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A dual sgRNA approach for functional genomics in Arabidopsis thaliana

Laurens Pauwels (UGent) , Rebecca De Clercq (UGent) , Jonas Goossens (UGent) , Sabrina Iñigo (UGent) , Clara Williams (UGent) , Mily Ron, Anne Britt and Alain Goossens (UGent)
(2018) G3-GENES GENOMES GENETICS. 8(8). p.2603-2615
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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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Chicago
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.
APA
Pauwels, Laurens, De Clercq, R., Goossens, J., Iñigo, S., Williams, C., Ron, M., Britt, A., et al. (2018). A dual sgRNA approach for functional genomics in Arabidopsis thaliana. G3-GENES GENOMES GENETICS, 8(8), 2603–2615.
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.
MLA
Pauwels, Laurens, Rebecca De Clercq, Jonas Goossens, et al. “A Dual sgRNA Approach for Functional Genomics in Arabidopsis Thaliana.” G3-GENES GENOMES GENETICS 8.8 (2018): 2603–2615. Print.
@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 I{\~n}igo, Sabrina and Williams, Clara and Ron, Mily and Britt, Anne and Goossens, Alain},
  issn         = {2160-1836},
  journal      = {G3-GENES GENOMES GENETICS},
  language     = {eng},
  number       = {8},
  pages        = {2603--2615},
  title        = {A dual sgRNA approach for functional genomics in Arabidopsis thaliana},
  url          = {http://dx.doi.org/10.1534/g3.118.200046},
  volume       = {8},
  year         = {2018},
}

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