Abstract
Bacterial cytidine deaminase fused to the DNA binding domains of transcription activator-like effector nucleases was recently reported to transiently substitute a targeted C to a T in mitochondrial DNA of mammalian cultured cells1. We applied this system to targeted base editing in the Arabidopsis thaliana plastid genome. The targeted Cs were homoplasmically substituted to Ts in some plantlets of the T1 generation and the mutations were inherited by their offspring independently of their nuclear-introduced vectors.
Highlights
Bacterial cytidine deaminase fused to the DNA binding domains of transcription activator-like effector nucleases was recently reported to transiently substitute a targeted C to a T in mitochondrial DNA of mammalian cultured cells[1]
We selected three genes whose modifications would be expected to lead to observable effects; 16S rRNA, whose modification was expected to confer resistance to an antibiotic and two genes whose modifications would lead to poor growth; rpoC1, which encodes a part of the DNA-directed RNA polymerase subunit beta′; and psbA, which encodes photosystem II (PSII) protein D1
The N terminus of the pTALECD was linked to a plastid-targeting signal peptide (PTP) of Arabidopsis thaliana RecA1 protein (51 aa)[7,8] (Fig. 1b), while the C terminus was linked to an uracil glycosylase inhibitor (UGI)[1,9] to inhibit hydrolysis of the generated uracil (Fig. 1b)
Summary
Plants with base change(s) in the target window on either day, some had bases heteroplasmically or chimaerically (h/c) substituted on both days with their mutation rate increased or decreased (27.5%, 14/51; Fig. 1i) and others had bases at which the mutation rate differed between the two time points (for example, homoplasmy (homo) to h/c, 5.9% (3/51); h/c to null, 15.7% (8/51); h/c to homo, 11.8% (6/51); or null to h/c, 2.0% (1/51); Fig. 1i). No dominant off-target mutations were detected in the mitochondrial genomes of these 17 lines, including 16S rRNA 1397CN 1 (Supplementary Table 2) These results indicate that ptpTALECD only infrequently introduced off-target point mutations into organelle genomes and can and homoplasmically (or dominantly) substitute C/G to T/A in the target windows. 23) on the transfer DNA (T-DNA; Fig. 1b) and/or a positive polymerase chain reaction (PCR) result showing the presence of the ptpTALECD reading frame (Fig. 1b and 3a) Both progeny stably inherited the homoplasmic mutations (Fig. 3a and Extended Data Fig. 5a). No major off-target mutations were detected in the three null-segregant T2 plants (16S rRNA 1397CN 8 lines 1, 2 and 8), whose genomes were sequenced by generation sequencing (Fig. 2a and Supplementary Table 2). These results potential start codons would shorten the protein by at least 10% but suggest that the AUA codon does not greatly affect the initiation a c
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
