Repositorium

What is a repositorium?

The repositorium is a searchable database that provides data on relevant articles from journals, company web pages and web pages of governmental agencies about studies/applications of genome-editing in model plants and agricultural crops in the period January 1996 to May 2018. Search options are article type, technique, plant, traits or free text. The repositorium is based on the systematic map of Dominik Modrzejewski et al., published in the journal environmental evidence. (Download article PDF).

Site-directed mutagenesis in Arabidopsis thaliana using dividing tissue-targeted RGEN of the CRISPR/Cas system to generate heritable null alleles


Typ / Jahr

Journal Article / 2015

Autoren

Hyun, Youbong; Kim, Jungeun; Cho, Seung Woo; Choi, Yeonhee; Kim, Jin-Soo; Coupland, George

Abstract

Dividing tissue-targeted site-directed mutagenesis using RGEN of CRISPR/Cas system produces heritable mutations in Arabidopsis thaliana. Site-directed genome engineering in higher plants has great potential for basic research and molecular breeding. Here, we describe a method for site-directed mutagenesis of the Arabidopsis nuclear genome that efficiently generates heritable mutations using the RNA-guided endonuclease (RGEN) derived from bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 (CRISPR associated) protein system. To induce mutagenesis in proliferating tissues during embryogenesis and throughout the plant life cycle, the single guide RNA (sgRNA) and Cas9 DNA endonuclease were expressed from the U6 snRNA and INCURVATA2 promoters, respectively. After Agrobacterium-mediated introduction of T-DNAs encoding RGENs that targets FLOWERING LOCUS T (FT) and SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 4 genes, somatic mutagenesis at the targeted loci was observed in T1 transformants. In the results of FT-RGEN, T1 plants often showed late flowering indicative of the presence of large somatic sectors in which the FT gene is mutated on both chromosomes. DNA sequencing analysis estimated that about 90 % of independent chromosomal DNA fragments carried mutations in the analyzed tissue of a T1 plant showing late flowering. The most frequently detected somatic polymorphism showed a high rate of inheritance in T2 plants, and inheritance of less frequent polymorphisms was also observed. As a result, late-flowering plants homozygous for novel, heritable null alleles of FT including a 1 bp insertion or short deletions were recovered in the following T2 and T3 generations. Our results demonstrate that dividing tissue-targeted mutagenesis using RGEN provides an efficient heritable genome engineering method in A. thaliana.

Keywords
CRISPR/Cas system; ICU2; RGEN; Site-directed mutagenesis
Periodical
Planta
Periodical Number
1
Page range
271–284
Volume
241
DOI
10.1007/s00425-014-2180-5

Techniques

ID Corresponding Author
Country
Plant Species GE Technique
Sequence Identifier
Trait
Type of Alteration
Progress in Research
Key Topic
153 Coupland, George
Germany
Arabidopsis thaliana CRISPR/Cas9
SPL4
No information
SDN1
Basic research
Basic research
154 Coupland, George
Germany
Arabidopsis thaliana CRISPR/Cas9
FT
Late flowering/ flowering time/ flowering induction
SDN1
Basic research
Basic research