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Strategies for Efficient Gene Editing in Protoplasts of Solanum tuberosum Theme: Determining gRNA Efficiency Design by Utilizing Protoplast (Research)
Potato (Solanum tuberosum) is a highly diverse tetraploid crop. Elite cultivars are extremely heterozygous with a high prevalence of small length polymorphisms (indels) and single nucleotide polymorphisms (SNPs) within and between cultivars, which must be considered in CRISPR/Cas gene editing strate...
Autores principales: | , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Frontiers Media S.A.
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8811252/ https://www.ncbi.nlm.nih.gov/pubmed/35128523 http://dx.doi.org/10.3389/fgeed.2021.795644 |
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author | Carlsen, Frida Meijer Johansen, Ida Elisabeth Yang, Zhang Liu, Ying Westberg, Ida Nøhr Kieu, Nam Phuong Jørgensen, Bodil Lenman, Marit Andreasson, Erik Nielsen, Kåre Lehmann Blennow, Andreas Petersen, Bent Larsen |
author_facet | Carlsen, Frida Meijer Johansen, Ida Elisabeth Yang, Zhang Liu, Ying Westberg, Ida Nøhr Kieu, Nam Phuong Jørgensen, Bodil Lenman, Marit Andreasson, Erik Nielsen, Kåre Lehmann Blennow, Andreas Petersen, Bent Larsen |
author_sort | Carlsen, Frida Meijer |
collection | PubMed |
description | Potato (Solanum tuberosum) is a highly diverse tetraploid crop. Elite cultivars are extremely heterozygous with a high prevalence of small length polymorphisms (indels) and single nucleotide polymorphisms (SNPs) within and between cultivars, which must be considered in CRISPR/Cas gene editing strategies and designs to obtain successful gene editing. In the present study, in-depth sequencing of the glucan water dikinase (GWD)1 and the downy mildew resistant 6 (DMR6-1) genes in the potato cultivars Saturna and Wotan, respectively, revealed both indels and a 1.3–2.8 higher SNP prevalence when compared to the heterozygous diploid RH genome sequence as expected for a tetraploid compared to a diploid. This complicates guide RNA (gRNA) and diagnostic PCR designs. High editing efficiencies at the cell pool (protoplast) level are pivotal for achieving full allelic knock-out in tetraploids and for reducing the downstream cumbersome and delicate ex-plant regeneration. Here, CRISPR/Cas ribonucleoprotein particles (RNP) were delivered transiently to protoplasts by polyethylene glycol (PEG) mediated transformation. For each of GWD1 and DMR6-1, 6–10 gRNAs were designed to target regions comprising the 5′ and the 3′ end of the two genes. Similar to other studies including several organisms, editing efficiency of the individual RNPs/gRNAs varied significantly, and some generated specific indel patterns. While RNPs targeting the 5′ end of GWD1 yielded significantly higher editing when compared to targeting the 3′ end, editing efficiencies in the 5′ and 3′ end of DMR6-1 appeared to be somewhat similar. Simultaneous targeting of either the 5′ or the 3′ end with two RNPs (multiplexing) yielded a clear positive synergistic effect on the total editing when targeting the 3′ end of the GWD1 gene only. Multiplexing of the two genes, residing on different chromosomes, yielded no or slightly negative effects on the individual RNP/gRNA editing efficiencies when compared to editing efficiencies obtained in the single RNP/gRNA transformations. These initial findings may instigate larger studies needed for facilitating and optimizing precision breeding in plants. |
format | Online Article Text |
