Cas9-Guide RNA Directed Genome Editing in Soybean

Abstract

Recently discovered bacteria and archaea adaptive immune system consisting of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) endonuclease has been explored in targeted genome editing in different species. Streptococcus pyogenes Cas9-guide RNA (gRNA) was successfully applied to generate targeted mutagenesis, gene integration, and gene editing in soybean (Glycine max). Two genomic sites, DD20 and DD43 on chromosome 4, were mutagenized with frequencies of 59% and 76%, respectively. Sequencing randomly selected transgenic events confirmed that the genome modifications were specific to the Cas9-gRNA cleavage sites and consisted of small deletions or insertions. Targeted gene integrations through homology-directed recombination were detected by border-specific polymerase chain reaction analysis for both sites at callus stage, and one DD43 homology-directed recombination event was transmitted to T1 generation. T1 progenies of the integration event segregated according to Mendelian laws and clean homozygous T1 plants with the donor gene precisely inserted at the DD43 target site were obtained. The Cas9-gRNA system was also successfully applied to make a directed P178S mutation of acetolactate synthase1 gene through in planta gene editing.

An Improved Variant of Soybean Type 1 Diacylglycerol Acyltransferase Increases the Oil Content and Decreases the Soluble Carbohydrate Content of Soybeans

“…As such, it could provide multiple, almost native, transgene-derived proteins with the ability to increase the oil content of seeds or vegetative tissues. Recently developed gene-editing technology (Li et al, 2015) also could be used to change the amino acid sequence of endogenous DGAT1 proteins from multiple plant species of interest to achieve similar goals.…”

“…FLP plasmid QC663 contained the yeast FLP recombinase under the control of the constitutive soybean EF1A2 promoter (Li et al, 2009(Li et al, , 2015 followed by the potato (Solanum tuberosum) proteinase inhibitor II transcription terminator, which provided the FLP recombinase for transient expression ( Supplemental Fig. S2C).…”

Accelerating genetic gains in legumes for the development of prosperous smallholder agriculture: integrating genomics, phenotyping, systems modelling and agronomy

“…This technology has already been optimized in three model legume crops, soybean (Sun et al, 2015;Li et al, 2015), Lotus japonicas and Medicago truncatula (Meng et al, 2017). The efficiency of genome editing using various endogenous and exogenous RNA polymerase III promoters has been compared (Sun et al, 2015;Du et al, 2016) and in soybean the Cas9-single guide RNA together with donor DNA fragments was successfully transformed into soybean using the particle bombardment method (Li et al, 2015). Medicago and Lotus, having smaller diploid genomes, are the model legume crops for conducting research in several areas including symbiotic nitrogen fixation.…”

New developments in green biotechnology - an inventory for RIVM

“…In maize and soybean (Svitashev et al 2015;Li et al 2015) the acetolactate synthase (ALS) gene was targeted using CRISPR-Cas9 and modified using a DNA repair template containing several nucleotide changes compared to the native sequence, thereby providing chlorsulfuron herbicide resistance. In another example using ZFNs and HDR, insertional disruption of the inositol-1,3,4,5,6-pentakisphosphate 2-kinase (IPK1) gene in maize was achieved (Shukla et al 2009).…”