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- Title
A Dual sgRNA Approach for Functional Genomics in Arabidopsis thaliana.
- Authors
Pauwels, Laurens; De Clercq, Rebecca; Goossens, Jonas; Iñigo, Sabrina; Williams, Clara; Ron, Mily; Britt, Anne; Goossens, Alain
- 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 θ (PolQ). We also demonstrate that our workflow is compatible with a dual sgRNA approach in which a gene is targeted by two sgRNAs simultane sly. 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.
- Subjects
CRISPRS; ARABIDOPSIS thaliana; ALLELES
- Publication
G3: Genes | Genomes | Genetics, 2018, Vol 8, Issue 8, p2603
- ISSN
2160-1836
- Publication type
Article
- DOI
10.1534/g3.118.200046