title: Genome editing in sea urchin embryos by using a CRISPR/Cas9 system.
abstract: Sea urchin embryos are a useful model system for investigating early developmental processes and the underlying gene regulatory networks. Most functional studies using sea urchin embryos rely on antisense morpholino oligonucleotides to knockdown gene functions. However, major concerns related to this technique include off-target effects, variations in morpholino efficiency, and potential morpholino toxicity; furthermore, such problems are difficult to discern. Recent advances in genome editing technologies have introduced the prospect of not only generating sequence-specific knockouts, but also providing genome-engineering applications. Two genome editing tools, zinc-finger nuclease (ZFN) and transcription activator-like effector nucleases (TALENs), have been utilized in sea urchin embryos, but the resulting efficiencies are far from satisfactory. The CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 (CRISPR-associated nuclease 9) system serves as an easy and efficient method with which to edit the genomes of several established and emerging model organisms in the field of developmental biology. Here, we apply the CRISPR/Cas9 system to the sea urchin embryo. We designed six guide RNAs (gRNAs) against the well-studied nodal gene and discovered that five of the gRNAs induced the expected phenotype in 60-80% of the injected embryos. In addition, we developed a simple method for isolating genomic DNA from individual embryos, enabling phenotype to be precisely linked to genotype, and revealed that the mutation rates were 67-100% among the sequenced clones. Of the two potential off-target sites we examined, no off-target effects were observed. The detailed procedures described herein promise to accelerate the usage of CRISPR/Cas9 system for genome editing in sea urchin embryos.
authors.affiliation: Institute of Cellular and Organismic Biology, Academia Sinica, Taipei 11529, Taiwan.
authors.forename: Che-Yi
authors.initials: CY
authors.lastname: Lin
authors.affiliation: Institute of Cellular and Organismic Biology, Academia Sinica, Taipei 11529, Taiwan. Electronic address: yhsu@gate.sinica.edu.tw.
authors.forename: Yi-Hsien
authors.initials: YH
authors.lastname: Su
mesh_terms: D000818:Animals; D001483:Base Sequence; D064113:CRISPR-Cas Systems / Q000235:genetics*; D004625:Embryo, Nonmammalian / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D016678:Genome; D005838:Genotype; D008969:Molecular Sequence Data; D060172:Morpholinos / Q000494:pharmacology; D055457:Nodal Protein / Q000378:metabolism; D009692:Nucleic Acid Heteroduplexes; D010641:Phenotype; D017393:RNA Editing / Q000187:drug effects / Q000235:genetics*; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012617:Sea Urchins / Q000187:drug effects / Q000196:embryology* / Q000235:genetics*; D015398:Signal Transduction / Q000187:drug effects
chemical_list: D060172:Morpholinos; D055457:Nodal Protein; D009692:Nucleic Acid Heteroduplexes; D000094704:RNA, Guide, CRISPR-Cas Systems; D012333:RNA, Messenger
keywords: CRISPR/Cas9; Nodal; Sea urchin