Rapid and efficient CRISPR/Cas9-mediated genome editing in potato via hairy root induction
Abstract Potato is one of the most important crops, and its trait improvement through genetic engineering is of utmost need. CRISPR/Cas9-mediated genome editing tool and its advancement revolutionized genetic and metabolic engineering. Cas9 endonuclease requires gRNA and PAM for targeted genome edit...
Ausführliche Beschreibung
Gespeichert in:
| Autor*in: |
Alok, Anshu [verfasserIn] |
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| Format: |
E-Artikel |
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| Sprache: |
Englisch |
| Erschienen: |
2022 |
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| Schlagwörter: |
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| Anmerkung: |
© The Society for In Vitro Biology 2022. Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. |
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| Übergeordnetes Werk: |
Enthalten in: In vitro cellular & developmental biology - Berlin : Springer, 1991, 59(2022), 1 vom: 08. Dez., Seite 83-94 |
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| Übergeordnetes Werk: |
volume:59 ; year:2022 ; number:1 ; day:08 ; month:12 ; pages:83-94 |
| Links: |
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| DOI / URN: |
10.1007/s11627-022-10318-0 |
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| Katalog-ID: |
SPR049595733 |
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| 520 | |a Abstract Potato is one of the most important crops, and its trait improvement through genetic engineering is of utmost need. CRISPR/Cas9-mediated genome editing tool and its advancement revolutionized genetic and metabolic engineering. Cas9 endonuclease requires gRNA and PAM for targeted genome editing. Delivery of CRISPR/Cas9 component via Rhizobium is most commonly used. Different factors affecting genetic transformation, for example bacterial strains, bacterial density, co-cultivation duration, acetosyringone, explants type, and cultivars, were optimized for Indian potato. Wild-type Rhizobium rhizogenes strain A4 showed the highest (60.7 ± 6.9%) hairy root induction followed by ATCC15834 (53.4 ± 10.7%). The highest transformation efficiency (68.8 ± 6.8%) was achieved onto Murashige and Skoog medium (MS) supplemented with 0.5 mg $ L^{−1} $ naphthaleneacetic acid (NAA). The putative transgenic hairy roots were induced within 8 to 10 d on hygromycin-containing media. A4 strain carrying CRISPR/Cas9 vector was used to target the StbHLH47. A total of 32 possible target sites near to PAM region were identified in the 500-bp sequence of StbHLH47. The target site adjacent to the ATG start codon was selected, and the construct was prepared into the pHSE401 vector. PCR amplification of rolB, HptII, and Cas9 gene–specific primers showed the integration of CRISPR/Cas9 components in the potato genome. Transgenic hairy root lines showed 2 to 7 nucleotide deletion at the target site of StbHLH. The editing frequency was calculated to be 15.3% for StbHLH47. This study reports a fast and efficient protocol for hairy root induction by Rhizobium rhizogenes to analyze editing in the potato genome within 18 to 20 d. The developed protocol will be very useful for studying gene function using CRISPR in the area of root biology. | ||
| 650 | 4 | |a Potato |7 (dpeaa)DE-He213 | |
| 650 | 4 | |a Hairy roots |7 (dpeaa)DE-He213 | |
| 650 | 4 | |a CRISPR/Cas9 |7 (dpeaa)DE-He213 | |
| 650 | 4 | |a Genome editing |7 (dpeaa)DE-He213 | |
| 650 | 4 | |a T7E1 assay |7 (dpeaa)DE-He213 | |
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| 700 | 1 | |a Kaushal, Nishant |4 aut | |
| 700 | 1 | |a Singh, Kashmir |0 (orcid)0000-0002-9646-9043 |4 aut | |
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10.1007/s11627-022-10318-0 doi (DE-627)SPR049595733 (SPR)s11627-022-10318-0-e DE-627 ger DE-627 rakwb eng Alok, Anshu verfasserin aut Rapid and efficient CRISPR/Cas9-mediated genome editing in potato via hairy root induction 2022 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Society for In Vitro Biology 2022. Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. Abstract Potato is one of the most important crops, and its trait improvement through genetic engineering is of utmost need. CRISPR/Cas9-mediated genome editing tool and its advancement revolutionized genetic and metabolic engineering. Cas9 endonuclease requires gRNA and PAM for targeted genome editing. Delivery of CRISPR/Cas9 component via Rhizobium is most commonly used. Different factors affecting genetic transformation, for example bacterial strains, bacterial density, co-cultivation duration, acetosyringone, explants type, and cultivars, were optimized for Indian potato. Wild-type Rhizobium rhizogenes strain A4 showed the highest (60.7 ± 6.9%) hairy root induction followed by ATCC15834 (53.4 ± 10.7%). The highest transformation efficiency (68.8 ± 6.8%) was achieved onto Murashige and Skoog medium (MS) supplemented with 0.5 mg $ L^{−1} $ naphthaleneacetic acid (NAA). The putative transgenic hairy roots were induced within 8 to 10 d on hygromycin-containing media. A4 strain carrying CRISPR/Cas9 vector was used to target the StbHLH47. A total of 32 possible target sites near to PAM region were identified in the 500-bp sequence of StbHLH47. The target site adjacent to the ATG start codon was selected, and the construct was prepared into the pHSE401 vector. PCR amplification of rolB, HptII, and Cas9 gene–specific primers showed the integration of CRISPR/Cas9 components in the potato genome. Transgenic hairy root lines showed 2 to 7 nucleotide deletion at the target site of StbHLH. The editing frequency was calculated to be 15.3% for StbHLH47. This study reports a fast and efficient protocol for hairy root induction by Rhizobium rhizogenes to analyze editing in the potato genome within 18 to 20 d. The developed protocol will be very useful for studying gene function using CRISPR in the area of root biology. Potato (dpeaa)DE-He213 Hairy roots (dpeaa)DE-He213 CRISPR/Cas9 (dpeaa)DE-He213 Genome editing (dpeaa)DE-He213 T7E1 assay (dpeaa)DE-He213 Chauhan, Hanny aut Kaushal, Nishant aut Singh, Kashmir (orcid)0000-0002-9646-9043 aut Enthalten in In vitro cellular & developmental biology Berlin : Springer, 1991 59(2022), 1 vom: 08. Dez., Seite 83-94 (DE-627)306365367 (DE-600)1497464-2 1475-2689 nnns volume:59 year:2022 number:1 day:08 month:12 pages:83-94 https://dx.doi.org/10.1007/s11627-022-10318-0 lizenzpflichtig Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_32 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_90 GBV_ILN_95 GBV_ILN_100 GBV_ILN_101 GBV_ILN_105 GBV_ILN_110 GBV_ILN_120 GBV_ILN_138 GBV_ILN_150 GBV_ILN_151 GBV_ILN_152 GBV_ILN_161 GBV_ILN_165 GBV_ILN_170 GBV_ILN_171 GBV_ILN_187 GBV_ILN_206 GBV_ILN_213 GBV_ILN_224 GBV_ILN_230 GBV_ILN_250 GBV_ILN_281 GBV_ILN_285 GBV_ILN_293 GBV_ILN_370 GBV_ILN_374 GBV_ILN_602 GBV_ILN_636 GBV_ILN_702 GBV_ILN_2001 GBV_ILN_2003 GBV_ILN_2004 GBV_ILN_2005 GBV_ILN_2006 GBV_ILN_2007 GBV_ILN_2008 GBV_ILN_2009 GBV_ILN_2010 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2015 GBV_ILN_2018 GBV_ILN_2020 GBV_ILN_2021 GBV_ILN_2025 GBV_ILN_2026 GBV_ILN_2027 GBV_ILN_2031 GBV_ILN_2034 GBV_ILN_2037 GBV_ILN_2038 GBV_ILN_2039 GBV_ILN_2044 GBV_ILN_2048 GBV_ILN_2049 GBV_ILN_2050 GBV_ILN_2055 GBV_ILN_2056 GBV_ILN_2057 GBV_ILN_2059 GBV_ILN_2061 GBV_ILN_2064 GBV_ILN_2065 GBV_ILN_2068 GBV_ILN_2088 GBV_ILN_2093 GBV_ILN_2106 GBV_ILN_2107 GBV_ILN_2108 GBV_ILN_2110 GBV_ILN_2111 GBV_ILN_2112 GBV_ILN_2113 GBV_ILN_2118 GBV_ILN_2122 GBV_ILN_2129 GBV_ILN_2143 GBV_ILN_2144 GBV_ILN_2147 GBV_ILN_2148 GBV_ILN_2152 GBV_ILN_2153 GBV_ILN_2188 GBV_ILN_2190 GBV_ILN_2232 GBV_ILN_2336 GBV_ILN_2446 GBV_ILN_2470 GBV_ILN_2472 GBV_ILN_2507 GBV_ILN_2522 GBV_ILN_2548 GBV_ILN_2939 GBV_ILN_2946 GBV_ILN_2949 GBV_ILN_2951 GBV_ILN_4012 GBV_ILN_4035 GBV_ILN_4037 GBV_ILN_4046 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4242 GBV_ILN_4246 GBV_ILN_4249 GBV_ILN_4251 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4326 GBV_ILN_4328 GBV_ILN_4333 GBV_ILN_4334 GBV_ILN_4335 GBV_ILN_4336 GBV_ILN_4338 GBV_ILN_4346 GBV_ILN_4393 GBV_ILN_4700 AR 59 2022 1 08 12 83-94 |
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10.1007/s11627-022-10318-0 doi (DE-627)SPR049595733 (SPR)s11627-022-10318-0-e DE-627 ger DE-627 rakwb eng Alok, Anshu verfasserin aut Rapid and efficient CRISPR/Cas9-mediated genome editing in potato via hairy root induction 2022 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Society for In Vitro Biology 2022. Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. Abstract Potato is one of the most important crops, and its trait improvement through genetic engineering is of utmost need. CRISPR/Cas9-mediated genome editing tool and its advancement revolutionized genetic and metabolic engineering. Cas9 endonuclease requires gRNA and PAM for targeted genome editing. Delivery of CRISPR/Cas9 component via Rhizobium is most commonly used. Different factors affecting genetic transformation, for example bacterial strains, bacterial density, co-cultivation duration, acetosyringone, explants type, and cultivars, were optimized for Indian potato. Wild-type Rhizobium rhizogenes strain A4 showed the highest (60.7 ± 6.9%) hairy root induction followed by ATCC15834 (53.4 ± 10.7%). The highest transformation efficiency (68.8 ± 6.8%) was achieved onto Murashige and Skoog medium (MS) supplemented with 0.5 mg $ L^{−1} $ naphthaleneacetic acid (NAA). The putative transgenic hairy roots were induced within 8 to 10 d on hygromycin-containing media. A4 strain carrying CRISPR/Cas9 vector was used to target the StbHLH47. A total of 32 possible target sites near to PAM region were identified in the 500-bp sequence of StbHLH47. The target site adjacent to the ATG start codon was selected, and the construct was prepared into the pHSE401 vector. PCR amplification of rolB, HptII, and Cas9 gene–specific primers showed the integration of CRISPR/Cas9 components in the potato genome. Transgenic hairy root lines showed 2 to 7 nucleotide deletion at the target site of StbHLH. The editing frequency was calculated to be 15.3% for StbHLH47. This study reports a fast and efficient protocol for hairy root induction by Rhizobium rhizogenes to analyze editing in the potato genome within 18 to 20 d. The developed protocol will be very useful for studying gene function using CRISPR in the area of root biology. Potato (dpeaa)DE-He213 Hairy roots (dpeaa)DE-He213 CRISPR/Cas9 (dpeaa)DE-He213 Genome editing (dpeaa)DE-He213 T7E1 assay (dpeaa)DE-He213 Chauhan, Hanny aut Kaushal, Nishant aut Singh, Kashmir (orcid)0000-0002-9646-9043 aut Enthalten in In vitro cellular & developmental biology Berlin : Springer, 1991 59(2022), 1 vom: 08. Dez., Seite 83-94 (DE-627)306365367 (DE-600)1497464-2 1475-2689 nnns volume:59 year:2022 number:1 day:08 month:12 pages:83-94 https://dx.doi.org/10.1007/s11627-022-10318-0 lizenzpflichtig Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_32 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_90 GBV_ILN_95 GBV_ILN_100 GBV_ILN_101 GBV_ILN_105 GBV_ILN_110 GBV_ILN_120 GBV_ILN_138 GBV_ILN_150 GBV_ILN_151 GBV_ILN_152 GBV_ILN_161 GBV_ILN_165 GBV_ILN_170 GBV_ILN_171 GBV_ILN_187 GBV_ILN_206 GBV_ILN_213 GBV_ILN_224 GBV_ILN_230 GBV_ILN_250 GBV_ILN_281 GBV_ILN_285 GBV_ILN_293 GBV_ILN_370 GBV_ILN_374 GBV_ILN_602 GBV_ILN_636 GBV_ILN_702 GBV_ILN_2001 GBV_ILN_2003 GBV_ILN_2004 GBV_ILN_2005 GBV_ILN_2006 GBV_ILN_2007 GBV_ILN_2008 GBV_ILN_2009 GBV_ILN_2010 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2015 GBV_ILN_2018 GBV_ILN_2020 GBV_ILN_2021 GBV_ILN_2025 GBV_ILN_2026 GBV_ILN_2027 GBV_ILN_2031 GBV_ILN_2034 GBV_ILN_2037 GBV_ILN_2038 GBV_ILN_2039 GBV_ILN_2044 GBV_ILN_2048 GBV_ILN_2049 GBV_ILN_2050 GBV_ILN_2055 GBV_ILN_2056 GBV_ILN_2057 GBV_ILN_2059 GBV_ILN_2061 GBV_ILN_2064 GBV_ILN_2065 GBV_ILN_2068 GBV_ILN_2088 GBV_ILN_2093 GBV_ILN_2106 GBV_ILN_2107 GBV_ILN_2108 GBV_ILN_2110 GBV_ILN_2111 GBV_ILN_2112 GBV_ILN_2113 GBV_ILN_2118 GBV_ILN_2122 GBV_ILN_2129 GBV_ILN_2143 GBV_ILN_2144 GBV_ILN_2147 GBV_ILN_2148 GBV_ILN_2152 GBV_ILN_2153 GBV_ILN_2188 GBV_ILN_2190 GBV_ILN_2232 GBV_ILN_2336 GBV_ILN_2446 GBV_ILN_2470 GBV_ILN_2472 GBV_ILN_2507 GBV_ILN_2522 GBV_ILN_2548 GBV_ILN_2939 GBV_ILN_2946 GBV_ILN_2949 GBV_ILN_2951 GBV_ILN_4012 GBV_ILN_4035 GBV_ILN_4037 GBV_ILN_4046 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4242 GBV_ILN_4246 GBV_ILN_4249 GBV_ILN_4251 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4326 GBV_ILN_4328 GBV_ILN_4333 GBV_ILN_4334 GBV_ILN_4335 GBV_ILN_4336 GBV_ILN_4338 GBV_ILN_4346 GBV_ILN_4393 GBV_ILN_4700 AR 59 2022 1 08 12 83-94 |
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10.1007/s11627-022-10318-0 doi (DE-627)SPR049595733 (SPR)s11627-022-10318-0-e DE-627 ger DE-627 rakwb eng Alok, Anshu verfasserin aut Rapid and efficient CRISPR/Cas9-mediated genome editing in potato via hairy root induction 2022 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Society for In Vitro Biology 2022. Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. Abstract Potato is one of the most important crops, and its trait improvement through genetic engineering is of utmost need. CRISPR/Cas9-mediated genome editing tool and its advancement revolutionized genetic and metabolic engineering. Cas9 endonuclease requires gRNA and PAM for targeted genome editing. Delivery of CRISPR/Cas9 component via Rhizobium is most commonly used. Different factors affecting genetic transformation, for example bacterial strains, bacterial density, co-cultivation duration, acetosyringone, explants type, and cultivars, were optimized for Indian potato. Wild-type Rhizobium rhizogenes strain A4 showed the highest (60.7 ± 6.9%) hairy root induction followed by ATCC15834 (53.4 ± 10.7%). The highest transformation