CRISPR/Cas9 and TALENs generate heritable mutations for genes involved in small RNA processing of Glycine max and Medicago truncatula

Shaun J. Curtin; Yer Xiong; Jean‐Michel Michno; Benjamin W. Campbell; Adrian O. Stec; Tomas Čermák; Colby Starker; Daniel F. Voytas; Andrew Eamens; Robert M. Stupar

doi:10.1111/pbi.12857

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CRISPR/Cas9 and TALENs generate heritable mutations for genes involved in small RNA processing of Glycine max and Medicago truncatula

Journal article

Open access

Peer reviewed

CRISPR/Cas9 and TALENs generate heritable mutations for genes involved in small RNA processing of Glycine max and Medicago truncatula

Shaun J. Curtin, Yer Xiong, Jean‐Michel Michno, Benjamin W. Campbell, Adrian O. Stec, Tomas Čermák, Colby Starker, Daniel F. Voytas, Andrew Eamens and Robert M. Stupar

Plant Biotechnology Journal, Vol.16(6), pp.1125-1137

2018

DOI: https://doi.org/10.1111/pbi.12857

PMCID: PMC5978873

PMID: 29087011

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Abstract

CRISPR/Cas9

Medicago

mutagenesis

small RNA

soya bean

Small RNA Biology

Molecular Manipulation

microRNAs

Plant Biology

Processing of double‐stranded RNA precursors into small RNAs is an essential regulator of gene expression in plant development and stress response. Small RNA processing requires the combined activity of a functionally diverse group of molecular components. However, in most of the plant species, there are insufficient mutant resources to functionally characterize each encoding gene. Here, mutations in loci encoding protein machinery involved in small RNA processing in soya bean and Medicago truncatula were generated using the CRISPR/Cas9 and TAL‐effector nuclease (TALEN) mutagenesis platforms. An efficient CRISPR/Cas9 reagent was used to create a bi‐allelic double mutant for the two soya bean paralogous Double‐stranded RNA‐binding2 (GmDrb2a and GmDrb2b) genes. These mutations, along with a CRISPR/Cas9‐generated mutation of the M. truncatula Hua enhancer1 (MtHen1) gene, were determined to be germ‐line transmissible. Furthermore, TALENs were used to generate a mutation within the soya bean Dicer‐like2 gene. CRISPR/Cas9 mutagenesis of the soya bean Dicer‐like3 gene and the GmHen1a gene was observed in the T0 generation, but these mutations failed to transmit to the T1 generation. The irregular transmission of induced mutations and the corresponding transgenes was investigated by whole‐genome sequencing to reveal a spectrum of non‐germ‐line‐targeted mutations and multiple transgene insertion events. Finally, a suite of combinatorial mutant plants were generated by combining the previously reported Gmdcl1a, Gmdcl1b and Gmdcl4b mutants with the Gmdrb2ab double mutant. Altogether, this study demonstrates the synergistic use of different genome engineering platforms to generate a collection of useful mutant plant lines for future study of small RNA processing in legume crops.

Details

Title: CRISPR/Cas9 and TALENs generate heritable mutations for genes involved in small RNA processing of Glycine max and Medicago truncatula
Authors: Shaun J. Curtin (Corresponding Author) - University of Minnesota
Yer Xiong (Author) - University of Minnesota
Jean‐Michel Michno (Author) - University of Minnesota
Benjamin W. Campbell (Author) - University of Minnesota
Adrian O. Stec (Author) - University of Minnesota
Tomas Čermák (Author) - University of Minnesota
Colby Starker (Author) - University of Minnesota
Daniel F. Voytas (Author) - University of Minnesota
Andrew Eamens (Author) - University of Minnesota
Robert M. Stupar (Corresponding Author) - University of Minnesota
Publication details: Plant Biotechnology Journal, Vol.16(6), pp.1125-1137
Publisher: Wiley-Blackwell Publishing Ltd.
Date published: 2018
DOI: 10.1111/pbi.12857
ISSN: 1467-7652; 1467-7644
PMID: 29087011; PMC5978873
Copyright note: (C) 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
Organisation Unit: School of Health - Biomedicine; University of the Sunshine Coast, Queensland; School of Health and Behavioural Sciences - Legacy
Language: English
Record Identifier: 99656591602621
Output Type: Journal article

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Collaboration types: Domestic collaboration; International collaboration
Web Of Science research areas: Biotechnology & Applied Microbiology; Plant Sciences

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