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CRISPR/Cas9-mediated gene targeting in Arabidopsis using sequential transformation
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CRISPR/Cas9-mediated gene targeting in Arabidopsis using sequential transformation
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Journal Article
CRISPR/Cas9-mediated gene targeting in Arabidopsis using sequential transformation
2018
Overview
Homologous recombination-based gene targeting is a powerful tool for precise genome modification and has been widely used in organisms ranging from yeast to higher organisms such as
Drosophila
and mouse. However, gene targeting in higher plants, including the most widely used model plant
Arabidopsis thaliana
, remains challenging. Here we report a sequential transformation method for gene targeting in
Arabidopsis
. We find that parental lines expressing the bacterial endonuclease Cas9 from the egg cell- and early embryo-specific
DD45
gene promoter can improve the frequency of single-guide RNA-targeted gene knock-ins and sequence replacements via homologous recombination at several endogenous sites in the
Arabidopsis
genome. These heritable gene targeting can be identified by regular PCR. Our approach enables routine and fine manipulation of the
Arabidopsis
genome.
Efficient gene targeting in higher plants remains challenging. Here, the authors develop a sequential transformation method for CRISPR/Cas9-mediated gene targeting in
Arabidopsis
and demonstrate its functionality at five genomic sites in two endogenous loci.
Publisher
Nature Publishing Group UK,Nature Publishing Group,Nature Portfolio
Subject
/ 45/23
/ 49
/ Amino Acid Substitution - genetics
/ Arabidopsis Proteins - genetics
/ CRISPR
/ Gene Knock-In Techniques - methods
/ Genomes
/ Homology
/ Humanities and Social Sciences
/ Plants, Genetically Modified - genetics
/ RNA
/ RNA, Guide, CRISPR-Cas Systems - genetics
/ Science
/ Transformation, Genetic - genetics
/ Yeast
