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The Obstacles and Potential Solution Clues of Prime Editing Applications in Tomato

Precision genome editing is highly desired for crop improvement. The recently emerged CRISPR/Cas technology offers great potential applications in precision plant genome engineering. A prime editing (PE) approach combining a reverse transcriptase (RT) with a Cas9 nickase and a “priming” extended gui...

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Autores principales: Vu, Tien Van, Nguyen, Ngan Thi, Kim, Jihae, Das, Swati, Lee, Jinsu, Kim, Jae-Yean
Formato: Online Artículo Texto
Lenguaje:English
Publicado: AAAS 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10593121/
https://www.ncbi.nlm.nih.gov/pubmed/37905201
http://dx.doi.org/10.34133/bdr.0001
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author Vu, Tien Van
Nguyen, Ngan Thi
Kim, Jihae
Das, Swati
Lee, Jinsu
Kim, Jae-Yean
author_facet Vu, Tien Van
Nguyen, Ngan Thi
Kim, Jihae
Das, Swati
Lee, Jinsu
Kim, Jae-Yean
author_sort Vu, Tien Van
collection PubMed
description Precision genome editing is highly desired for crop improvement. The recently emerged CRISPR/Cas technology offers great potential applications in precision plant genome engineering. A prime editing (PE) approach combining a reverse transcriptase (RT) with a Cas9 nickase and a “priming” extended guide RNA (gRNA) has shown a high frequency for precise genome modification in mammalian cells and several plant species. Nevertheless, the applications of the PE approach in dicot plants are still limited and inefficient. We designed and tested prime editors for precision editing of a synthetic sequence in a transient assay and for desirable alleles of 10 loci in tomato by stable transformation. Our data obtained by targeted deep sequencing also revealed only low PE efficiencies in both the tobacco and tomato systems. Further assessment of the activities of the PE components uncovered that the fusion of RT to Cas9 and the structure of PE gRNAs (pegRNAs) negatively affected the cleaving activity of the Cas9 nuclease. The self-complementarity between the primer binding sequences (PBSs) and spacer sequence might pose risks to the activity of the Cas9 complex. However, modifying the pegRNA sequences by shortening or introducing mismatches to the PBSs to reduce their melting temperatures did not enhance the PE efficiency at the MADS-box protein (SlMBP21), alcobaca (SlALC), and acetolactate synthase 1 (SlALS1) loci. Our data show challenges of the PE approach in tomato, indicating that a further improvement of the PE system for successful applications is demanded, such as the use of improved expression systems for enriching active PE complexes.
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spelling pubmed-105931212023-10-30 The Obstacles and Potential Solution Clues of Prime Editing Applications in Tomato Vu, Tien Van Nguyen, Ngan Thi Kim, Jihae Das, Swati Lee, Jinsu Kim, Jae-Yean Biodes Res Research Article Precision genome editing is highly desired for crop improvement. The recently emerged CRISPR/Cas technology offers great potential applications in precision plant genome engineering. A prime editing (PE) approach combining a reverse transcriptase (RT) with a Cas9 nickase and a “priming” extended guide RNA (gRNA) has shown a high frequency for precise genome modification in mammalian cells and several plant species. Nevertheless, the applications of the PE approach in dicot plants are still limited and inefficient. We designed and tested prime editors for precision editing of a synthetic sequence in a transient assay and for desirable alleles of 10 loci in tomato by stable transformation. Our data obtained by targeted deep sequencing also revealed only low PE efficiencies in both the tobacco and tomato systems. Further assessment of the activities of the PE components uncovered that the fusion of RT to Cas9 and the structure of PE gRNAs (pegRNAs) negatively affected the cleaving activity of the Cas9 nuclease. The self-complementarity between the primer binding sequences (PBSs) and spacer sequence might pose risks to the activity of the Cas9 complex. However, modifying the pegRNA sequences by shortening or introducing mismatches to the PBSs to reduce their melting temperatures did not enhance the PE efficiency at the MADS-box protein (SlMBP21), alcobaca (SlALC), and acetolactate synthase 1 (SlALS1) loci. Our data show challenges of the PE approach in tomato, indicating that a further improvement of the PE system for successful applications is demanded, such as the use of improved expression systems for enriching active PE complexes. AAAS 2022-12-15 /pmc/articles/PMC10593121/ /pubmed/37905201 http://dx.doi.org/10.34133/bdr.0001 Text en Copyright © 2022 Tien Van Vu et al. https://creativecommons.org/licenses/by/4.0/Exclusive licensee Nanjing Agricultural University. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY 4.0) (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Research Article
Vu, Tien Van
Nguyen, Ngan Thi
Kim, Jihae
Das, Swati
Lee, Jinsu
Kim, Jae-Yean
The Obstacles and Potential Solution Clues of Prime Editing Applications in Tomato
title The Obstacles and Potential Solution Clues of Prime Editing Applications in Tomato
title_full The Obstacles and Potential Solution Clues of Prime Editing Applications in Tomato
title_fullStr The Obstacles and Potential Solution Clues of Prime Editing Applications in Tomato
title_full_unstemmed The Obstacles and Potential Solution Clues of Prime Editing Applications in Tomato
title_short The Obstacles and Potential Solution Clues of Prime Editing Applications in Tomato
title_sort obstacles and potential solution clues of prime editing applications in tomato
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10593121/
https://www.ncbi.nlm.nih.gov/pubmed/37905201
http://dx.doi.org/10.34133/bdr.0001
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