A dual sgRNA approach for functional genomics in Arabidopsis thaliana
- Author
- Laurens Pauwels (UGent) , Rebecca De Clercq (UGent) , Jonas Goossens (UGent) , Sabrina Iñigo (UGent) , Clara Williams, Mily Ron, Anne Britt and Alain Goossens (UGent)
- Organization
- Abstract
- Reverse genetics uses loss-of-function alleles to interrogate gene function. The advent of CRISPR/Cas9-based gene editing now allows the generation of knock-out alleles for any gene and entire gene families. Even in the model plant Arabidopsis thaliana, gene editing is welcomed as T-DNA insertion lines do not always generate null alleles. Here, we show efficient generation of heritable mutations in Arabidopsis using CRISPR/Cas9 with a workload similar to generating overexpression lines. We obtain for several different genes Cas9 null-segregants with bi-allelic mutations in the T2 generation. While somatic mutations were predominantly generated by the canonical non-homologous end joining (cNHEJ) pathway, we observed inherited mutations that were the result of synthesis-dependent microhomology-mediated end joining (SD-MMEJ), a repair pathway linked to polymerase theta (PolQ). We also demonstrate that our workflow is compatible with a dual sgRNA approach in which a gene is targeted by two sgRNAs simultaneously. This paired nuclease method results in more reliable loss-of-function alleles that lack a large essential part of the gene. The ease of the CRISPR/Cas9 workflow should help in the eventual generation of true null alleles of every gene in the Arabidopsis genome, which will advance both basic and applied plant research.
- Keywords
- STRAND BREAK REPAIR, SOMATIC PLANT-CELLS, MESSENGER-RNA DECAY, HIGH-EFFICIENCY, TARGETED MUTAGENESIS, JASMONATE RESPONSES, CRISPR/CAS9, SYSTEM, GENE MODIFICATIONS, OFF-TARGET, IN-PLANTA, Arabidopsis thaliana, genome engineering, genome editing, RNA-guided, nuclease, alt-EJ, polymerase theta, CRISPR/Cas9
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Citation
Please use this url to cite or link to this publication: http://hdl.handle.net/1854/LU-8577908
- MLA
- Pauwels, Laurens, et al. “A Dual SgRNA Approach for Functional Genomics in Arabidopsis Thaliana.” G3-GENES GENOMES GENETICS, vol. 8, no. 8, 2018, pp. 2603–15, doi:10.1534/g3.118.200046.
- APA
- Pauwels, L., De Clercq, R., Goossens, J., Iñigo, S., Williams, C., Ron, M., … Goossens, A. (2018). A dual sgRNA approach for functional genomics in Arabidopsis thaliana. G3-GENES GENOMES GENETICS, 8(8), 2603–2615. https://doi.org/10.1534/g3.118.200046
- Chicago author-date
- Pauwels, Laurens, Rebecca De Clercq, Jonas Goossens, Sabrina Iñigo, Clara Williams, Mily Ron, Anne Britt, and Alain Goossens. 2018. “A Dual SgRNA Approach for Functional Genomics in Arabidopsis Thaliana.” G3-GENES GENOMES GENETICS 8 (8): 2603–15. https://doi.org/10.1534/g3.118.200046.
- Chicago author-date (all authors)
- Pauwels, Laurens, Rebecca De Clercq, Jonas Goossens, Sabrina Iñigo, Clara Williams, Mily Ron, Anne Britt, and Alain Goossens. 2018. “A Dual SgRNA Approach for Functional Genomics in Arabidopsis Thaliana.” G3-GENES GENOMES GENETICS 8 (8): 2603–2615. doi:10.1534/g3.118.200046.
- Vancouver
- 1.Pauwels L, De Clercq R, Goossens J, Iñigo S, Williams C, Ron M, et al. A dual sgRNA approach for functional genomics in Arabidopsis thaliana. G3-GENES GENOMES GENETICS. 2018;8(8):2603–15.
- IEEE
- [1]L. Pauwels et al., “A dual sgRNA approach for functional genomics in Arabidopsis thaliana,” G3-GENES GENOMES GENETICS, vol. 8, no. 8, pp. 2603–2615, 2018.
@article{8577908,
abstract = {{Reverse genetics uses loss-of-function alleles to interrogate gene function. The advent of CRISPR/Cas9-based gene editing now allows the generation of knock-out alleles for any gene and entire gene families. Even in the model plant Arabidopsis thaliana, gene editing is welcomed as T-DNA insertion lines do not always generate null alleles. Here, we show efficient generation of heritable mutations in Arabidopsis using CRISPR/Cas9 with a workload similar to generating overexpression lines. We obtain for several different genes Cas9 null-segregants with bi-allelic mutations in the T2 generation. While somatic mutations were predominantly generated by the canonical non-homologous end joining (cNHEJ) pathway, we observed inherited mutations that were the result of synthesis-dependent microhomology-mediated end joining (SD-MMEJ), a repair pathway linked to polymerase theta (PolQ). We also demonstrate that our workflow is compatible with a dual sgRNA approach in which a gene is targeted by two sgRNAs simultaneously. This paired nuclease method results in more reliable loss-of-function alleles that lack a large essential part of the gene. The ease of the CRISPR/Cas9 workflow should help in the eventual generation of true null alleles of every gene in the Arabidopsis genome, which will advance both basic and applied plant research.}},
author = {{Pauwels, Laurens and De Clercq, Rebecca and Goossens, Jonas and Iñigo, Sabrina and Williams, Clara and Ron, Mily and Britt, Anne and Goossens, Alain}},
issn = {{2160-1836}},
journal = {{G3-GENES GENOMES GENETICS}},
keywords = {{STRAND BREAK REPAIR,SOMATIC PLANT-CELLS,MESSENGER-RNA DECAY,HIGH-EFFICIENCY,TARGETED MUTAGENESIS,JASMONATE RESPONSES,CRISPR/CAS9,SYSTEM,GENE MODIFICATIONS,OFF-TARGET,IN-PLANTA,Arabidopsis thaliana,genome engineering,genome editing,RNA-guided,nuclease,alt-EJ,polymerase theta,CRISPR/Cas9}},
language = {{eng}},
number = {{8}},
pages = {{2603--2615}},
title = {{A dual sgRNA approach for functional genomics in Arabidopsis thaliana}},
url = {{http://doi.org/10.1534/g3.118.200046}},
volume = {{8}},
year = {{2018}},
}
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