Li Jian-Feng, Zhang Dandan, Sheen Jen
Massachusetts General Hospital, Boston, MA, USA,
Methods Mol Biol. 2015;1284:239-55. doi: 10.1007/978-1-4939-2444-8_12.
Targeted modification of plant genome is key for elucidating and manipulating gene functions in basic and applied plant research. The CRISPR (clustered regularly interspaced short palindromic repeats)/CRISPR-associated protein (Cas) technology is emerging as a powerful genome editing tool in diverse organisms. This technology utilizes an easily reprogrammable guide RNA (gRNA) to guide Streptococcus pyogenes Cas9 endonuclease to generate a DNA double-strand break (DSB) within an intended genomic sequence and subsequently stimulate chromosomal mutagenesis or homologous recombination near the DSB site through cellular DNA repair machineries. In this chapter, we describe the detailed procedure to design, construct, and evaluate dual gRNAs for plant codon-optimized Cas9 (pcoCas9)-mediated genome editing using Arabidopsis thaliana and Nicotiana benthamiana protoplasts as model cellular systems. We also discuss strategies to apply the CRISPR/Cas9 system to generating targeted genome modifications in whole plants.
在基础和应用植物研究中，对植物基因组进行靶向修饰是阐明和操纵基因功能的关键。CRISPR（成簇规律间隔短回文重复序列）/CRISPR相关蛋白（Cas）技术正成为一种在多种生物体中强大的基因组编辑工具。该技术利用易于重新编程的向导RNA（gRNA）引导化脓性链球菌Cas9核酸内切酶在预期的基因组序列内产生DNA双链断裂（DSB），随后通过细胞DNA修复机制刺激DSB位点附近的染色体诱变或同源重组。在本章中，我们描述了以拟南芥和本氏烟草原生质体作为模型细胞系统，设计、构建和评估用于植物密码子优化的Cas9（pcoCas9）介导的基因组编辑的双gRNA的详细程序。我们还讨论了将CRISPR/Cas9系统应用于在整株植物中产生靶向基因组修饰的策略。
