdCBE. Interestingly, a cytosine (C7) in a 5′-AC context was changed to thymine at a frequency of 4.2% in lettuce protoplasts by another cp-DdCBE (Left-G1333-N + Right-G1333-C). We also investigated the persistence of cp-DdCBE-mediated base editing in lettuce protoplasts over 14 days of cultivation ( Supplementary Fig 2). Editing efficiencies continuously increased for up to 10 days and were maintained throughout the period of cultivation. We also tested base editing in two additional chloroplast genes, psbA and psbB, which encode the photosynthetic proteins, D1 and CP-47, respectively, of Photosystem II (Fig. 2c,d and Supplementary  Fig. 3). Among four cp-DdCBEs targeted to the psbA gene, the most active one (Left-G1397-C + Right-G1397-N) was able to induce C•G-to-T•A conversions in lettuce protoplasts with frequencies of up to 25% (Fig. 2d). Only the two cytosines (C11 and C12) in a 5′-TCC context were efficiently converted to thymines by this base editor. It is possible that 5′-TCC was first converted to 5′-TTC and then to 5′-TTT. In rapeseed protoplasts, the other split pair (Left-G1333-N + Right-G1333-C) was most active at four cytosine positions (C3, C4, C11 and C12) with editing efficiencies of up to 3.5% (C3). Note that C3 and C4 are in a 5′-TCC context in the rapeseed gene, whereas they are in a 5′-ACC context in the lettuce counterpart, owing to a single nucleotide polymorphism, which is responsible for efficient editing of the two cytosines (C3 and C4) in the rapeseed gene but not in the lettuce gene by this DdCBE. Likewise, the cp-DdCBE pair targeted to the psbB gene catalysed the conversion of two cytosines in a TCC context at editing frequencies of 0.36% to 4.1% in rapeseed protoplasts ( Supplementary Fig. 3). Taken together, these results suggest that editing efficiencies depend on cytosine positions and contexts within a spacer region as well as DddA tox split positions (G1333 versus G1397) and orientations (Left-G1333-N versus Left-G1333-C) and demonstrate that our cp-DdCBEs enable efficient base editing in the chloroplast genome in plants.
Next, we sought to achieve base editing in plant mitochondrial DNA using our custom-designed mt-DdCBEs. To this end, we constructed mt-DdCBE-encoding plasmids (using our Golden Gate cloning system) targeted to the atp6 gene in lettuce and rapeseed and the rps14 gene in rapeseed, transfected the resulting plasmids into lettuce and rapeseed protoplasts, and measured base editing frequencies using targeted deep sequencing at day 7 post-transfection (Fig. 2e,f and Supplementary Fig. 4). The most active mt-DdCBE pairs (Left-G1397-N + Right-G1397-C in lettuce and Left-G1397-C + Right-G1397-N in rapeseed) were able to catalyse C•G-to-T•A conversions at the atp6 target site with a frequency of 23% in lettuce protoplasts and 23% in rapeseed protoplasts (Fig. 2f). Also, the mt-DdCBE pair induced C•G-to-T•A conversions at the rps14 target site with frequencies of 11% in rapeseed protoplasts ( Supplementary Fig. 4). These results show that mitochondrial DNA in plants is amenable to base editing with mt-DdCBEs.
To investigate whether DdCBE-mediated edits in cpDNA and mtDNA were maintained during regeneration, we collected lettuce and rapeseed calli regenerated from DdCBE-treated protoplasts, four weeks after transfection (Fig. 3a), and measured base editing efficiencies in each callus using targeted deep sequencing and Sanger sequencing ( Fig. 3b and Supplementary Fig. 5). Base edits induced by the DdCBE specific to the chloroplast or mitochondrial genes were detected in 22 out of 26 lettuce calli and 7 out of 14 rapeseed calli with frequencies of up to 38% and 25%, respectively (Fig. 3c). Also, base edits in the chloroplast psbA gene were observed with frequencies of up to 3.9% in lettuce calli ( Supplementary  Fig. 5). Likewise, mitochondrial base edits were detected in rapeseed calli with frequencies of up to 25% and 1.9% in the atp6 and rps14 target sites, respectively ( Supplementary Fig. 5). These results show that DdCBE expression in plant protoplasts can be tolerated and that organelle base edits induced by DdCBEs in protoplasts remain intact during regeneration.
We then sought to demonstrate DNA-free base editing in organelles using in vitro transcribed cp-DdCBE mRNA rather than expression plasmids. We transfected in vitro transcripts encoding the cp-DdCBE targeted to the 16S rRNA gene into lettuce c d e f

