High-efficiency plastome base editing in rice with TAL cytosine deaminase

Abstract

Genome editing has been widely applied in nuclei but not so in organelles such as the mitochondria and chloroplasts. The challenge for organelle genome editing is the limited accessibility of editing reagents to the compartments of organelles (Zhu et al., 2020Zhu H. Li C. Gao C. Applications of CRISPR-Cas in agriculture and plant biotechnology.Nat. Rev. Mol. Cell Biol. 2020; 21: 661-677Crossref PubMed Scopus (182) Google Scholar). Recently, fusion proteins of cytidine deaminase domain (DddAtox) and transcription activator-like effectors (TALEs) have been developed for DNA base editing in mitochondria of human cells and mice (Mok et al., 2020Mok B.Y. de Moraes M.H. Zeng J. Bosch D.E. Kotrys A.V. Raguram A. Hsu F. Radey M.C. Peterson S.B. Mootha V.K. et al.A bacterial cytidine deaminase toxin enables CRISPR-free mitochondrial base editing.Nature. 2020; 583: 631-637Crossref PubMed Scopus (195) Google Scholar; Lee et al., 2021Lee H. Lee S. Baek G. Kim A. Kang B.C. Seo H. Kim J.S. Mitochondrial DNA editing in mice with DddA-TALE fusion deaminases.Nat. Commun. 2021; 12: 1190Crossref PubMed Scopus (31) Google Scholar). The DddAtox-derived base editors (DdCBEs) function in pairs, each consisting of a mitochondrial targeting sequence, TALE, a split half of DddAtox, and an uracil glycosylase inhibitor (UGI). The split halves of DddAtox heterodimerize to catalyze the deamination of cytosines of double-stranded DNA, resulting in efficient C⋅G to T⋅A conversions (Mok et al., 2020Mok B.Y. de Moraes M.H. Zeng J. Bosch D.E. Kotrys A.V. Raguram A. Hsu F. Radey M.C. Peterson S.B. Mootha V.K. et al.A bacterial cytidine deaminase toxin enables CRISPR-free mitochondrial base editing.Nature. 2020; 583: 631-637Crossref PubMed Scopus (195) Google Scholar). Chloroplasts (plastids) have their own genomes (plastome), albeit small (110–200 kb in size), which encode about 130 proteins and RNA molecules essential for photosynthesis in plants (Daniell et al., 2016Daniell H. Lin C.S. Yu M. Chang W.J. Chloroplast genomes: diversity, evolution, and applications in genetic engineering.Genome Biol. 2016; 17: 134Crossref PubMed Scopus (583) Google Scholar). Number of chloroplasts per plant cell and copy number of genomes per chloroplast vary greatly during leaf development. Nevertheless, plastomes are allopolyploid genomes (Zoschke et al., 2007Zoschke R. Liere K. Borner T. From seedling to mature plant: Arabidopsis plastidial genome copy number, RNA accumulation and transcription are differentially regulated during leaf development.Plant J. 2007; 50: 710-722Crossref PubMed Scopus (143) Google Scholar). Plastome editing lags behind the nuclear genome editing (Daniell et al., 2016Daniell H. Lin C.S. Yu M. Chang W.J. Chloroplast genomes: diversity, evolution, and applications in genetic engineering.Genome Biol. 2016; 17: 134Crossref PubMed Scopus (583) Google Scholar). In the present study, we report the adaptation of DdCBEs tailored for chloroplast genomes. The system includes a series of DNA vectors and a library of TALE modular repeats. To validate the efficacy of this system, we chose a conserved chloroplast gene, psaA, that encodes the chlorophyll a of photosystem I, for C⋅G to T⋅A changes in rice. We first designed an architecture of the TALE DNA binding domain from a Xanthomonas oryzae TAL effector. The repeatless TALE scaffold contains an N terminus of 135 amino acids (aa), a 60-aa C terminus, and a cloning site between them for an insertion of designer TALE repeats of 34 aa, corresponding to the user-chosen target sequences (Supplemental Figure 1). A chloroplast transition peptide (cTP; Supplemental Figure 2) was fused N terminally to the TALE scaffold. The split halves (G1397-N and G1397-C) of DddAtox (Mok et al., 2020Mok B.Y. de Moraes M.H. Zeng J. Bosch D.E. Kotrys A.V. Raguram A. Hsu F. Radey M.C. Peterson S.B. Mootha V.K. et al.A bacterial cytidine deaminase toxin enables CRISPR-free mitochondrial base editing.Nature. 2020; 583: 631-637Crossref PubMed Scopus (195) Google Scholar) each together with the UGI were rice codon optimized and C terminally fused to the TALE scaffold (Supplemental Figure 2). The resulting paired scaffolds are referred to as cpDdCBE-L and cpDdCBE-R. We next chose psaA for gene inactivation, one potential application in plastome editing. Inactivation of the psaA gene led to the white/yellow leaves due to reduced chlorophyll production but not lethality in sunflower (Azarin et al., 2020Azarin K. Usatov A. Makarenko M. Kozel N. Kovalevich A. Dremuk I. Yemelyanova A. Logacheva M. Fedorenko A. Averina N. A point mutation in the photosystem I P700 chlorophyll a apoprotein A1 gene confers variegation in Helianthus annuus L.Plant Mol. Biol. 2020; 103: 373-389Crossref PubMed Scopus (7) Google Scholar) and tobacco (Leelavathi et al., 2011Leelavathi S. Bhardwaj A. Kumar S. Dass A. Pathak R. Pandey S.S. Tripathy B.C. Padmalatha K.V. Dhandapani G. Kanakachari M. et al.Genome-wide transcriptome and proteome analyses of tobacco psaA and psbA deletion mutants.Plant Mol. Biol. 2011; 76: 407-423Crossref PubMed Scopus (20) Google Scholar). We scanned the coding sequence of rice psaA (Supplemental Figure 