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Further analysis of barley MORC1 using a highly efficient RNA-guided Cas9 gene editing system.
Saturday, 2018/03/31 | 06:29:43

Kumar N, Galli M, Ordon J, Stuttmann J, Kogel KH, Imani J.

Plant Biotechnol J. 2018 Mar 25. doi: 10.1111/pbi.12924.

Abstract

Microrchidia (MORC) proteins comprise a family of proteins that have been identified in prokaryotes and eukaryotes. They are defined by two hallmark domains: a GHKL-type ATPase and an S5 fold. In plants, MORC proteins were first discovered in a genetic screen for Arabidopsis thaliana mutants compromised for resistance to a viral pathogen. Subsequent studies expanded their role in plant immunity and revealed their involvement in gene silencing and genome stabilization. Little is known about the role of MORC proteins of cereals, especially because knock-out (KO) mutants were not available and assessment of loss of function relied only on RNAi strategies, which were arguable, given that MORC proteins in itself are influencing gene silencing. Here we used a Streptococcus pyogenes Cas9 (SpCas9)-mediated KO strategy to functionally study HvMORC1, one of the currently seven MORC members of barley. Using a novel barley RNA Pol III-dependent U3 small nuclear RNA (snRNA) promoter to drive expression of the synthetic single guide RNA (sgRNA), we achieved a very high mutation frequency in HvMORC1. High frequencies of mutations were detectable by target sequencing in the callus, the T0 generation (77%) and T1 (70%-100%), which constitutes an important improvement of the gene editing technology in cereals. Corroborating and extending earlier findings, SpCas9-edited hvmorc1-KO barley, in clear contrast to Arabidopsis atmorc1 mutants, had a distinct phenotype of increased disease resistance to fungal pathogens, while morc1 mutants of either plant showed de-repressed expression of transposable elements (TEs), substantiating that plant MORC proteins contribute to genome stabilization in mono- and dicotyledonous plants.

 

See https://onlinelibrary.wiley.com/doi/epdf/10.1111/pbi.12924

 

U3 promoter sequences from rice (a); wheat (b) and barley (c). Sequence motifs are underlined: TATA-box, upstream sequence element (USE), and monocot-specific promoter (MSP) element.

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