efficiency (68.8 ± 6.8%) was achieved onto Murashige and Skoog medium (MS) supplemented with 0.5 mg $ L^{−1} $ naphthaleneacetic acid (NAA). The putative transgenic hairy roots were induced within 8 to 10 d on hygromycin-containing media. A4 strain carrying CRISPR/Cas9 vector was used to target the StbHLH47. A total of 32 possible target sites near to PAM region were identified in the 500-bp sequence of StbHLH47. The target site adjacent to the ATG start codon was selected, and the construct was prepared into the pHSE401 vector. PCR amplification of rolB, HptII, and Cas9 gene–specific primers showed the integration of CRISPR/Cas9 components in the potato genome. Transgenic hairy root lines showed 2 to 7 nucleotide deletion at the target site of StbHLH. The editing frequency was calculated to be 15.3% for StbHLH47. This study reports a fast and efficient protocol for hairy root induction by Rhizobium rhizogenes to analyze editing in the potato genome within 18 to 20 d. The developed protocol will be very useful for studying gene function using CRISPR in the area of root biology. Potato (dpeaa)DE-He213 Hairy roots (dpeaa)DE-He213 CRISPR/Cas9 (dpeaa)DE-He213 Genome editing (dpeaa)DE-He213 T7E1 assay (dpeaa)DE-He213 Chauhan, Hanny aut Kaushal, Nishant aut Singh, Kashmir (orcid)0000-0002-9646-9043 aut Enthalten in In vitro cellular & developmental biology Berlin : Springer, 1991 59(2022), 1 vom: 08. Dez., Seite 83-94 (DE-627)306365367 (DE-600)1497464-2 1475-2689 nnns volume:59 year:2022 number:1 day:08 month:12 pages:83-94 https://dx.doi.org/10.1007/s11627-022-10318-0 lizenzpflichtig Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_32 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_90 GBV_ILN_95 GBV_ILN_100 GBV_ILN_101 GBV_ILN_105 GBV_ILN_110 GBV_ILN_120 GBV_ILN_138 GBV_ILN_150 GBV_ILN_151 GBV_ILN_152 GBV_ILN_161 GBV_ILN_165 GBV_ILN_170 GBV_ILN_171 GBV_ILN_187 GBV_ILN_206 GBV_ILN_213 GBV_ILN_224 GBV_ILN_230 GBV_ILN_250 GBV_ILN_281 GBV_ILN_285 GBV_ILN_293 GBV_ILN_370 GBV_ILN_374 GBV_ILN_602 GBV_ILN_636 GBV_ILN_702 GBV_ILN_2001 GBV_ILN_2003 GBV_ILN_2004 GBV_ILN_2005 GBV_ILN_2006 GBV_ILN_2007 GBV_ILN_2008 GBV_ILN_2009 GBV_ILN_2010 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2015 GBV_ILN_2018 GBV_ILN_2020 GBV_ILN_2021 GBV_ILN_2025 GBV_ILN_2026 GBV_ILN_2027 GBV_ILN_2031 GBV_ILN_2034 GBV_ILN_2037 GBV_ILN_2038 GBV_ILN_2039 GBV_ILN_2044 GBV_ILN_2048 GBV_ILN_2049 GBV_ILN_2050 GBV_ILN_2055 GBV_ILN_2056 GBV_ILN_2057 GBV_ILN_2059 GBV_ILN_2061 GBV_ILN_2064 GBV_ILN_2065 GBV_ILN_2068 GBV_ILN_2088 GBV_ILN_2093 GBV_ILN_2106 GBV_ILN_2107 GBV_ILN_2108 GBV_ILN_2110 GBV_ILN_2111 GBV_ILN_2112 GBV_ILN_2113 GBV_ILN_2118 GBV_ILN_2122 GBV_ILN_2129 GBV_ILN_2143 GBV_ILN_2144 GBV_ILN_2147 GBV_ILN_2148 GBV_ILN_2152 GBV_ILN_2153 GBV_ILN_2188 GBV_ILN_2190 GBV_ILN_2232 GBV_ILN_2336 GBV_ILN_2446 GBV_ILN_2470 GBV_ILN_2472 GBV_ILN_2507 GBV_ILN_2522 GBV_ILN_2548 GBV_ILN_2939 GBV_ILN_2946 GBV_ILN_2949 GBV_ILN_2951 GBV_ILN_4012 GBV_ILN_4035 GBV_ILN_4037 GBV_ILN_4046 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4242 GBV_ILN_4246 GBV_ILN_4249 GBV_ILN_4251 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4326 GBV_ILN_4328 GBV_ILN_4333 GBV_ILN_4334 GBV_ILN_4335 GBV_ILN_4336 GBV_ILN_4338 GBV_ILN_4346 GBV_ILN_4393 GBV_ILN_4700 AR 59 2022 1 08 12 83-94 |
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10.1007/s11627-022-10318-0 doi (DE-627)SPR049595733 (SPR)s11627-022-10318-0-e DE-627 ger DE-627 rakwb eng Alok, Anshu verfasserin aut Rapid and efficient CRISPR/Cas9-mediated genome editing in potato via hairy root induction 2022 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Society for In Vitro Biology 2022. Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. Abstract Potato is one of the most important crops, and its trait improvement through genetic engineering is of utmost need. CRISPR/Cas9-mediated genome editing tool and its advancement revolutionized genetic and metabolic engineering. Cas9 endonuclease requires gRNA and PAM for targeted genome editing. Delivery of CRISPR/Cas9 component via Rhizobium is most commonly used. Different factors affecting genetic transformation, for example bacterial strains, bacterial density, co-cultivation duration, acetosyringone, explants type, and cultivars, were optimized for Indian potato. Wild-type Rhizobium rhizogenes strain A4 showed the highest (60.7 ± 6.9%) hairy root induction followed by ATCC15834 (53.4 ± 10.7%). The highest transformation efficiency (68.8 ± 6.8%) was achieved onto Murashige and Skoog medium (MS) supplemented with 0.5 mg $ L^{−1} $ naphthaleneacetic acid (NAA). The putative