title: Targeted mutagenesis in cotton (Gossypium hirsutum L.) using the CRISPR/Cas9 system.
abstract: The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 system has been widely used for genome editing in various plants because of its simplicity, high efficiency and design flexibility. However, to our knowledge, there is no report on the application of CRISPR/Cas9-mediated targeted mutagenesis in cotton. Here, we report the genome editing and targeted mutagenesis in upland cotton (Gossypium hirsutum L., hereafter cotton) using the CRISPR/Cas9 system. We designed two guide RNAs to target distinct sites of the cotton Cloroplastos alterados 1 (GhCLA1) and vacuolar H+-pyrophosphatase (GhVP) genes. Mutations in these two genes were detected in cotton protoplasts. Most of the mutations were nucleotide substitutions, with one nucleotide insertion and one substitution found in GhCLA1 and one deletion found in GhVP in cotton protoplasts. Subsequently, the two vectors were transformed into cotton shoot apexes through Agrobacterium-mediated transformation, resulting in efficient target gene editing. Most of the mutations were nucleotide deletions, and the mutation efficiencies were 47.6-81.8% in transgenic cotton plants. Evaluation using restriction-enzyme-PCR assay and sequence analysis detected no off-target mutations. Our results indicated that the CRISPR/Cas9 system was an efficient and specific tool for targeted mutagenesis of the cotton genome.
authors.affiliation: State Key Laboratory of Cotton Biology/Institute of Cotton Research of Chinese Academy of Agricultural Sciences, Anyang, Henan 455000, China.|National Key Laboratory of Crop Genetic Improvement, College of Plant Sciences and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, China.
authors.forename: Xiugui
authors.initials: X
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Cotton Biology/Institute of Cotton Research of Chinese Academy of Agricultural Sciences, Anyang, Henan 455000, China.
authors.forename: Xuke
authors.initials: X
authors.lastname: Lu
authors.affiliation: State Key Laboratory of Cotton Biology/Institute of Cotton Research of Chinese Academy of Agricultural Sciences, Anyang, Henan 455000, China.
authors.forename: Na
authors.initials: N
authors.lastname: Shu
authors.affiliation: State Key Laboratory of Cotton Biology/Institute of Cotton Research of Chinese Academy of Agricultural Sciences, Anyang, Henan 455000, China.
authors.forename: Shuai
authors.initials: S
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Cotton Biology/Institute of Cotton Research of Chinese Academy of Agricultural Sciences, Anyang, Henan 455000, China.
authors.forename: Junjuan
authors.initials: J
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Cotton Biology/Institute of Cotton Research of Chinese Academy of Agricultural Sciences, Anyang, Henan 455000, China.
authors.forename: Delong
authors.initials: D
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Cotton Biology/Institute of Cotton Research of Chinese Academy of Agricultural Sciences, Anyang, Henan 455000, China.
authors.forename: Lixue
authors.initials: L
authors.lastname: Guo
authors.affiliation: State Key Laboratory of Cotton Biology/Institute of Cotton Research of Chinese Academy of Agricultural Sciences, Anyang, Henan 455000, China.
authors.forename: Wuwei
authors.initials: W
authors.lastname: Ye
mesh_terms: D001483:Base Sequence; D064113:CRISPR-Cas Systems; D000072669:Gene Editing / Q000379:methods*; D018390:Gene Targeting / Q000379:methods*; D018745:Genome, Plant / Q000235:genetics; D003368:Gossypium / Q000235:genetics*; D008957:Models, Genetic; D016296:Mutagenesis; D009154:Mutation; D010940:Plant Proteins / Q000235:genetics; D030821:Plants, Genetically Modified; D011523:Protoplasts / Q000378:metabolism; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid
chemical_list: D010940:Plant Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems