A Dual sgRNA Approach for Functional Genomics in Arabidopsis thaliana

G3: Genes, Genomes, Genetics

article

Laurens Pauwels¹  Rebecca De Clercq¹  Jonas Goossens¹  Sabrina Iñigo¹  Clara Williams¹  Mily Ron³  Anne Britt³  Alain Goossens¹ 

[1] Ghent University, Department of Plant Biotechnology and Bioinformatics, 9052 Ghent, Belgium;VIB Center for Plant Systems Biology, 9052 Ghent, Belgium;UC Davis, Department of Plant Biology, Davis, CA 95616, US

关键词: Keywords: Arabidopsis thaliana;    genome engineering;    genome editing;    RNA-guided nuclease;    alt-EJ;    polymerase theta;    CRISPR/Cas9;   

DOI  :  10.1534/g3.118.200046

学科分类：社会科学、人文和艺术（综合）

来源: Genetics Society of America

【 摘 要 】

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.

【 授权许可】

CC BY|CC BY-NC