id | pubmed-8811252 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-88112522022-02-04 Strategies for Efficient Gene Editing in Protoplasts of Solanum tuberosum Theme: Determining gRNA Efficiency Design by Utilizing Protoplast (Research) Carlsen, Frida Meijer Johansen, Ida Elisabeth Yang, Zhang Liu, Ying Westberg, Ida Nøhr Kieu, Nam Phuong Jørgensen, Bodil Lenman, Marit Andreasson, Erik Nielsen, Kåre Lehmann Blennow, Andreas Petersen, Bent Larsen Front Genome Ed Genome Editing Potato (Solanum tuberosum) is a highly diverse tetraploid crop. Elite cultivars are extremely heterozygous with a high prevalence of small length polymorphisms (indels) and single nucleotide polymorphisms (SNPs) within and between cultivars, which must be considered in CRISPR/Cas gene editing strategies and designs to obtain successful gene editing. In the present study, in-depth sequencing of the glucan water dikinase (GWD)1 and the downy mildew resistant 6 (DMR6-1) genes in the potato cultivars Saturna and Wotan, respectively, revealed both indels and a 1.3–2.8 higher SNP prevalence when compared to the heterozygous diploid RH genome sequence as expected for a tetraploid compared to a diploid. This complicates guide RNA (gRNA) and diagnostic PCR designs. High editing efficiencies at the cell pool (protoplast) level are pivotal for achieving full allelic knock-out in tetraploids and for reducing the downstream cumbersome and delicate ex-plant regeneration. Here, CRISPR/Cas ribonucleoprotein particles (RNP) were delivered transiently to protoplasts by polyethylene glycol (PEG) mediated transformation. For each of GWD1 and DMR6-1, 6–10 gRNAs were designed to target regions comprising the 5′ and the 3′ end of the two genes. Similar to other studies including several organisms, editing efficiency of the individual RNPs/gRNAs varied significantly, and some generated specific indel patterns. While RNPs targeting the 5′ end of GWD1 yielded significantly higher editing when compared to targeting the 3′ end, editing efficiencies in the 5′ and 3′ end of DMR6-1 appeared to be somewhat similar. Simultaneous targeting of either the 5′ or the 3′ end with two RNPs (multiplexing) yielded a clear positive synergistic effect on the total editing when targeting the 3′ end of the GWD1 gene only. Multiplexing of the two genes, residing on different chromosomes, yielded no or slightly negative effects on the individual RNP/gRNA editing efficiencies when compared to editing efficiencies obtained in the single RNP/gRNA transformations. These initial findings may instigate larger studies needed for facilitating and optimizing precision breeding in plants. Frontiers Media S.A. 2022-01-20 /pmc/articles/PMC8811252/ /pubmed/35128523 http://dx.doi.org/10.3389/fgeed.2021.795644 Text en Copyright © 2022 Carlsen, Johansen, Yang, Liu, Westberg, Kieu, Jørgensen, Lenman, Andreasson, Nielsen, Blennow and Petersen. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Genome Editing Carlsen, Frida Meijer Johansen, Ida Elisabeth Yang, Zhang Liu, Ying Westberg, Ida Nøhr Kieu, Nam Phuong Jørgensen, Bodil Lenman, Marit Andreasson, Erik Nielsen, Kåre Lehmann Blennow, Andreas Petersen, Bent Larsen Strategies for Efficient Gene Editing in Protoplasts of Solanum tuberosum Theme: Determining gRNA Efficiency Design by Utilizing Protoplast (Research) |
title | Strategies for Efficient Gene Editing in Protoplasts of Solanum tuberosum Theme: Determining gRNA Efficiency Design by Utilizing Protoplast (Research) |
title_full | Strategies for Efficient Gene Editing in Protoplasts of Solanum tuberosum Theme: Determining gRNA Efficiency Design by Utilizing Protoplast (Research) |
title_fullStr | Strategies for Efficient Gene Editing in Protoplasts of Solanum tuberosum Theme: Determining gRNA Efficiency Design by Utilizing Protoplast (Research) |
title_full_unstemmed | Strategies for Efficient Gene Editing in Protoplasts of Solanum tuberosum Theme: Determining gRNA Efficiency Design by Utilizing Protoplast (Research) |
title_short | Strategies for Efficient Gene Editing in Protoplasts of Solanum tuberosum Theme: Determining gRNA Efficiency Design by Utilizing Protoplast (Research) |
title_sort | strategies for efficient gene editing in protoplasts of solanum tuberosum theme: determining grna efficiency design by utilizing protoplast (research) |
topic | Genome Editing |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8811252/ https://www.ncbi.nlm.nih.gov/pubmed/35128523 http://dx.doi.org/10.3389/fgeed.2021.795644 |
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