3) for target regions that could accommodate two adjacent 23-nt target sites separated by a spacer of 16 base pairs wherein certain codons (CAA, CAG, CGA, and ACC of TGG), when C to T transition occurs, become stop codons. A T precedes each of the TALE binding sequences at the 5′ end (Figure 1A). Two arrays of 23-nt TALE repeats were assembled using a kit that contained 51 single repeat units (Li et al., 2011Li T. Huang S. Zhao X. Wright D.A. Carpenter S. Spalding M.H. Weeks D.P. Yang B. Modularly assembled designer TAL effector nucleases for targeted gene knockout and gene replacement in eukaryotes.Nucleic Acids Res. 2011; 39: 6315-6325Crossref PubMed Scopus (314) Google Scholar) and cloned into the scaffolds of cpDdCBE-L and cpDdCBE-R, resulting in two TALE deaminase genes, cpDdCBE_psaA-L and cpDdCBE_psaA-R (Supplemental Figure 4). cpDdCBE_psaA-L and cpDdCBE_psaA-R, under the ubiquitin promoters of maize and rice, respectively, to maximize DNA heterogeneity of two cassettes, were combined, resulting in cpDdCBE_psaA-L+R for rice transformation (Figure 1A and Supplemental methods). cpDdCBE_psaA-L+R was introduced into the rice cultivar Kitaake through Agrobacterium-mediated gene transfer. Fifteen of 49 lines were genotyped for psaA by Sanger sequencing the PCR amplicons. Fourteen callus lines contained C to T transitions at positions 1874 and 1875. Among them, one line contained one uniform C to T conversion; six lines contained the uniform C to T changes at 1875 and heteroplasmic edits at 1874; seven lines contained heteroplasmic edits for the two Cs. The two Cs were in the context of 5′-TCC-3′ with the C next to T preferable to the second C, with no sign of editing for the other five Cs in the spacer region. The callus lines were regenerated into whole plants that were genotyped for presence of cpDdCBEs. Thirty-three independent lines were positive for cpDdCBEs. Among them, 17 lines had multiple plants that showed pale yellow in their leaves and stems, four lines had some plantlets yellowish and some plants light green, while 12 lines had most plantlets green (Figure 1B and Supplemental Figure 5). All 33 lines were genotyped for editing in the target region of psaA. Directly Sanger sequencing the PCR amplicons showed consistency of genotypes and phenotypes of those plants. The sequencing chromatograms demonstrated the editing efficiency reached up to 64% among the 33 stable T0 transgenic lines. Twelve and six lines had two Cs and one C changes, respectively; three lines had the C next to the T uniformly changed while the second C was partially edited; one line had both Cs partially edited (Supplemental Figure 6). In addition, among those lines with two Cs changed, three lines each carried an additional C partially edited (Supplemental Figure 6). Among those 21 edited plants, 17 lines appeared homoplasmic for psaA edits that constituted a premature stop codon (TAA or TGA) from the tryptophan codon (TGG) and showed the albino phenotype (Supplemental Figure 6). Similarly, another pair of cpDdCBEs were made to target psaA in rice. Some transgenic shoots carried the C⋅G to T⋅A edits and were albino (Supplemental Figure 7). We further sequenced the psaA PCR amplicons from 13 lines using Illumina technology. All 13 lines carried the C⋅G to T⋅A changes in the TGG codon (positions 1874 and 1875 for the two Gs) ranging between 94% and 98% (Figure 1C and Supplemental Table 2), and four lines carried edits at either 1886 or 1888 or both. Measurement of chlorophyll a and b contents in three psaA edited lines revealed remarkable reduction of both forms of chlorophyll in the edited lines (Figure 1D). Gene expression was also investigated for three photosynthetic genes (psaA, psaB, and psbA) for six lines (three albino and three green lines). The three genes showed significantly lower expression in albino lines (Supplemental Figure 8). Finally, we examined the morphology of chloroplasts in edited lines. The chloroplasts in edited lines were impaired as they lacked stacked grana and were smaller compared with wild-type, indicating the abnormality of mesophyll chloroplasts in edited lines (Figure 1E). In conclusion, we report an efficient cytosine base editing system in rice chloroplasts. Multiple rice lines contained the near-homoplasmic psaA edits that were corroborated by their albinism. The DddAtox in this system is used as split halves to prevent its toxicity and has preference for a 5′-TC-3′ context (Mok et al., 2020Mok B.Y. de Moraes M.H. Zeng J. Bosch D.E. Kotrys A.V. Raguram A. Hsu F. Radey M.C. Peterson S.B. Mootha V.K. et al.A bacterial cytidine deaminase toxin enables CRISPR-free mitochondrial base editing.Nature. 2020; 583: 631-637Crossref PubMed Scopus (195) Google Scholar). Future research will include engineering DddAtox variants or identifying novel deaminases to develop single TAL-deaminase, relax the restricted preference, and expand the scope of base editing in chloroplast genomes. Plastome editing will be an exciting technology for basic and applied research in agriculture. This work was partially supported by the Startup funds from the University of Missouri and Danforth Plant Science Center , United States.

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