Application of protoplast technology to CRISPR/Cas9 mutagenesis: from single-cell mutation detection to mutant plant regeneration

Choun Sea Lin; Chen Tran Hsu; Ling Hung Yang; Lan Ying Lee; Jin Yuan Fu; Qiao Wei Cheng; Fu Hui Wu; Han C.W. Hsiao; Yesheng Zhang; Ru Zhang; Wan Jung Chang; Chen Ting Yu; Wen Wang; Li Jen Liao; Stanton B. Gelvin; Ming Che Shih

doi:10.1111/pbi.12870

Application of protoplast technology to CRISPR/Cas9 mutagenesis: from single-cell mutation detection to mutant plant regeneration

Choun Sea Lin, Chen Tran Hsu, Ling Hung Yang, Lan Ying Lee, Jin Yuan Fu, Qiao Wei Cheng, Fu Hui Wu, Han C.W. Hsiao, Yesheng Zhang, Ru Zhang, Wan Jung Chang, Chen Ting Yu, Wen Wang, Li Jen Liao, Stanton B. Gelvin, Ming Che Shih

Research output: Contribution to journal › Article › peer-review

265 Scopus citations

Abstract

Plant protoplasts are useful for assessing the efficiency of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) mutagenesis. We improved the process of protoplast isolation and transfection of several plant species. We also developed a method to isolate and regenerate single mutagenized Nicotianna tabacum protoplasts into mature plants. Following transfection of protoplasts with constructs encoding Cas9 and sgRNAs, target gene DNA could be amplified for further analysis to determine mutagenesis efficiency. We investigated N. tabacum protoplasts and derived regenerated plants for targeted mutagenesis of the phytoene desaturase (NtPDS) gene. Genotyping of albino regenerants indicated that all four NtPDS alleles were mutated in amphidiploid tobacco, and no Cas9 DNA could be detected in most regenerated plants.

Original language	English
Pages (from-to)	1295-1310
Number of pages	16
Journal	Plant Biotechnology Journal
Volume	16
Issue number	7
DOIs	https://doi.org/10.1111/pbi.12870
State	Published - Jul 2018
Externally published	Yes

Keywords

CRISPR/Cas9
protoplast isolation
protoplast regeneration
single-cell analysis

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10.1111/pbi.12870

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Lin, C. S., Hsu, C. T., Yang, L. H., Lee, L. Y., Fu, J. Y., Cheng, Q. W., Wu, F. H., Hsiao, H. C. W., Zhang, Y., Zhang, R., Chang, W. J., Yu, C. T., Wang, W., Liao, L. J., Gelvin, S. B., & Shih, M. C. (2018). Application of protoplast technology to CRISPR/Cas9 mutagenesis: from single-cell mutation detection to mutant plant regeneration. Plant Biotechnology Journal, 16(7), 1295-1310. https://doi.org/10.1111/pbi.12870

@article{15ea34f8bc97483691ce42019e1d4a47,

title = "Application of protoplast technology to CRISPR/Cas9 mutagenesis: from single-cell mutation detection to mutant plant regeneration",

abstract = "Plant protoplasts are useful for assessing the efficiency of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) mutagenesis. We improved the process of protoplast isolation and transfection of several plant species. We also developed a method to isolate and regenerate single mutagenized Nicotianna tabacum protoplasts into mature plants. Following transfection of protoplasts with constructs encoding Cas9 and sgRNAs, target gene DNA could be amplified for further analysis to determine mutagenesis efficiency. We investigated N. tabacum protoplasts and derived regenerated plants for targeted mutagenesis of the phytoene desaturase (NtPDS) gene. Genotyping of albino regenerants indicated that all four NtPDS alleles were mutated in amphidiploid tobacco, and no Cas9 DNA could be detected in most regenerated plants.",

keywords = "CRISPR/Cas9, protoplast isolation, protoplast regeneration, single-cell analysis",