transgenic hairy roots were induced within 8 to 10 d on hygromycin-containing media. A4 strain carrying CRISPR/Cas9 vector was used to target the StbHLH47. A total of 32 possible target sites near to PAM region were identified in the 500-bp sequence of StbHLH47. The target site adjacent to the ATG start codon was selected, and the construct was prepared into the pHSE401 vector. PCR amplification of rolB, HptII, and Cas9 gene–specific primers showed the integration of CRISPR/Cas9 components in the potato genome. Transgenic hairy root lines showed 2 to 7 nucleotide deletion at the target site of StbHLH. The editing frequency was calculated to be 15.3% for StbHLH47. This study reports a fast and efficient protocol for hairy root induction by Rhizobium rhizogenes to analyze editing in the potato genome within 18 to 20 d. The developed protocol will be very useful for studying gene function using CRISPR in the area of root biology. Potato (dpeaa)DE-He213 Hairy roots (dpeaa)DE-He213 CRISPR/Cas9 (dpeaa)DE-He213 Genome editing (dpeaa)DE-He213 T7E1 assay (dpeaa)DE-He213 Chauhan, Hanny aut Kaushal, Nishant aut Singh, Kashmir (orcid)0000-0002-9646-9043 aut Enthalten in In vitro cellular & developmental biology Berlin : Springer, 1991 59(2022), 1 vom: 08. Dez., Seite 83-94 (DE-627)306365367 (DE-600)1497464-2 1475-2689 nnns volume:59 year:2022 number:1 day:08 month:12 pages:83-94 https://dx.doi.org/10.1007/s11627-022-10318-0 lizenzpflichtig Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_32 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_90 GBV_ILN_95 GBV_ILN_100 GBV_ILN_101 GBV_ILN_105 GBV_ILN_110 GBV_ILN_120 GBV_ILN_138 GBV_ILN_150 GBV_ILN_151 GBV_ILN_152 GBV_ILN_161 GBV_ILN_165 GBV_ILN_170 GBV_ILN_171 GBV_ILN_187 GBV_ILN_206 GBV_ILN_213 GBV_ILN_224 GBV_ILN_230 GBV_ILN_250 GBV_ILN_281 GBV_ILN_285 GBV_ILN_293 GBV_ILN_370 GBV_ILN_374 GBV_ILN_602 GBV_ILN_636 GBV_ILN_702 GBV_ILN_2001 GBV_ILN_2003 GBV_ILN_2004 GBV_ILN_2005 GBV_ILN_2006 GBV_ILN_2007 GBV_ILN_2008 GBV_ILN_2009 GBV_ILN_2010 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2015 GBV_ILN_2018 GBV_ILN_2020 GBV_ILN_2021 GBV_ILN_2025 GBV_ILN_2026 GBV_ILN_2027 GBV_ILN_2031 GBV_ILN_2034 GBV_ILN_2037 GBV_ILN_2038 GBV_ILN_2039 GBV_ILN_2044 GBV_ILN_2048 GBV_ILN_2049 GBV_ILN_2050 GBV_ILN_2055 GBV_ILN_2056 GBV_ILN_2057 GBV_ILN_2059 GBV_ILN_2061 GBV_ILN_2064 GBV_ILN_2065 GBV_ILN_2068 GBV_ILN_2088 GBV_ILN_2093 GBV_ILN_2106 GBV_ILN_2107 GBV_ILN_2108 GBV_ILN_2110 GBV_ILN_2111 GBV_ILN_2112 GBV_ILN_2113 GBV_ILN_2118 GBV_ILN_2122 GBV_ILN_2129 GBV_ILN_2143 GBV_ILN_2144 GBV_ILN_2147 GBV_ILN_2148 GBV_ILN_2152 GBV_ILN_2153 GBV_ILN_2188 GBV_ILN_2190 GBV_ILN_2232 GBV_ILN_2336 GBV_ILN_2446 GBV_ILN_2470 GBV_ILN_2472 GBV_ILN_2507 GBV_ILN_2522 GBV_ILN_2548 GBV_ILN_2939 GBV_ILN_2946 GBV_ILN_2949 GBV_ILN_2951 GBV_ILN_4012 GBV_ILN_4035 GBV_ILN_4037 GBV_ILN_4046 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4242 GBV_ILN_4246 GBV_ILN_4249 GBV_ILN_4251 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4326 GBV_ILN_4328 GBV_ILN_4333 GBV_ILN_4334 GBV_ILN_4335 GBV_ILN_4336 GBV_ILN_4338 GBV_ILN_4346 GBV_ILN_4393 GBV_ILN_4700 AR 59 2022 1 08 12 83-94 |
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10.1007/s11627-022-10318-0 doi (DE-627)SPR049595733 (SPR)s11627-022-10318-0-e DE-627 ger DE-627 rakwb eng Alok, Anshu verfasserin aut Rapid and efficient CRISPR/Cas9-mediated genome editing in potato via hairy root induction 2022 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Society for In Vitro Biology 2022. Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. Abstract Potato is one of the most important crops, and its trait improvement through genetic engineering is of utmost need. CRISPR/Cas9-mediated genome editing tool and its advancement revolutionized genetic and metabolic engineering. Cas9 endonuclease requires gRNA and PAM for targeted genome editing. Delivery of CRISPR/Cas9 component via Rhizobium is most commonly used. Different factors affecting genetic transformation, for example bacterial strains, bacterial density, co-cultivation duration, acetosyringone, explants type, and cultivars, were optimized for Indian potato. Wild-type Rhizobium rhizogenes strain A4 showed the highest (60.7 ± 6.9%) hairy root induction followed by ATCC15834 (53.4 ± 10.7%). The highest transformation efficiency (68.8 ± 6.8%) was achieved onto Murashige and Skoog medium (MS) supplemented with 0.5 mg $ L^{−1} $ naphthaleneacetic acid (NAA). The putative transgenic hairy roots were induced within 8 to 10 d on hygromycin-containing media. A4 strain carrying CRISPR/Cas9 vector was used to target the StbHLH47. A