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

Abstract

SummaryProcessing 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.

Multiplex CRISPR/Cas9‐mediated metabolic engineering increases soya bean isoflavone content and resistance to soya bean mosaic virus

“…The multiplex CRISPR/Cas9 platform provides an opportunity to simultaneously manipulate different agronomic traits. Previously, a CRISPR/Cas9 system that allowed two sgRNA targeting sites was used to create a biallelic double mutant for the two soya bean paralogous double‐stranded RNA‐binding2 ( GmDrb2a and GmDrb2b ) genes (Curtin et al , ). Recently, the CRISPR/Cas9‐mediated targeted mutagenesis of GmSPL9 , which were expressed using Arabidopsis U3 and U6 promoters, altered plant architecture in soya bean (Bao et al , ).…”

The CRISPR/Cas revolution continues: From efficient gene editing for crop breeding to plant synthetic biology

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Construction and comparison of three reference‐quality genome assemblies for soybean

“…These genome assemblies are being used to further understand soybean biology and to accelerate breeding. A small sampling of the many studies that have used the soybean reference assembly includes: population structure and ancestry (Bandillo et al ., ; Zhou et al ., ; Valliyodan et al ., ); identification of the locus determining pod shattering (Dong et al ., ; Funatsuki et al ., ); seed protein content (Hwang et al ., ; Vaughn et al ., ; Bandillo et al ., ); plant architecture (Prince et al ., ); precision gene editing (Curtin et al ., ); and genomic selection (Desta and Ortiz, ). Recent advances in next‐generation sequencing technologies, including long‐read sequencing, long‐range scaffolding and advanced bioinformatics for short read assembly (Burton et al ., ), have led to the production of improved assemblies for even large and highly complex genomes (Jiao et al ., ; Ling et al ., ; Raymond et al ., ).…”