Dataset Information

A Dual sgRNA Approach for Functional Genomics in Arabidopsis thaliana.

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 ? (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.

SUBMITTER: Pauwels L

PROVIDER: S-EPMC6071589 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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Publications

A Dual sgRNA Approach for Functional Genomics in <i>Arabidopsis thaliana</i>.

Pauwels Laurens L De Clercq Rebecca R Goossens Jonas J Iñigo Sabrina S Williams Clara C Ron Mily M Britt Anne A Goossens Alain A

G3 (Bethesda, Md.) 20180731 8

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 <i>Arabidopsis thaliana</i>, 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. W ...[more]

PMID: 29884615

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Project description:Organellar gene expression (OGE) is crucial for plant development, photosynthesis, and respiration, but our understanding of the mechanisms that control it is still relatively poor. Thus, OGE requires various nucleus-encoded proteins that promote transcription, splicing, trimming, and editing of organellar RNAs, and regulate translation. In metazoans, proteins of the mitochondrial Transcription tERmination Factor (mTERF) family interact with the mitochondrial chromosome and regulate transcriptional initiation and termination. Sequencing of the Arabidopsis thaliana genome led to the identification of a diversified MTERF gene family but, in contrast to mammalian mTERFs, knowledge about the function of these proteins in photosynthetic organisms is scarce. In this hypothesis article, I show that tandem duplications and one block duplication contributed to the large number of MTERF genes in A. thaliana, and propose that the expansion of the family is related to the evolution of land plants. The MTERF genes-especially the duplicated genes-display a number of distinct mRNA accumulation patterns, suggesting functional diversification of mTERF proteins to increase adaptability to environmental changes. Indeed, hypothetical functions for the different mTERF proteins can be predicted using co-expression analysis and gene ontology (GO) annotations. On this basis, mTERF proteins can be sorted into five groups. Members of the "chloroplast" and "chloroplast-associated" clusters are principally involved in chloroplast gene expression, embryogenesis, and protein catabolism, while representatives of the "mitochondrial" cluster seem to participate in DNA and RNA metabolism in that organelle. Moreover, members of the "mitochondrion-associated" cluster and the "low expression" group may act in the nucleus and/or the cytosol. As proteins involved in OGE and presumably nuclear gene expression (NGE), mTERFs are ideal candidates for the coordination of the expression of organelle and nuclear genomes.

Dataset Information

A Dual sgRNA Approach for Functional Genomics in Arabidopsis thaliana.

Publications

A Dual sgRNA Approach for Functional Genomics in <i>Arabidopsis thaliana</i>.

Similar Datasets

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