total of 32 possible target sites near to PAM region were identified in the 500-bp sequence of StbHLH47. The target site adjacent to the ATG start codon was selected, and the construct was prepared into the pHSE401 vector. PCR amplification of rolB, HptII, and Cas9 gene–specific primers showed the integration of CRISPR/Cas9 components in the potato genome. Transgenic hairy root lines showed 2 to 7 nucleotide deletion at the target site of StbHLH. The editing frequency was calculated to be 15.3% for StbHLH47. This study reports a fast and efficient protocol for hairy root induction by Rhizobium rhizogenes to analyze editing in the potato genome within 18 to 20 d. The developed protocol will be very useful for studying gene function using CRISPR in the area of root biology. Potato (dpeaa)DE-He213 Hairy roots (dpeaa)DE-He213 CRISPR/Cas9 (dpeaa)DE-He213 Genome editing (dpeaa)DE-He213 T7E1 assay (dpeaa)DE-He213 Chauhan, Hanny aut Kaushal, Nishant aut Singh, Kashmir (orcid)0000-0002-9646-9043 aut Enthalten in In vitro cellular & developmental biology Berlin : Springer, 1991 59(2022), 1 vom: 08. Dez., Seite 83-94 (DE-627)306365367 (DE-600)1497464-2 1475-2689 nnns volume:59 year:2022 number:1 day:08 month:12 pages:83-94 https://dx.doi.org/10.1007/s11627-022-10318-0 lizenzpflichtig Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_32 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_90 GBV_ILN_95 GBV_ILN_100 GBV_ILN_101 GBV_ILN_105 GBV_ILN_110 GBV_ILN_120 GBV_ILN_138 GBV_ILN_150 GBV_ILN_151 GBV_ILN_152 GBV_ILN_161 GBV_ILN_165 GBV_ILN_170 GBV_ILN_171 GBV_ILN_187 GBV_ILN_206 GBV_ILN_213 GBV_ILN_224 GBV_ILN_230 GBV_ILN_250 GBV_ILN_281 GBV_ILN_285 GBV_ILN_293 GBV_ILN_370 GBV_ILN_374 GBV_ILN_602 GBV_ILN_636 GBV_ILN_702 GBV_ILN_2001 GBV_ILN_2003 GBV_ILN_2004 GBV_ILN_2005 GBV_ILN_2006 GBV_ILN_2007 GBV_ILN_2008 GBV_ILN_2009 GBV_ILN_2010 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2015 GBV_ILN_2018 GBV_ILN_2020 GBV_ILN_2021 GBV_ILN_2025 GBV_ILN_2026 GBV_ILN_2027 GBV_ILN_2031 GBV_ILN_2034 GBV_ILN_2037 GBV_ILN_2038 GBV_ILN_2039 GBV_ILN_2044 GBV_ILN_2048 GBV_ILN_2049 GBV_ILN_2050 GBV_ILN_2055 GBV_ILN_2056 GBV_ILN_2057 GBV_ILN_2059 GBV_ILN_2061 GBV_ILN_2064 GBV_ILN_2065 GBV_ILN_2068 GBV_ILN_2088 GBV_ILN_2093 GBV_ILN_2106 GBV_ILN_2107 GBV_ILN_2108 GBV_ILN_2110 GBV_ILN_2111 GBV_ILN_2112 GBV_ILN_2113 GBV_ILN_2118 GBV_ILN_2122 GBV_ILN_2129 GBV_ILN_2143 GBV_ILN_2144 GBV_ILN_2147 GBV_ILN_2148 GBV_ILN_2152 GBV_ILN_2153 GBV_ILN_2188 GBV_ILN_2190 GBV_ILN_2232 GBV_ILN_2336 GBV_ILN_2446 GBV_ILN_2470 GBV_ILN_2472 GBV_ILN_2507 GBV_ILN_2522 GBV_ILN_2548 GBV_ILN_2939 GBV_ILN_2946 GBV_ILN_2949 GBV_ILN_2951 GBV_ILN_4012 GBV_ILN_4035 GBV_ILN_4037 GBV_ILN_4046 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4242 GBV_ILN_4246 GBV_ILN_4249 GBV_ILN_4251 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4326 GBV_ILN_4328 GBV_ILN_4333 GBV_ILN_4334 GBV_ILN_4335 GBV_ILN_4336 GBV_ILN_4338 GBV_ILN_4346 GBV_ILN_4393 GBV_ILN_4700 AR 59 2022 1 08 12 83-94 |
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Alok, Anshu |
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Alok, Anshu misc Potato misc Hairy roots misc CRISPR/Cas9 misc Genome editing misc T7E1 assay Rapid and efficient CRISPR/Cas9-mediated genome editing in potato via hairy root induction |
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Rapid and efficient CRISPR/Cas9-mediated genome editing in potato via hairy root induction Potato (dpeaa)DE-He213 Hairy roots (dpeaa)DE-He213 CRISPR/Cas9 (dpeaa)DE-He213 Genome editing (dpeaa)DE-He213 T7E1 assay (dpeaa)DE-He213 |
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Alok, Anshu Chauhan, Hanny Kaushal, Nishant Singh, Kashmir |
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rapid and efficient crispr/cas9-mediated genome editing in potato via hairy root induction |
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Rapid and efficient CRISPR/Cas9-mediated genome editing in potato via hairy root induction |
| abstract |
