Biolistic Delivery of CRISPR/Cas9 with Ribonucleoprotein Complex in Wheat

Zhen Liang; Kunling Chen; Caixia Gao

doi:10.1007/978-1-4939-8991-1_24

Biolistic Delivery of CRISPR/Cas9 with Ribonucleoprotein Complex in Wheat

Methods Mol Biol. 2019:1917:327-335. doi: 10.1007/978-1-4939-8991-1_24.

Authors

Zhen Liang^#^{1

2}, Kunling Chen^#¹, Caixia Gao³

Affiliations

¹ State Key Laboratory of Plant Cell and Chromosome Engineering, Center for Genome Editing, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China.
² University of Chinese Academy of Sciences, Beijing, China.
³ State Key Laboratory of Plant Cell and Chromosome Engineering, Center for Genome Editing, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China. cxgao@genetics.ac.cn.

^# Contributed equally.

PMID: 30610647
DOI: 10.1007/978-1-4939-8991-1_24

Abstract

The great advances in exploiting the CRISPR/Cas9 system are paving the way for targeted genome engineering in plants. Genome editing by direct delivery of CRISPR/Cas9 ribonucleoprotein complexes (RNPs) into plant cells reduces off-target mutations and avoids the integration of foreign DNA fragments, thus providing an efficient and accurate method for precision crop breeding. Here we describe an RNP-based genome editing protocol for wheat. The protocol covers the in vitro transcription of sgRNA, purification of Cas9 protein, biolistic delivery of CRISPR/Cas9 RNPs, and tissue culture procedures for regenerating testable seedlings.

Keywords: Biolistic delivery; CRISPR/Cas9; Ribonucleoprotein; Wheat.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Biolistics / methods*
CRISPR-Cas Systems / genetics
CRISPR-Cas Systems / physiology*
Gene Editing / methods
Seedlings / genetics
Triticum / genetics*