Efficient scar-free knock-ins of several kilobases in plants by engineered CRISPR-Cas endonucleases

Schreiber, Tom; Prange, Anja; Schäfer, Petra; Iwen, Thomas; Grützner, Ramona; Marillonnet, Sylvestre; Lepage, Aurélie; Javelle, Marie; Paul, Wyatt; Tissier, Alain

doi:10.1016/j.molp.2024.03.013

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Molecular Plant

2024

DOI: 10.1016/j.molp.2024.03.013

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Efficient scar-free knock-ins of several kilobases in plants by engineered CRISPR-Cas endonucleases

Tom Schreiber,

Anja Prange,

Petra Schäfer

et al.

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2024

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Cited by 2 publications

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Amphicarpic development in Cardamine chenopodiifolia

Emonet,

Pérez‐Antón,

Neumann

et al. 2024

New Phytologist

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Summary Amphicarpy is an unusual trait where two fruit types develop on the same plant: one above and the other belowground. This trait is not found in conventional model species. Therefore, its development and molecular genetics remain under‐studied. Here, we establish the allooctoploid Cardamine chenopodiifolia as an emerging experimental system to study amphicarpy. We characterized C. chenopodiifolia development, focusing on differences in morphology and cell wall histochemistry between above‐ and belowground fruit. We generated a reference transcriptome with PacBio full‐length transcript sequencing and analysed differential gene expression between above‐ and belowground fruit valves. Cardamine chenopodiifolia has two contrasting modes of seed dispersal. The main shoot fails to bolt and initiates floral primordia that grow underground where they self‐pollinate and set seed. By contrast, axillary shoots bolt and develop exploding seed pods aboveground. Morphological differences between aerial explosive fruit and subterranean nonexplosive fruit were reflected in a large number of differentially regulated genes involved in photosynthesis, secondary cell wall formation and defence responses. Tools established in C. chenopodiifolia, such as a reference transcriptome, draft genome assembly and stable plant transformation, pave the way to study amphicarpy and trait evolution via allopolyploidy.

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Amphicarpic development in Cardamine chenopodiifolia

Emonet,

Pérez‐Antón,

Neumann

et al. 2024

New Phytologist

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A quantitative assay for the efficiency of RNA‐guided genome editing in plants

Bircheneder,

Schreiber,

Tissier

et al. 2024

The Plant Journal

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SUMMARYRNA‐guided endonucleases originating from the bacterial CRISPR/Cas system are a versatile tool for targeted gene editing. To determine the functional relevance of a gene of interest, deletion of the entire open reading frame (ORF) by two independent double‐strand breaks (DSBs) is particularly attractive. This strategy greatly benefits from high editing efficiency, which is strongly influenced by the Cas endonuclease version used. We developed two reporter switch‐on assays, for quantitative comparison and optimization of Cas constructs. The assays are based on four components: (i) A reporter gene, the mRNA of which carries a hairpin (HP) loop targeted by (ii) the endoribonuclease Csy4. Cleavage of the mRNA at the HP loop by Csy4 abolishes the translation of the reporter. Csy4 was used as the target for full deletion. (iii) A Cas system targeting sites flanking the Csy4 ORF with a 20‐bp spacer either side to preferentially detect full‐deletion events. Loss of functional Csy4 would lead to reporter gene expression, allowing indirect quantification of Cas‐mediated deletion events. (iv) A reference gene for normalization. We tested these assays on Nicotiana benthamiana leaves and Lotus japonicus calli induced on hypocotyl sections, using Firefly luciferase and mCitrine as reporter genes and Renilla luciferase and hygromycin phosphotransferase II as reference genes, respectively. We observed a >90% correlation between reporter expression and full Csy4 deletion events, demonstrating the validity of these assays. The principle of using the Csy4–HP module as Cas target should be applicable to other editing goals including single DSBs in all organisms.

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Efficient scar-free knock-ins of several kilobases in plants by engineered CRISPR-Cas endonucleases

Cited by 2 publications

References 63 publications

Amphicarpic development in Cardamine chenopodiifolia

Amphicarpic development in Cardamine chenopodiifolia

A quantitative assay for the efficiency of RNA‐guided genome editing in plants

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