Targeted plant genome editing via the CRISPR/Cas9 technology

Methods in Molecular Biology
Jian-Feng LiJen Sheen

Abstract

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.

Citations

Feb 24, 2016·Bioengineered·Huimin FangJi Huang
Sep 18, 2017·Functional & Integrative Genomics·Dongjin KimHikmet Budak
Nov 19, 2015·The Plant Journal : for Cell and Molecular Biology·Jeannette SteinertHolger Puchta
Aug 18, 2018·The Biochemical Journal·Shuguo HouPing He
Mar 5, 2017·Plant Molecular Biology·Madhusudhana R JangaKeerti S Rathore

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