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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...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
AAAS
2022
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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. |
format | Online Article Text |
id | pubmed-10593121 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | AAAS |
record_format | MEDLINE/PubMed |
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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