Abstract Potato is one of the most important crops, and its trait improvement through genetic engineering is of utmost need. CRISPR/Cas9-mediated genome editing tool and its advancement revolutionized genetic and metabolic engineering. Cas9 endonuclease requires gRNA and PAM for targeted genome editing. Delivery of CRISPR/Cas9 component via Rhizobium is most commonly used. Different factors affecting genetic transformation, for example bacterial strains, bacterial density, co-cultivation duration, acetosyringone, explants type, and cultivars, were optimized for Indian potato. Wild-type Rhizobium rhizogenes strain A4 showed the highest (60.7 ± 6.9%) hairy root induction followed by ATCC15834 (53.4 ± 10.7%). The highest transformation efficiency (68.8 ± 6.8%) was achieved onto Murashige and Skoog medium (MS) supplemented with 0.5 mg $ L^{−1} $ naphthaleneacetic acid (NAA). The putative transgenic hairy roots were induced within 8 to 10 d on hygromycin-containing media. A4 strain carrying CRISPR/Cas9 vector was used to target the StbHLH47. A total of 32 possible target sites near to PAM region were identified in the 500-bp sequence of StbHLH47. The target site adjacent to the ATG start codon was selected, and the construct was prepared into the pHSE401 vector. PCR amplification of rolB, HptII, and Cas9 gene–specific primers showed the integration of CRISPR/Cas9 components in the potato genome. Transgenic hairy root lines showed 2 to 7 nucleotide deletion at the target site of StbHLH. The editing frequency was calculated to be 15.3% for StbHLH47. This study reports a fast and efficient protocol for hairy root induction by Rhizobium rhizogenes to analyze editing in the potato genome within 18 to 20 d. The developed protocol will be very useful for studying gene function using CRISPR in the area of root biology. © The Society for In Vitro Biology 2022. Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. |
| abstractGer |
Abstract Potato is one of the most important crops, and its trait improvement through genetic engineering is of utmost need. CRISPR/Cas9-mediated genome editing tool and its advancement revolutionized genetic and metabolic engineering. Cas9 endonuclease requires gRNA and PAM for targeted genome editing. Delivery of CRISPR/Cas9 component via Rhizobium is most commonly used. Different factors affecting genetic transformation, for example bacterial strains, bacterial density, co-cultivation duration, acetosyringone, explants type, and cultivars, were optimized for Indian potato. Wild-type Rhizobium rhizogenes strain A4 showed the highest (60.7 ± 6.9%) hairy root induction followed by ATCC15834 (53.4 ± 10.7%). The highest transformation efficiency (68.8 ± 6.8%) was achieved onto Murashige and Skoog medium (MS) supplemented with 0.5 mg $ L^{−1} $ naphthaleneacetic acid (NAA). The putative transgenic hairy roots were induced within 8 to 10 d on hygromycin-containing media. A4 strain carrying CRISPR/Cas9 vector was used to target the StbHLH47. A total of 32 possible target sites near to PAM region were identified in the 500-bp sequence of StbHLH47. The target site adjacent to the ATG start codon was selected, and the construct was prepared into the pHSE401 vector. PCR amplification of rolB, HptII, and Cas9 gene–specific primers showed the integration of CRISPR/Cas9 components in the potato genome. Transgenic hairy root lines showed 2 to 7 nucleotide deletion at the target site of StbHLH. The editing frequency was calculated to be 15.3% for StbHLH47. This study reports a fast and efficient protocol for hairy root induction by Rhizobium rhizogenes to analyze editing in the potato genome within 18 to 20 d. The developed protocol will be very useful for studying gene function using CRISPR in the area of root biology. © The Society for In Vitro Biology 2022. Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. |
| abstract_unstemmed |
Abstract Potato is one of the most important crops, and its trait improvement through genetic engineering is of utmost need. CRISPR/Cas9-mediated genome editing tool and its advancement revolutionized genetic and metabolic engineering. Cas9 endonuclease requires gRNA and PAM for targeted genome editing. Delivery of CRISPR/Cas9 component via Rhizobium is most commonly used. Different factors affecting genetic transformation, for example bacterial strains, bacterial density, co-cultivation duration, acetosyringone, explants type, and cultivars, were optimized for Indian potato. Wild-type Rhizobium rhizogenes strain A4 showed the highest (60.7 ± 6.9%) hairy root induction followed by ATCC15834 (53.4 ± 10.7%). The highest transformation efficiency (68.8 ± 6.8%) was achieved onto Murashige and Skoog medium (MS) supplemented with 0.5 mg $ L^{−1} $ naphthaleneacetic acid (NAA). The putative transgenic hairy roots were induced within 8 to 10 d on hygromycin-containing media. A4 strain carrying CRISPR/Cas9 vector was used to target the StbHLH47. A total of 32 possible target sites near to PAM region were identified in the 500-bp sequence of StbHLH47. The target site adjacent to the ATG start codon was selected, and the construct was prepared into the pHSE401 vector. PCR amplification of rolB, HptII, and Cas9 gene–specific primers showed the integration of CRISPR/Cas9 components in the potato genome. Transgenic hairy root lines showed 2 to 7 nucleotide deletion at the target site of StbHLH. The editing frequency was calculated to be 15.3% for StbHLH47. This study reports a fast and efficient protocol for hairy root induction by Rhizobium rhizogenes to analyze editing in the potato genome within 18 to 20 d. The developed protocol will be very useful for studying gene function using CRISPR in the area of root biology. © The Society for In Vitro Biology 2022. Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. |
