Cas9-Guide RNA Directed Genome Editing in Soybean.

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.

Tissue-specific expression of a soybean hypersensitive-induced response (HIR) protein gene promoter.

A Glycine max gene encoding a putative protein similar to hypersensitive-induced response proteins (HIR) was identified as a gene with preferred expressions in flowers and developing seeds by whole transcriptome gene expression profiling. Its promoter gm-hir1 was cloned and revealed to strongly express a fluorescence reporter gene primarily in integuments, anther tapetum, and seed coat with unique tissue-specificity. Expression in the inner integument was apparent prior to pollination, while expression in the outer integument started to develop from the micropylar end outward as the embryo matured. A 5'-deletion study showed that the promoter can be truncated to 600 bp long relative to the translation start site without affecting expression. A positive regulatory element was identified between 600 and 481 bp that controls expression in the inner integument, with no noticeable effect on expression in the outer integument or tapetum. Additionally, removal of the 5'UTR intron had no effect on levels or location of gm-hir1 expression while truncation to 370 bp resulted in a complete loss of expression suggesting that elements controlling both the outer integument and tapetum expression are located within the 481-370 bp region.