Engineering Plastid Genomes: Methods, Tools, and Applications in Basic Research and Biotechnology

Bock, Ralph

doi:10.1146/annurev-arplant-050213-040212

Cited by 291 publications

(222 citation statements)

References 196 publications

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“…The results in this study reveal that SOT1 can achieve programmable RNA recognition and cleavage, suggesting that SOT1, similar to the CRISPR/Cas system, can be used as a tool for RNA manipulation. Currently, chloroplast and mitochondrial transformation is technically very difficult, labor intensive, and time consuming and achieved only in very few organisms (58,59). It is unclear whether CRISPR/Cas9 could cleave or edit the mitochondrial genome because it is challenging to import the guide RNA component into mitochondria (60).…”

Section: Discussionmentioning

confidence: 99%

PPR-SMR protein SOT1 has RNA endonuclease activity

Zhou

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Numerous attempts have been made to identify and engineer sequence-specific RNA endonucleases, as these would allow for efficient RNA manipulation. However, no natural RNA endonuclease that recognizes RNA in a sequence-specific manner has been described to date. Here, we report that SUPPRESSOR OF THYLAKOID FORMATION 1 (SOT1), an Arabidopsis pentatricopeptide repeat (PPR) protein with a small MutS-related (SMR) domain, has RNA endonuclease activity. We show that the SMR moiety of SOT1 performs the endonucleolytic maturation of 23S and 4.5S rRNA through the PPR domain, specifically recognizing a 13-nucleotide RNA sequence in the 5′ end of the chloroplast 23S-4.5S rRNA precursor. In addition, we successfully engineered the SOT1 protein with altered PPR motifs to recognize and cleave a predicted RNA substrate. Our findings point to SOT1 as an exciting tool for RNA manipulation.photosynthesis | PPR-SMR protein | RNA endonuclease | rRNA biogenesis S equence-specific RNA endonucleases are crucial to establishing RNA manipulation technology (1). Compared with DNA editing, RNA manipulation could be more useful and reversible because it does not result in permanent changes to the genome. In addition, sequence-specific RNA endonucleases could potentially be used as an RNA silencing tool to complement RNAi, because RNAi is sometimes ineffective in certain organisms and RNAi machinery is not present in cellular compartments such as chloroplasts and mitochondria. Despite extensive investigations, a natural RNA endonuclease that recognizes RNA in an intrinsic sequence-specific manner has not yet been identified.Pentatricopeptide repeat (PPR) proteins exist in eukaryotes, have greatly expanded in terrestrial plants, and take part in most RNA metabolic processes in organelles (2-5). The PPR domain can specifically recognize RNAs in an intrinsic sequence-specific manner (5-8). The 2nd, 5th, and 35th (or 1st, 4th, and 34th or 3rd, 6th, and 1st in other numbering systems) residues at each repeat are considered to be RNA selection "codes" (9-11). Based on these codes, several PPR proteins have been successfully modified to recognize predictable RNA targets (9,(12)(13)(14)(15)(16)(17).The small MutS-related (SMR) domain was originally identified at the C terminus of MutS2 in the cyanobacterium Synechocystis (18). SMR proteins are widely distributed in almost all organisms (19). Recent studies demonstrated the SMR domain exhibits DNA nicking nuclease activity in vitro (20-25). Furthermore, a C-terminal SMR domain in Leishmania donovani S-phase mRNA cycling sequence binding protein (CSBP) has RNA cleavage activity in vitro (26). These findings suggest that the SMR domain has nuclease activity.Interestingly, a small protein family containing both PPR and SMR domains was recently described (27). PPR-SMR proteins are found mainly in land plants. Arabidopsis thaliana contains eight PPR-SMR proteins localized to the organelles, including mitochondria and chloroplasts (27). Currently, four PPR-SMR proteins have been characterized. They play ...

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Section: Discussionmentioning

confidence: 99%

PPR-SMR protein SOT1 has RNA endonuclease activity

Zhou

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…However, the progress to date in the development of the algal chloroplast as a platform has almost exclusively focused on C. reinhardtii, with over 100 reports in the literature of the production of recombinant proteins in this species. Chloroplast transformation is also feasible for a number of plant species, with advanced genetic engineering technologies available for tobacco (Nicotiana tabacum) and several other plants such as tomato, potato and petunia [21]. Although plant chloroplasts represent an attractive low-cost and easily scalable platform for the synthesis of biopharmaceuticals [22], there are fundamental challenges associated with the use of crop plants for drug production.…”

Section: The Algal Chloroplast As a New Bio-factorymentioning

confidence: 99%

The algal chloroplast as a synthetic biology platform for production of therapeutic proteins

2018

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The chloroplast of Chlamydomonas reinhardtii and other microalgae represents an attractive new platform for the synthesis of recombinant therapeutics using synthetic biology (synbio) approaches. Transgenes can be designed in silico, assembled from validated DNA parts and inserted at precise and predetermined locations within the chloroplast genome to give stable synthesis of a desired recombinant protein. Numerous recent examples of different therapeutic proteins produced successfully in the C. reinhardtii chloroplast highlight the potential of this green alga as a simple, low-cost and benign host. Furthermore, some of the features of the alga may offer additional advantages over more-established microbial, mammalian or plant-based systems. These include efficient folding and accumulation of the product in the chloroplast; a lack of contaminating toxins or infectious agents; reduced downstream processing requirements; the possibility to make complex therapeutics such as immunotoxins; and the opportunity to use the whole alga as a low-cost oral vaccine. In this paper we review the current status of algal chloroplast engineering with respect to therapeutic proteins. We also consider future advances in synbio tools, together with improvements to recipient strains, which will allow the design of bespoke strains with high levels of productivity.

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“…Marchantia is one of few plant species for which chloroplast transformation protocols have been confirmed by at least two independent studies (Bock 2015). Stable transplastomic lines of Marchantia encoding dual spectinomycin and streptomycin resistance were generated through particle bombardment of sporelings…”

Section: Marchantia As a Basal Model Chassis For Plant Synthetic Biologymentioning

confidence: 99%

Synthetic Botany

Boehm

Pollak

Purswani³

et al. 2017

Cold Spring Harb Perspect Biol

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Plants are attractive platforms for synthetic biology and metabolic engineering. Plants' modular and plastic body plans, capacity for photosynthesis, extensive secondary metabolism, and agronomic systems for large-scale production make them ideal targets for genetic reprogramming. However, efforts in this area have been constrained by slow growth, long life cycles, the requirement for specialized facilities, a paucity of efficient tools for genetic manipulation, and the complexity of multicellularity. There is a need for better experimental and theoretical frameworks to understand the way genetic networks, cellular populations, and tissue-wide physical processes interact at different scales. We highlight new approaches to the DNA-based manipulation of plants and the use of advanced quantitative imaging techniques in simple plant models such as Marchantia polymorpha. These offer the prospects of improved understanding of plant dynamics and new approaches to rational engineering of plant traits.

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Engineering Plastid Genomes: Methods, Tools, and Applications in Basic Research and Biotechnology

Cited by 291 publications

References 196 publications

PPR-SMR protein SOT1 has RNA endonuclease activity

PPR-SMR protein SOT1 has RNA endonuclease activity

The algal chloroplast as a synthetic biology platform for production of therapeutic proteins

Synthetic Botany

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