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

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 simple 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 the 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 that no Cas9 DNA could be detected in the majority of the resulting plants.

同研究人員:Choung-Sea Lin

Ming-Che Shih

Corresponding Research Fellow

(02) 2787-2041

mcshih@gate.sinica.edu.tw

Lab.

Tel: (02) 2787-2039

2017 ~ Current, Distinguished Research Fellow and Chair, Academia Sinica Southern Campus Planning Commission
2016 ~ 2017, Distinguished Research Fellow and Secretary General of Academia Sinica
2008 ~ 2016, Distinguished Research Fellow and Director
2003 ~ 2008, Professor, Dept. of Biological Sciences, University of Iowa, USA
2005 ~ 2007, Director, Center for Comparative Genomics, University of Iowa, USA
1994 ~ 2003, Associate Professor, Dept. of Biological Sciences, University of Iowa, USA
1988 ~ 1994, Assistant Professor, Dept. of Biological Sciences, University of Iowa, USA
1984 ~ 1988, Postdoctoral Fellow Dept. of Genetics, Harvard Medical School, USA
1978 ~ 1983, Ph.D. Genetics Ph.D. Program, University of Iowa, USA