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


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Article Info

DATE

2019-02-23

AUTHORS

Jana Ordon, Mauro Bressan, Carola Kretschmer, Luca Dall’Osto, Sylvestre Marillonnet, Roberto Bassi, Johannes 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 lines lacking detectable amounts of Lhcb1 protein represented approximately 30% of T1 transformants. These results show how improved genome editing systems facilitate the isolation of complex mutant alleles, previously considered impossible to generate, at high frequency even in a single (T1) generation. More... »

PAGES

151-162

References to SciGraph publications

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  • 2013-08-20. Efficient genome editing in plants using a CRISPR/Cas system in CELL RESEARCH
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  • 2017-04-24. Plant genome editing with TALEN and CRISPR in CELL & BIOSCIENCE
  • 2015-07-21. Egg cell-specific promoter-controlled CRISPR/Cas9 efficiently generates homozygous mutants for multiple target genes in Arabidopsis in a single generation in GENOME BIOLOGY
  • 2014-09-03. Rational design of highly active sgRNAs for CRISPR-Cas9–mediated gene inactivation in NATURE BIOTECHNOLOGY
  • 2016-01-18. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9 in NATURE BIOTECHNOLOGY
  • 2017-01-18. Efficient DNA-free genome editing of bread wheat using CRISPR/Cas9 ribonucleoprotein complexes in NATURE COMMUNICATIONS
  • 2014-10-01. Site-directed mutagenesis in Arabidopsis thaliana using dividing tissue-targeted RGEN of the CRISPR/Cas system to generate heritable null alleles in PLANTA
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  • 2004-03. Crystal structure of spinach major light-harvesting complex at 2.72 Å resolution in NATURE
  • Identifiers

    URI

    http://scigraph.springernature.com/pub.10.1007/s10142-019-00665-4

    DOI

    http://dx.doi.org/10.1007/s10142-019-00665-4

    DIMENSIONS

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    PUBMED

    https://www.ncbi.nlm.nih.gov/pubmed/30796544


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