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

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.

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Acknowledgements

We acknowledge Bianca Rosinsky for taking care of plant growth facilities and growing plants. Ulla Bonas is acknowledged for generous support.

Funding information

This work was funded by GRC grant STU 642-1/1 (Deutsche Forschungsgemeinschaft, DFG) and seed funding by the CRC 648 (DFG) to Johannes Stuttmann. Mauro Bressan and Luca Dall’Osto were supported by University of Verona (Program CooperInt2017 and HuntingLight Ricerca di Base 2015).

Author information

JS and JO conceived the work, performed experiments, and analyzed data. CK performed experiments. MB, LD’O and RB conceived Lhcb1-related experiments, and MB performed experiments. JS wrote the manuscript with contributions from all authors.

Correspondence to Johannes Stuttmann.

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Online Resource 1
figure5

Architecture and functional verification of an adaptable, GUS-based nuclease activity reporter. (a) T-DNA region of the adaptable reporter plasmid, and cloning of user-defined target sequences. The “empty” plasmid contains a 35S-driven GUS, with a BsmBI-excisable ccdB cassette inserted between the initiating ATG and the GUS coding sequence. In a BsmBI Golden Gate reaction, the ccdB cassette is exchanged for a user-defined target sequence introduced as hybridzed oligonucleotides. A configuration in which the reporter detects a -1 nt repair event (or e.g. -4 or +2 nt events) is shown as example. The reporter may be adapted to detect different events by varying the length of the introduced target site. It should be noted that the introduced target site may not, after repair, contain an in-frame STOP codon. (b) Functional verification of the GUS-based reporter. Two different target sites were introduced into the adaptable plasmid to obtain Reporters 1/2, reporters were co-expressed with respective nucleases in N. benth., and GUS activity visualized by X-Gluc 3 dpi. Nuclease/reporter combination 2 consistently showed stronger GUS activity. (PNG 501 kb)

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Online Resource 2

Sequence details on nuclease and reporter constructs used in this study (annotated GenBank files) (GBK 969 kb)

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Ordon, J., Bressan, M., Kretschmer, C. et al. Optimized Cas9 expression systems for highly efficient Arabidopsis genome editing facilitate isolation of complex alleles in a single generation. Funct Integr Genomics 20, 151–162 (2020). https://doi.org/10.1007/s10142-019-00665-4

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Keywords

  • SpCas9
  • Arabidopsis
  • Promoter
  • FAST marker
  • Lhcb1
  • RPS5a