Cas9-Guide RNA Directed Genome Editing in Soybean

• Published in: Plant physiology (Bethesda) Vol. 169; no. 2; pp. 960 - 970
• Main Authors: Li, Zhongsen, Liu, Zhan-Bin, Xing, Aiqiu, Moon, Bryan P., Koellhoffer, Jessica P., Huang, Lingxia, Ward, R. Timothy, Clifton, Elizabeth, Falco, S. Carl, Cigan, A. Mark
• Format: Journal Article
• Language: English
• Published: United States American Society of Plant Biologists 01-10-2015
• Subjects: Alleles; Bacterial Proteins - genetics; Breakthrough Technologies; CRISPR-Associated Protein 9; DNA; DNA End-Joining Repair; Endonucleases - genetics; Genetic Engineering - methods; Genetic mutation; Genome, Plant; Genomes; Genomics; Glycine max - drug effects; Glycine max - genetics; Guide RNA; Homologous Recombination; Mutagenesis, Insertional; Plant Proteins - genetics; Plants; Plants, Genetically Modified; Polymerase chain reaction; RNA Editing; RNA, Guide, CRISPR-Cas Systems; Segregation; Soybeans; Sulfonamides - pharmacology; Triazines - pharmacology
• ISSN: 0032-0889; 1532-2548
• DOI: 10.1104/pp.15.00783

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 pyogenesCas9-guide RNA (gRNA) was successfully applied to generate targeted mutagenesis, gene integration, and gene editing in soybean (Glycine max). Two genomic sites,DD20andDD43on 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 oneDD43homology-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 theDD43target 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.