Development of germ-line-specific CRISPR-Cas9 systems to improve the production of heritable gene modifications in Arabidopsis

Mao, Yanfei, Zhang, Zhengjing, Feng, Zhengyan, Wei, Pengliang, Zhang, Hui, Botella, Jose Ramon and Zhu, Jian-Kang (2015) Development of germ-line-specific CRISPR-Cas9 systems to improve the production of heritable gene modifications in Arabidopsis. Plant Biotechnology Journal, 14 2: 519-532. doi:10.1111/pbi.12468


Author Mao, Yanfei
Zhang, Zhengjing
Feng, Zhengyan
Wei, Pengliang
Zhang, Hui
Botella, Jose Ramon
Zhu, Jian-Kang
Title Development of germ-line-specific CRISPR-Cas9 systems to improve the production of heritable gene modifications in Arabidopsis
Journal name Plant Biotechnology Journal   Check publisher's open access policy
ISSN 1467-7644
1467-7652
Publication date 2015-01-01
Year available 2015
Sub-type Article (original research)
DOI 10.1111/pbi.12468
Open Access Status DOI
Volume 14
Issue 2
Start page 519
End page 532
Total pages 14
Place of publication Oxford, United Kingdom
Publisher Wiley-Blackwell Publishing
Language eng
Subject 1305 Biotechnology
1102 Agronomy and Crop Science
1110 Plant Science
Abstract The Streptococcus-derived CRISPR/Cas9 system is being widely used to perform targeted gene modifications in plants. This customized endonuclease system has two components, the single-guide RNA (sgRNA) for target DNA recognition and the CRISPR-associated protein 9 (Cas9) for DNA cleavage. Ubiquitously expressed CRISPR/Cas9 systems (UC) generate targeted gene modifications with high efficiency but only those produced in reproductive cells are transmitted to the next generation. We report the design and characterization of a germ-line-specific Cas9 system (GSC) for Arabidopsis gene modification in male gametocytes, constructed using a SPOROCYTELESS (SPL) genomic expression cassette. Four loci in two endogenous genes were targeted by both systems for comparative analysis. Mutations generated by the GSC system were rare in T1 plants but were abundant (30%) in the T2 generation. The vast majority (70%) of the T2 mutant population generated using the UC system were chimeras while the newly developed GSC system produced only 29% chimeras, with 70% of the T2 mutants being heterozygous. Analysis of two loci in the T2 population showed that the abundance of heritable gene mutations was 37% higher in the GSC system compared to the UC system and the level of polymorphism of the mutations was also dramatically increased with the GSC system. Two additional systems based on germ-line-specific promoters (pDD45-GT and pLAT52-GT) were also tested, and one of them was capable of generating heritable homozygous T1 mutant plants. Our results suggest that future application of the described GSC system will facilitate the screening for targeted gene modifications, especially lethal mutations in the T2 population.
Keyword Arabidopsis
CRISPR Cas9
Gene modification
Germ line specific
Q-Index Code C1
Q-Index Status Confirmed Code
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
Collections: School of Agriculture and Food Sciences
Official 2016 Collection
 
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