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CRISPR-finder: A high throughput and cost effective method for identifying successfully edited A. thaliana individuals

View ORCID ProfileEfthymia Symeonidi, View ORCID ProfileJulian Regalado, View ORCID ProfileRebecca Schwab, View ORCID ProfileDetlef Weigel
doi: https://doi.org/10.1101/2020.06.25.171538
Efthymia Symeonidi
1Max Planck Institute for Developmental Biology, Department of Molecular Biology, Max-Planck-Ring 5, 72076 Tübingen, Germany
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Julian Regalado
1Max Planck Institute for Developmental Biology, Department of Molecular Biology, Max-Planck-Ring 5, 72076 Tübingen, Germany
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Rebecca Schwab
1Max Planck Institute for Developmental Biology, Department of Molecular Biology, Max-Planck-Ring 5, 72076 Tübingen, Germany
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Detlef Weigel
1Max Planck Institute for Developmental Biology, Department of Molecular Biology, Max-Planck-Ring 5, 72076 Tübingen, Germany
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  • For correspondence: weigel@weigelworld.org
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Abstract

Background Genome editing with the CRISPR/Cas9 system allows the user to mutate a targeted region of the genome using an endonuclease (Cas9) and an artificial single-guide RNA (sgRNA). Both because of variable efficiency with which such mutations arise and because the repair process produces a spectrum of mutations, one needs to ascertain the genome sequence at the targeted locus for many individuals that have been subjected to CRISPR/Cas9 mutagenesis. This process can be laborious, expensive and inefficient with conventional methods such as the T7E1 assay or Sanger sequencing. An alternative comprises methods for amplicon sequencing, but most available protocols do not include a facile way for high throughput generation of the samples for sequencing.

Results In this study we provide a full pipeline based on amplicon sequencing, CRISPR-finder. We provide a complete protocol for the generation of amplicons up until the identification of the exact mutations in the targeted region. CRISPR-finder can be used to process thousands of individuals in a single sequencing run. For example, we were able to analyze in one sequencing reaction over 900 Arabidopsis thaliana individuals whose genomes had been targeted with the CRISPR/Cas9 system.

Conclusions In order to validate the potential of CRISPR-finder, we targeted the ISOCHORISMATE SYNTHASE 1 gene in A. thaliana using the CRISPR/Cas9 system. We successfully identified a mutant line in which the production of salicylic acid was impaired compared to the wild type, as expected. These features establish CRISPR-finder as a high-throughput, cost-effective and -efficient genotyping method of individuals whose genomes have been targeted using the CRISPR/Cas9 system.

Competing Interest Statement

The authors have declared no competing interest.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.
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Posted June 25, 2020.
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CRISPR-finder: A high throughput and cost effective method for identifying successfully edited A. thaliana individuals
Efthymia Symeonidi, Julian Regalado, Rebecca Schwab, Detlef Weigel
bioRxiv 2020.06.25.171538; doi: https://doi.org/10.1101/2020.06.25.171538
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CRISPR-finder: A high throughput and cost effective method for identifying successfully edited A. thaliana individuals
Efthymia Symeonidi, Julian Regalado, Rebecca Schwab, Detlef Weigel
bioRxiv 2020.06.25.171538; doi: https://doi.org/10.1101/2020.06.25.171538

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