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protoplasts and analysed base editing frequencies at the target site (Fig. 3a). C-to-T mutations were detected in protoplasts with frequencies of up to 25% ( Fig. 3d and Supplementary Fig. 6). As expected, DdCBE mRNA or DNA sequences were absent in protoplasts at day 7 post-transfection ( Supplementary Fig. 7). This method can avoid potential integration of plasmid DNA fragments in the host genome. Encouraged by the stable maintenance of organelle edits in calli regenerated from protoplasts, we investigated whether the chloroplast DNA edits in the 16S rRNA gene could confer resistance     and 3b,d). We transferred lettuce calli regenerated from DdCBE-treated protoplasts to medium containing streptomycin or spectinomycin. Mock-treated calli turned white, indicative of protoplast dysfunction, upon exposure to antibiotics ( Supplementary  Fig. 8). In contrast, DdCBE-treated calli remained greenish, showing resistance to these antibiotics. We analysed DdCBE editing efficiencies in the resulting antibiotic-resistant lettuce calli and plantlets. C-to-T conversions at the C9 position, corresponding to the C860T mutation, were observed at high frequencies of up to 98.6% in calli and shoots regenerated from the drug-resistant calli (Fig. 3e,f). Interestingly, bystander C-to-T edits at the nearby C13 position were detected at frequencies of up to 20% in the absence of spectinomycin but not at all in the presence of the antibiotic, demonstrating selection against this mutation upon drug treatment. Taken together, these results show that plant organelle mutations induced by DdCBEs in protoplasts can be maintained after cell division and plant development and that near homoplasy of chloroplast edits can be achieved by drug selection.
We also analysed the off-target activity of the TALE deaminase targeted to the 16S rRNA site in protoplasts, calli and shoots. No off-target mutations were detectably induced in antibiotic-resistant calli or shoots, which were derived from single cells, in the vicinity (±50 base pairs) of the target site ( Supplementary Fig. 9) or at the top five candidate off-target sites in the chloroplast genome, which were chosen on the basis of sequence homology (Supplementary Fig. 10). In contrast, when plasmids encoding the DdCBE pair were used to transfect protoplasts, off-target TC-to-TT mutations were induced in the proximity of the target site and at three of the five candidate off-target sites with low frequencies that ranged from 1.2% to 4.1% ( Supplementary Fig. 10). The use of in vitro transcripts (mRNA) instead of plasmids encoding the TALE deaminase largely avoided these off-target activities in protoplasts (Fig. 4). These results suggest that overexpression or prolonged, plasmid-based expression of DdCBEs can give rise to off-target mutations and that transient, mRNA-based expression using mRNA is desirable for avoiding off-target base editing.
In summary, we have developed a Golden Gate cloning system, which employs a total of 424 TALE subarray plasmids and 16 expression plasmids, to assemble DdCBE-encoding plasmids for organelle base editing in plants. Our DdCBEs custom-designed to target three genes in chloroplast DNA and two genes in mitochondrial DNA achieved C-to-T conversions at high frequencies in lettuce and rapeseed protoplasts. Importantly, the edits in plant organelles were maintained during cell division and plant development. Furthermore, we were able to obtain antibiotic-resistant lettuce calli and plantlets with near homoplasy (99%) by inducing a mutation in the chloroplast 16S rRNA gene. Even without antibiotic selection, edit frequencies were as high as 25% in mitochondria and 38% in chloroplasts. Further studies are warranted to investigate whether DdCBE-induced heteroplasy gives rise to phenotypic effects and whether organelle editing efficiencies can be enhanced by engineering DdCBEs. We expect that our Golden Gate cloning system will be a valuable resource for organelle DNA editing in plants.

Fig. 4 | Comparison of off-target activity in the vicinity of the target site in DdCBe plasmid-transfected or DdCBe mrNA-transfected lettuce protoplasts.
Plasmids or mRNAs encoding the cp-DdCBE pair targeted to the chloroplast 16S rRNA gene were transfected into lettuce protoplasts.
Off-target TC-to-TT edits were detected in the immediate proximity of the target site. Editing efficiencies were measured by targeted deep sequencing seven days post-transfection. Frequencies (mean ± s.d.) were obtained from three independent experiments. Student's unpaired two-tailed t-test was applied. **P < 0.01; *P < 0.05; NS, not significant (P > 0.05).

