Chloroplast and mitochondrial DNA editing in plants

Abstract

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.

TALEN‐mediated depletion of the mitochondrial gene orf312 proves that it is a Tadukan‐type cytoplasmic male sterility‐causative gene in rice

“…The stable inheritance of mitoTALEN‐mediated mitochondrial genome reorganization in null segregants paves the way to mitochondrial genome breeding. A recently developed tool for mitochondrial base editing, mitoTALECD, in which the mitoTALEN nuclease is replaced with a cytidine deaminase (Mok et al., 2020; Kang et al., 2021), would also be effective for use in mitochondrial genome breeding.…”

Challenges Facing CRISPR/Cas9-Based Genome Editing in Plants

“…To allow for their use in the modification of the mitochondrial and chloroplast genomes, sequence-specific nucleases were redesigned by adding a targeting peptide such as a mitochondrial targeting sequence (MTS) or chloroplast transit peptide (CTP) specific to each organelle. Most recently, Kang et al (2021) reported TALEN plasmids based on bacterial cytidine deaminase (CD) rather than FokI or TevI for mtDNA/cpDNA editing. They generated double-stranded DNA deaminase (DddA) toxin A-derived cytosine base editor (DdCBE) plasmids targeting mtDNA (mt-DdCBE) and cpDNA (cp-DdCBE) via fusion of MTS and CTP, resulting in the efficient introduction of point mutations in the relevant organellar genome, up to 25% in mitochondria and 38% in chloroplast, by mt-DdCBEs and cp-DdCBEs in lettuce ( Lactuca sativa ) and rapeseed, respectively.…”

Genome editing techniques in plants: a comprehensive review and future prospects toward zero hunger

“… 40 , 41 Also, genome editing could be used to induce point mutation in the 16S rRNA gene in the chloroplast genome to enhance antibiotic resistance. 42 , 43 …”