title: Site-directed mutagenesis in Petunia × hybrida protoplast system using direct delivery of purified recombinant Cas9 ribonucleoproteins.
abstract: Site-directed mutagenesis of nitrate reductase genes using direct delivery of purified Cas9 protein preassembled with guide RNA produces mutations efficiently in Petunia × hybrida protoplast system. The clustered, regularly interspaced, short palindromic repeat (CRISPR)-CRISPR associated endonuclease 9 (CRISPR/Cas9) system has been recently announced as a powerful molecular breeding tool for site-directed mutagenesis in higher plants. Here, we report a site-directed mutagenesis method targeting Petunia nitrate reductase (NR) gene locus. This method could create mutations efficiently using direct delivery of purified Cas9 protein and single guide RNA (sgRNA) into protoplast cells. After transient introduction of RNA-guided endonuclease (RGEN) ribonucleoproteins (RNPs) with different sgRNAs targeting NR genes, mutagenesis at the targeted loci was detected by T7E1 assay and confirmed by targeted deep sequencing. T7E1 assay showed that RGEN RNPs induced site-specific mutations at frequencies ranging from 2.4 to 21 % at four different sites (NR1, 2, 4 and 6) in the PhNR gene locus with average mutation efficiency of 14.9 ± 2.2 %. Targeted deep DNA sequencing revealed mutation rates of 5.3-17.8 % with average mutation rate of 11.5 ± 2 % at the same NR gene target sites in DNA fragments of analyzed protoplast transfectants. Further analysis from targeted deep sequencing showed that the average ratio of deletion to insertion produced collectively by the four NR-RGEN target sites (NR1, 2, 4, and 6) was about 63:37. Our results demonstrated that direct delivery of RGEN RNPs into protoplast cells of Petunia can be exploited as an efficient tool for site-directed mutagenesis of genes or genome editing in plant systems.
authors.affiliation: Department of Horticultural Science, Chungnam National University, Daejeon, 305-764, South Korea.
authors.forename: Saminathan
authors.initials: S
authors.lastname: Subburaj
authors.affiliation: Department of Horticultural Science, Chungnam National University, Daejeon, 305-764, South Korea.
authors.forename: Sung Jin
authors.initials: SJ
authors.lastname: Chung
authors.affiliation: Department of Chemistry, Seoul National University, Seoul, 151-742, South Korea.|Center for Genome Engineering, Institute for Basic Science, Seoul, 151-742, South Korea.
authors.forename: Choongil
authors.initials: C
authors.lastname: Lee
authors.affiliation: Department of Chemistry, Seoul National University, Seoul, 151-742, South Korea.|Center for Genome Engineering, Institute for Basic Science, Seoul, 151-742, South Korea.
authors.forename: Seuk-Min
authors.initials: SM
authors.lastname: Ryu
authors.affiliation: Department of Chemistry, Hanyang University, Seoul, 133-791, South Korea.
authors.forename: Duk Hyoung
authors.initials: DH
authors.lastname: Kim
authors.affiliation: Department of Chemistry, Seoul National University, Seoul, 151-742, South Korea.|Center for Genome Engineering, Institute for Basic Science, Seoul, 151-742, South Korea.
authors.forename: Jin-Soo
authors.initials: JS
authors.lastname: Kim
authors.affiliation: Department of Chemistry, Hanyang University, Seoul, 133-791, South Korea. sangsubae@hanyang.ac.kr.|Research Institute for Convergence of Basic Sciences, Hanyang University, Seoul, 133-791, South Korea. sangsubae@hanyang.ac.kr.
authors.forename: Sangsu
authors.initials: S
authors.lastname: Bae
authors.affiliation: Department of Horticultural Science, Chungnam National University, Daejeon, 305-764, South Korea. gjlee@cnu.ac.kr.
authors.forename: Geung-Joo
authors.initials: GJ
authors.lastname: Lee
mesh_terms: D001483:Base Sequence; D064113:CRISPR-Cas Systems / Q000235:genetics*; D005818:Genetic Engineering / Q000379:methods; D018745:Genome, Plant / Q000235:genetics; D059014:High-Throughput Nucleotide Sequencing; D008856:Microscopy, Fluorescence; D008957:Models, Genetic; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D032306:Petunia / Q000166:cytology / Q000235:genetics*; D010940:Plant Proteins / Q000235:genetics; D011523:Protoplasts / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012261:Ribonucleoproteins / Q000235:genetics* / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid
chemical_list: D010940:Plant Proteins; D011994:Recombinant Proteins; D012261:Ribonucleoproteins
keywords: CRISPR/Cas system; Nitrate reductase; Petunia; Protoplast; RGEN; Ribonucleoproteins; Site-directed mutagenesis
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