Cas9-based genome editing in Arabidopsis and tobacco.
Targeted modification of plant genome is key to elucidating and manipulating gene functions in plant research and biotechnology. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) technology is emerging as a powerful genome-editing method in diverse plants that traditionally lacked facile and versatile tools for targeted genetic engineering. This technology utilizes easily reprogrammable guide RNAs (sgRNAs) to direct Streptococcus pyogenes Cas9 endonuclease to generate DNA double-stranded breaks in targeted genome sequences, which facilitates efficient mutagenesis by error-prone nonhomologous end-joining (NHEJ) or sequence replacement by homology-directed repair (HDR). In this chapter, we describe the procedure to design and evaluate dual sgRNAs for plant codon-optimized Cas9-mediated genome editing using mesophyll protoplasts as model cell systems in Arabidopsis thaliana and Nicotiana benthamiana. We also discuss future directions in sgRNA/Cas9 applications for generating targeted genome modifications and gene regulations in plants.
Department of Molecular Biology, Centre for Computational and Integrative Biology, Massachusetts General Hospital, Boston, Massachusetts, USA; Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA. Electronic address: lijfeng3@mail.sysu.edu.cn.
Jian-Feng
JF
Li
Department of Molecular Biology, Centre for Computational and Integrative Biology, Massachusetts General Hospital, Boston, Massachusetts, USA; Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.
Dandan
D
Zhang
Department of Molecular Biology, Centre for Computational and Integrative Biology, Massachusetts General Hospital, Boston, Massachusetts, USA; Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.
Jen
J
Sheen
D017360:Arabidopsis / Q000235:genetics; D001483:Base Sequence; D064130:CRISPR-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism*; D005818:Genetic Engineering / Q000379:methods; D018745:Genome, Plant; D059765:Homologous Recombination; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011523:Protoplasts / Q000378:metabolism; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics / Q000378:metabolism; D013297:Streptococcus pyogenes / Q000201:enzymology* / Q000235:genetics; D014026:Nicotiana / Q000235:genetics
D064130:CRISPR-Associated Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems; D004720:Endonucleases
Arabidopsis thaliana; CRISPR/Cas9; Chromosomal mutagenesis; Dual guide RNAs; Homologous recombination; Nicotiana benthamiana; Plant genome editing; Protoplast transient expression assay