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 L. Eamens, Robert M Stupar

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

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 T 0 generation, but these mutations failed to transmit to the T 1 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.

Original languageEnglish (US)
Pages (from-to)1125-1137
Number of pages13
JournalPlant Biotechnology Journal
Volume16
Issue number6
DOIs
StatePublished - Jun 1 2018

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Clustered Regularly Interspaced Short Palindromic Repeats
Medicago truncatula
Soybeans
Glycine max
RNA
mutation
mutants
Mutation
beans
mutagenesis
Genes
genes
double-stranded RNA
Double-Stranded RNA
transgenes
Transgenes
Mutagenesis
Genome
genome
nucleases

Keywords

  • CRISPR/Cas9
  • Drb2
  • Medicago
  • mutagenesis
  • small RNA
  • soya bean

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CRISPR/Cas9 and TALENs generate heritable mutations for genes involved in small RNA processing of Glycine max and Medicago truncatula. / Curtin, Shaun J.; Xiong, Yer; Michno, Jean Michel; Campbell, Benjamin W.; Stec, Adrian O.; Čermák, Tomas; Starker, Colby; Voytas, Daniel F; Eamens, Andrew L.; Stupar, Robert M.

In: Plant Biotechnology Journal, Vol. 16, No. 6, 01.06.2018, p. 1125-1137.

Research output: Contribution to journalArticle

Curtin, Shaun J. ; Xiong, Yer ; Michno, Jean Michel ; Campbell, Benjamin W. ; Stec, Adrian O. ; Čermák, Tomas ; Starker, Colby ; Voytas, Daniel F ; Eamens, Andrew L. ; Stupar, Robert M. / CRISPR/Cas9 and TALENs generate heritable mutations for genes involved in small RNA processing of Glycine max and Medicago truncatula. In: Plant Biotechnology Journal. 2018 ; Vol. 16, No. 6. pp. 1125-1137.
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AU - Stec, Adrian O.

AU - Čermák, Tomas

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