title: A high-efficiency CRISPR/Cas9 system for targeted mutagenesis in Cotton (Gossypium hirsutum L.).
abstract: The complex allotetraploid genome is one of major challenges in cotton for repressing gene expression. Developing site-specific DNA mutation is the long-term dream for cotton breeding scientists. The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system is emerging as a robust biotechnology for targeted-DNA mutation. In this study, two sgRNAs, GhMYB25-like-sgRNA1 and GhMYB25-like-sgRNA2, were designed in the identical genomic regions of GhMYB25-like A and GhMYB25-like D, which were encoded by cotton A subgenome and the D subgenome, respectively, was assembled to direct Cas9-mediated allotetraploid cotton genome editing. High proportion (14.2-21.4%) CRISPR/Cas9-induced specific truncation events, either from GhMYB25-like A DNA site or from GhMYB25-like D DNA site, were detected in 50% examined transgenic cotton through PCR amplification assay and sequencing analyses. Sequencing results also demonstrated that 100% and 98.8% mutation frequency were occurred on GhMYB25-like-sgRNA1 and GhMYB25-like-sgRNA2 target site respectively. The off-target effect was evaluated by sequencing two putative off-target sites, which have 3 and 1 mismatched nucleotides with GhMYB25-like-sgRNA1 and GhMYB25-like-sgRNA2, respectively; all the examined samples were not detected any off-target-caused mutation events. Thus, these results demonstrated that CRISPR/Cas9 is qualified for generating DNA level mutations on allotetraploid cotton genome with high-efficiency and high-specificity.
authors.affiliation: Department of Biology, East Carolina University, Greenville, NC 27858, USA.
authors.forename: Chao
authors.initials: C
authors.lastname: Li
authors.affiliation: Izmir International Biomedicine and Genome Institute (iBG-izmir), Dokuz Eylul University, Balcova 35340, Izmir, Turkey.
authors.forename: Turgay
authors.initials: T
authors.lastname: Unver
authors.affiliation: Department of Biology, East Carolina University, Greenville, NC 27858, USA.
authors.forename: Baohong
authors.initials: B
authors.lastname: Zhang
mesh_terms: D064113:CRISPR-Cas Systems; D018744:DNA, Plant / Q000235:genetics / Q000378:metabolism; D018390:Gene Targeting / Q000379:methods*; D003368:Gossypium / Q000235:genetics*; D011995:Recombination, Genetic
chemical_list: D018744:DNA, Plant