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<?xml version="1.0" encoding="UTF-8"?><collection xmlns="http://www.loc.gov/MARC21/slim"><record><leader>01000naa a22002652 4500</leader><controlfield tag="001">SPR049595733</controlfield><controlfield tag="003">DE-627</controlfield><controlfield tag="005">20230323103957.0</controlfield><controlfield tag="007">cr uuu---uuuuu</controlfield><controlfield tag="008">230323s2022 xx |||||o 00| ||eng c</controlfield><datafield tag="024" ind1="7" ind2=" "><subfield code="a">10.1007/s11627-022-10318-0</subfield><subfield code="2">doi</subfield></datafield><datafield tag="035" ind1=" " ind2=" "><subfield code="a">(DE-627)SPR049595733</subfield></datafield><datafield tag="035" ind1=" " ind2=" "><subfield code="a">(SPR)s11627-022-10318-0-e</subfield></datafield><datafield tag="040" ind1=" " ind2=" "><subfield code="a">DE-627</subfield><subfield code="b">ger</subfield><subfield code="c">DE-627</subfield><subfield code="e">rakwb</subfield></datafield><datafield tag="041" ind1=" " ind2=" "><subfield code="a">eng</subfield></datafield><datafield tag="100" ind1="1" ind2=" "><subfield code="a">Alok, Anshu</subfield><subfield code="e">verfasserin</subfield><subfield code="4">aut</subfield></datafield><datafield tag="245" ind1="1" ind2="0"><subfield code="a">Rapid and efficient CRISPR/Cas9-mediated genome editing in potato via hairy root induction</subfield></datafield><datafield tag="264" ind1=" " ind2="1"><subfield code="c">2022</subfield></datafield><datafield tag="336" ind1=" " ind2=" "><subfield code="a">Text</subfield><subfield code="b">txt</subfield><subfield code="2">rdacontent</subfield></datafield><datafield tag="337" ind1=" " ind2=" "><subfield code="a">Computermedien</subfield><subfield code="b">c</subfield><subfield code="2">rdamedia</subfield></datafield><datafield tag="338" ind1=" " ind2=" "><subfield code="a">Online-Ressource</subfield><subfield code="b">cr</subfield><subfield code="2">rdacarrier</subfield></datafield><datafield tag="500" ind1=" " ind2=" "><subfield code="a">© The Society for In Vitro Biology 2022. Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.</subfield></datafield><datafield tag="520" ind1=" " ind2=" "><subfield code="a">Abstract Potato is one of the most important crops, and its trait improvement through genetic engineering is of utmost need. CRISPR/Cas9-mediated genome editing tool and its advancement revolutionized genetic and metabolic engineering. Cas9 endonuclease requires gRNA and PAM for targeted genome editing. Delivery of CRISPR/Cas9 component via Rhizobium is most commonly used. Different factors affecting genetic transformation, for example bacterial strains, bacterial density, co-cultivation duration, acetosyringone, explants type, and cultivars, were optimized for Indian potato. Wild-type Rhizobium rhizogenes strain A4 showed the highest (60.7 ± 6.9%) hairy root induction followed by ATCC15834 (53.4 ± 10.7%). The highest transformation efficiency (68.8 ± 6.8%) was achieved onto Murashige and Skoog medium (MS) supplemented with 0.5 mg $ L^{−1} $ naphthaleneacetic acid (NAA). The putative transgenic hairy roots were induced within 8 to 10 d on hygromycin-containing media. A4 strain carrying CRISPR/Cas9 vector was used to target the StbHLH47. A total of 32 possible target sites near to PAM region were identified in the 500-bp sequence of StbHLH47. The target site adjacent to the ATG start codon was selected, and the construct was prepared into the pHSE401 vector. PCR amplification of rolB, HptII, and Cas9 gene–specific primers showed the integration of CRISPR/Cas9 components in the potato genome. Transgenic hairy root lines showed 2 to 7 nucleotide deletion at the target site of StbHLH. The editing frequency was calculated to be 15.3% for StbHLH47. This study reports a fast and efficient protocol for hairy root induction by Rhizobium rhizogenes to analyze editing in the potato genome within 18 to 20 d. 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