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amplified from A. thaliana cDNAs. The PcUbi promoter and pea3A terminator were used to replace the mammalian CMV promoter in a backbone plasmid for plant expression 1 . To construct the vector for in vitro DdCBE mRNA transcription, a T7 promoter cassette was cloned into the DdCBE Golden Gate destination vector between the PcUbi promoter and the DdCBE coding region. TALE array genes were cloned by one-way Golden Gate assembly 1 . Plasmids expressing DdCBE were constructed by BsaI digestion and T4 DNA ligation of Golden Gate assembly products using 424 TALE array plasmids and destination vectors. One-way Golden Gate cloning was performed using the following steps: 20 cycles of 37 °C and 50 °C for 5 min each, followed by final incubations at 50 °C for 15 min and 80 °C for 5 min. All vectors for plant protoplast transfection were purified using Plasmid Plus Midiprep kits (Qiagen). The DNA and amino acid sequences used in vector construction are provided in Supplementary Sequences 1-3. mRNA in vitro transcription. DdCBE DNA templates were prepared by PCR using Phusion High-Fidelity DNA Polymerase (Thermo Scientific). DdCBE mRNAs were synthesized and purified using an in vitro mRNA synthesis kit (Enzynomics). The primers for DNA template PCR amplification are listed in Supplementary Table 1.
Protoplast isolation and transfection. Lettuce (Lactuca sativa cv. Cheongchima) seeds were surface sterilized in 70% ethanol for 30 s and in a 0.4% hypochlorite solution for 15 min and were washed three times in distilled water. The lettuce seeds were germinated on 0.5× Murashige and Skoog (MS) medium supplemented with 2% sucrose under conditions of 16 h light and 8 h dark at 25 °C. Rapeseed (Brassica napus cv. Halla) seeds were surface sterilized in 70% ethanol for 3 min and in a 1.0% hypochlorite solution for 30 min, after which they were washed three times with distilled water. The rapeseed seeds were germinated on 1× MS medium supplemented with 3% sucrose under conditions of 16 h light and 8 h dark at 23 °C.
Lettuce protoplasts (5 × 10 5 ) and rapeseed protoplasts ( Rapeseed protoplasts transfected with DdCBE-encoding plasmids were resuspended in rapeseed protoplast culture medium 20 (RPCM) (1× B5 culture medium supplemented with 0.6 g l −1 CaCl 2 , 20 g l −1 glucose, 70 g l −1 mannitol, 1 mg l −1 NAA, 1 mg l −1 BAP and 0.25 mg l −1 2.4-D). The protoplast-RPCM mixture was transferred into a six-well plate and incubated at 25 °C for two weeks in the dark. After two weeks, the protoplasts were incubated at 25 °C under conditions of 16 h dim light and 8 h dark for three weeks. The RPCM was replaced with fresh RPCM every week.
DNA and RNA extraction. Total DNAs or RNAs were extracted from cultured cells in liquid medium and transgenic calli using a DNeasy Plant Mini Kit or RNeasy Plant Mini Kit (Qiagen). Cultured cells and calli were harvested by centrifugation at 10,000 r.p.m. for 1 min. The cDNA from total RNA was reverse-transcribed using RNA to cDNA EcoDry Premix (Oligo dT) (Takara).
Targeted deep sequencing. Target regions were amplified using Phusion High-Fidelity DNA Polymerase with the appropriate primers (Supplementary  Table 1). Three rounds of PCR were performed (first, nested PCR; second, PCR; and third, indexing PCR) to make a DNA sequencing library. Equal amounts of the DNA libraries were pooled and sequenced using MiniSeq (Illumina). The paired-end sequencing files were analysed by the Cas-analyzer (http://www. rgenome.net) 21 and source code of the computer program at https://github.com/ ibs-cge/maund.
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The data that support the findings of this study are available from the corresponding author upon request. The high-throughput sequencing data from this study have been deposited in the NCBI BioProject database under the accession codes PRJNA727868 and PRJNA727869.

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The source code used to calculate the substitution frequencies can be accessed at https://github.com/ibs-cge/maund.

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April 2020 Corresponding author(s): Jin-Soo Kim Last updated by author(s): May 8, 2021 Reporting Summary Nature Research wishes to improve the reproducibility of the work that we publish. This form provides structure for consistency and transparency in reporting. For further information on Nature Research policies, see our Editorial Policies and the Editorial Policy Checklist.

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