title: 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.
authors: DuPont Pioneer Agricultural Biotechnology, Wilmington, Delaware 19803 zhongsenli97@gmail.com.
authors: Zhongsen
authors: http://orcid.org/0000-0001-8697-3388
authors: Z
authors: Li
authors: DuPont Pioneer Agricultural Biotechnology, Wilmington, Delaware 19803.
authors: Zhan-Bin
authors: ZB
authors: Liu
authors: DuPont Pioneer Agricultural Biotechnology, Wilmington, Delaware 19803.
authors: Aiqiu
authors: A
authors: Xing
authors: DuPont Pioneer Agricultural Biotechnology, Wilmington, Delaware 19803.
authors: Bryan P
authors: BP
authors: Moon
authors: DuPont Pioneer Agricultural Biotechnology, Wilmington, Delaware 19803.
authors: Jessica P
authors: JP
authors: Koellhoffer
authors: DuPont Pioneer Agricultural Biotechnology, Wilmington, Delaware 19803.
authors: Lingxia
authors: L
authors: Huang
authors: DuPont Pioneer Agricultural Biotechnology, Wilmington, Delaware 19803.
authors: R Timothy
authors: RT
authors: Ward
authors: DuPont Pioneer Agricultural Biotechnology, Wilmington, Delaware 19803.
authors: Elizabeth
authors: E
authors: Clifton
authors: DuPont Pioneer Agricultural Biotechnology, Wilmington, Delaware 19803.
authors: S Carl
authors: SC
authors: Falco
authors: DuPont Pioneer Agricultural Biotechnology, Wilmington, Delaware 19803.
authors: A Mark
authors: http://orcid.org/0000-0002-5983-5235
authors: AM
authors: Cigan
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D000076987:CRISPR-Associated Protein 9; D059766:DNA End-Joining Repair; D004720:Endonucleases / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods*; D018745:Genome, Plant; D059765:Homologous Recombination; D016254:Mutagenesis, Insertional; D010940:Plant Proteins / Q000235:genetics; D030821:Plants, Genetically Modified; D017393:RNA Editing; D000094704:RNA, Guide, CRISPR-Cas Systems; D013025:Glycine max / Q000187:drug effects / Q000235:genetics*; D013449:Sulfonamides / Q000494:pharmacology; D014227:Triazines / Q000494:pharmacology
chemical_list: D001426:Bacterial Proteins; D010940:Plant Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems; D013449:Sulfonamides; D014227:Triazines; D000076987:CRISPR-Associated Protein 9; C000606107:Cas9 endonuclease Streptococcus pyogenes; D004720:Endonucleases; C037137:chlorsulfuron