author = "Lin, {Choun Sea} and Hsu, {Chen Tran} and Yang, {Ling Hung} and Lee, {Lan Ying} and Fu, {Jin Yuan} and Cheng, {Qiao Wei} and Wu, {Fu Hui} and Hsiao, {Han C.W.} and Yesheng Zhang and Ru Zhang and Chang, {Wan Jung} and Yu, {Chen Ting} and Wen Wang and Liao, {Li Jen} and Gelvin, {Stanton B.} and Shih, {Ming Che}",

note = "Publisher Copyright: {\textcopyright} 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.",

year = "2018",

month = jul,

doi = "10.1111/pbi.12870",

language = "英语",

volume = "16",

pages = "1295--1310",

journal = "Plant Biotechnology Journal",

issn = "1467-7644",

publisher = "Wiley-Blackwell Publishing Ltd",

number = "7",

}

Lin, CS, Hsu, CT, Yang, LH, Lee, LY, Fu, JY, Cheng, QW, Wu, FH, Hsiao, HCW, Zhang, Y, Zhang, R, Chang, WJ, Yu, CT, Wang, W, Liao, LJ, Gelvin, SB & Shih, MC 2018, 'Application of protoplast technology to CRISPR/Cas9 mutagenesis: from single-cell mutation detection to mutant plant regeneration', Plant Biotechnology Journal, vol. 16, no. 7, pp. 1295-1310. https://doi.org/10.1111/pbi.12870

TY - JOUR

T1 - Application of protoplast technology to CRISPR/Cas9 mutagenesis

T2 - from single-cell mutation detection to mutant plant regeneration

AU - Lin, Choun Sea

AU - Hsu, Chen Tran

AU - Yang, Ling Hung

AU - Lee, Lan Ying

AU - Fu, Jin Yuan

AU - Cheng, Qiao Wei

AU - Wu, Fu Hui

AU - Hsiao, Han C.W.

AU - Zhang, Yesheng

AU - Zhang, Ru

AU - Chang, Wan Jung

AU - Yu, Chen Ting

AU - Wang, Wen

AU - Liao, Li Jen

AU - Gelvin, Stanton B.

AU - Shih, Ming Che

PY - 2018/7

Y1 - 2018/7

N2 - Plant protoplasts are useful for assessing the efficiency of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) mutagenesis. We improved the process of protoplast isolation and transfection of several plant species. We also developed a method to isolate and regenerate single mutagenized Nicotianna tabacum protoplasts into mature plants. Following transfection of protoplasts with constructs encoding Cas9 and sgRNAs, target gene DNA could be amplified for further analysis to determine mutagenesis efficiency. We investigated N. tabacum protoplasts and derived regenerated plants for targeted mutagenesis of the phytoene desaturase (NtPDS) gene. Genotyping of albino regenerants indicated that all four NtPDS alleles were mutated in amphidiploid tobacco, and no Cas9 DNA could be detected in most regenerated plants.

AB - Plant protoplasts are useful for assessing the efficiency of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) mutagenesis. We improved the process of protoplast isolation and transfection of several plant species. We also developed a method to isolate and regenerate single mutagenized Nicotianna tabacum protoplasts into mature plants. Following transfection of protoplasts with constructs encoding Cas9 and sgRNAs, target gene DNA could be amplified for further analysis to determine mutagenesis efficiency. We investigated N. tabacum protoplasts and derived regenerated plants for targeted mutagenesis of the phytoene desaturase (NtPDS) gene. Genotyping of albino regenerants indicated that all four NtPDS alleles were mutated in amphidiploid tobacco, and no Cas9 DNA could be detected in most regenerated plants.

KW - CRISPR/Cas9

KW - protoplast isolation

KW - protoplast regeneration

KW - single-cell analysis

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U2 - 10.1111/pbi.12870

DO - 10.1111/pbi.12870

M3 - 文章

C2 - 29230929

AN - SCOPUS:85040249952

SN - 1467-7644

VL - 16

SP - 1295

EP - 1310

JO - Plant Biotechnology Journal

JF - Plant Biotechnology Journal

IS - 7

ER -

Application of protoplast technology to CRISPR/Cas9 mutagenesis: from single-cell mutation detection to mutant plant regeneration

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