Comparison of genotyping assays for detection of targeted CRISPR/Cas mutagenesis in highly polyploid sugarcane
Sugarcane (Saccharum spp.) is an important biofuel feedstock and a leading source of global table sugar. Saccharum hybrid cultivars are highly polyploid (2n = 100–130), containing large numbers of functionally redundant hom(e)ologs in their genomes. Genome editing with sequence-specific nucleases ho...
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Frontiers Media S.A.
2024-12-01
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| Series: | Frontiers in Genome Editing |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fgeed.2024.1505844/full |
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| author | Eleanor J. Brant Eleanor J. Brant David May David May Ayman Eid Ayman Eid Fredy Altpeter Fredy Altpeter |
| author_facet | Eleanor J. Brant Eleanor J. Brant David May David May Ayman Eid Ayman Eid Fredy Altpeter Fredy Altpeter |
| author_sort | Eleanor J. Brant |
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| description | Sugarcane (Saccharum spp.) is an important biofuel feedstock and a leading source of global table sugar. Saccharum hybrid cultivars are highly polyploid (2n = 100–130), containing large numbers of functionally redundant hom(e)ologs in their genomes. Genome editing with sequence-specific nucleases holds tremendous promise for sugarcane breeding. However, identification of plants with the desired level of co-editing within a pool of primary transformants can be difficult. While DNA sequencing provides direct evidence of targeted mutagenesis, it is cost-prohibitive as a primary screening method in sugarcane and most other methods of identifying mutant lines have not been optimized for use in highly polyploid species. In this study, non-sequencing methods of mutant screening, including capillary electrophoresis (CE), Cas9 RNP assay, and high-resolution melt analysis (HRMA), were compared to assess their potential for CRISPR/Cas9-mediated mutant screening in sugarcane. These assays were used to analyze sugarcane lines containing mutations at one or more of six sgRNA target sites. All three methods distinguished edited lines from wild type, with co-mutation frequencies ranging from 2% to 100%. Cas9 RNP assays were able to identify mutant sugarcane lines with as low as 3.2% co-mutation frequency, and samples could be scored based on undigested band intensity. CE was highlighted as the most comprehensive assay, delivering precise information on both mutagenesis frequency and indel size to a 1 bp resolution across all six targets. This represents an economical and comprehensive alternative to sequencing-based genotyping methods which could be applied in other polyploid species. |
| format | Article |
| id | doaj-art-e5fdcb7fdfdd439ab4bcae33e0f14816 |
| institution | OA Journals |
| issn | 2673-3439 |
| language | English |
| publishDate | 2024-12-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Genome Editing |
| spelling | doaj-art-e5fdcb7fdfdd439ab4bcae33e0f148162025-08-20T02:34:20ZengFrontiers Media S.A.Frontiers in Genome Editing2673-34392024-12-01610.3389/fgeed.2024.15058441505844Comparison of genotyping assays for detection of targeted CRISPR/Cas mutagenesis in highly polyploid sugarcaneEleanor J. Brant0Eleanor J. Brant1David May2David May3Ayman Eid4Ayman Eid5Fredy Altpeter6Fredy Altpeter7Agronomy Department, Plant Molecular and Cellular Biology Program, Genetics Institute, University of Florida, IFAS-Institute of Food and Agricultural Science, Gainesville, FL, United StatesDOE Center for Advanced Bioenergy and Bioproducts Innovation, Gainesville, FL, United StatesAgronomy Department, Plant Molecular and Cellular Biology Program, Genetics Institute, University of Florida, IFAS-Institute of Food and Agricultural Science, Gainesville, FL, United StatesDOE Center for Advanced Bioenergy and Bioproducts Innovation, Gainesville, FL, United StatesAgronomy Department, Plant Molecular and Cellular Biology Program, Genetics Institute, University of Florida, IFAS-Institute of Food and Agricultural Science, Gainesville, FL, United StatesDOE Center for Advanced Bioenergy and Bioproducts Innovation, Gainesville, FL, United StatesAgronomy Department, Plant Molecular and Cellular Biology Program, Genetics Institute, University of Florida, IFAS-Institute of Food and Agricultural Science, Gainesville, FL, United StatesDOE Center for Advanced Bioenergy and Bioproducts Innovation, Gainesville, FL, United StatesSugarcane (Saccharum spp.) is an important biofuel feedstock and a leading source of global table sugar. Saccharum hybrid cultivars are highly polyploid (2n = 100–130), containing large numbers of functionally redundant hom(e)ologs in their genomes. Genome editing with sequence-specific nucleases holds tremendous promise for sugarcane breeding. However, identification of plants with the desired level of co-editing within a pool of primary transformants can be difficult. While DNA sequencing provides direct evidence of targeted mutagenesis, it is cost-prohibitive as a primary screening method in sugarcane and most other methods of identifying mutant lines have not been optimized for use in highly polyploid species. In this study, non-sequencing methods of mutant screening, including capillary electrophoresis (CE), Cas9 RNP assay, and high-resolution melt analysis (HRMA), were compared to assess their potential for CRISPR/Cas9-mediated mutant screening in sugarcane. These assays were used to analyze sugarcane lines containing mutations at one or more of six sgRNA target sites. All three methods distinguished edited lines from wild type, with co-mutation frequencies ranging from 2% to 100%. Cas9 RNP assays were able to identify mutant sugarcane lines with as low as 3.2% co-mutation frequency, and samples could be scored based on undigested band intensity. CE was highlighted as the most comprehensive assay, delivering precise information on both mutagenesis frequency and indel size to a 1 bp resolution across all six targets. This represents an economical and comprehensive alternative to sequencing-based genotyping methods which could be applied in other polyploid species.https://www.frontiersin.org/articles/10.3389/fgeed.2024.1505844/fullpolyploidgenotypingmutationsCRISPR/Cas9high resolution meltcapillary electrophoresis |
| spellingShingle | Eleanor J. Brant Eleanor J. Brant David May David May Ayman Eid Ayman Eid Fredy Altpeter Fredy Altpeter Comparison of genotyping assays for detection of targeted CRISPR/Cas mutagenesis in highly polyploid sugarcane Frontiers in Genome Editing polyploid genotyping mutations CRISPR/Cas9 high resolution melt capillary electrophoresis |
| title | Comparison of genotyping assays for detection of targeted CRISPR/Cas mutagenesis in highly polyploid sugarcane |
| title_full | Comparison of genotyping assays for detection of targeted CRISPR/Cas mutagenesis in highly polyploid sugarcane |
| title_fullStr | Comparison of genotyping assays for detection of targeted CRISPR/Cas mutagenesis in highly polyploid sugarcane |
| title_full_unstemmed | Comparison of genotyping assays for detection of targeted CRISPR/Cas mutagenesis in highly polyploid sugarcane |
| title_short | Comparison of genotyping assays for detection of targeted CRISPR/Cas mutagenesis in highly polyploid sugarcane |
| title_sort | comparison of genotyping assays for detection of targeted crispr cas mutagenesis in highly polyploid sugarcane |
| topic | polyploid genotyping mutations CRISPR/Cas9 high resolution melt capillary electrophoresis |
| url | https://www.frontiersin.org/articles/10.3389/fgeed.2024.1505844/full |
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