A Dual sgRNA Approach for Functional Genomics in Arabidopsis thaliana. Issue 8 (1st August 2018)

• Record Type: Journal Article
• Authors: Pauwels, Laurens; De Clercq, Rebecca; Goossens, Jonas; Iñigo, Sabrina; Williams, Clara; Ron, Mily; Britt, Anne; Goossens, Alain
• Is Part Of: G3. Volume 8:Issue 8(2018)
• Page Start: 2603
• Page End: 2615
• Publication Date: 2018-08-01
• Subjects: Arabidopsis thaliana -- genome engineering -- genome editing -- RNA-guided nuclease -- alt-EJ -- polymerase theta -- CRISPR/Cas9; Genetics -- Research -- Periodicals; Genomics -- Periodicals; Genetics; Genomics; Genes; Genetics -- Research; Genomics; Electronic journals; Periodical; Periodicals; Fulltext; Internet Resources; Periodicals; 572.8
• DOI: 10.1534/g3.118.200046
• Languages: English
• ISSNs: 2160-1836

Abstract

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 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.