Optimized Cas9 expression systems for highly efficient Arabidopsis genome editing facilitate isolation of complex alleles in a single generation

Functional & Integrative Genomics
Jana OrdonJohannes Stuttmann

Abstract

Genetic resources for the model plant Arabidopsis comprise mutant lines defective in almost any single gene in reference accession Columbia. However, gene redundancy and/or close linkage often render it extremely laborious or even impossible to isolate a desired line lacking a specific function or set of genes from segregating populations. Therefore, we here evaluated strategies and efficiencies for the inactivation of multiple genes by Cas9-based nucleases and multiplexing. In first attempts, we succeeded in isolating a mutant line carrying a 70 kb deletion, which occurred at a frequency of ~ 1.6% in the T2 generation, through PCR-based screening of numerous individuals. However, we failed to isolate a line lacking Lhcb1 genes, which are present in five copies organized at two loci in the Arabidopsis genome. To improve efficiency of our Cas9-based nuclease system, regulatory sequences controlling Cas9 expression levels and timing were systematically compared. Indeed, use of DD45 and RPS5a promoters improved efficiency of our genome editing system by approximately 25-30-fold in comparison to the previous ubiquitin promoter. Using an optimized genome editing system with RPS5a promoter-driven Cas9, putatively quintuple mutant lin...Continue Reading

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Citations

Feb 26, 2021·The Plant Journal : for Cell and Molecular Biology·Jana OrdonJohannes Stuttmann
Feb 13, 2021·The Plant Journal : for Cell and Molecular Biology·Johannes StuttmannUlla Bonas
Apr 27, 2021·Plant Communications·Ramona GrütznerSylvestre Marillonnet
Aug 4, 2021·Trends in Plant Science·Md Mahmudul HassanXiaohan Yang
Oct 1, 2021·Plant Cell Reports·Zhifen ZhangPeggy Ozias-Akins

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Methods Mentioned

BETA
PCR
transgenic

Software Mentioned

Modular
sgRNA

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