Efficient generation of knock-in transgenic zebrafish carrying reporter/driver genes by CRISPR/Cas9-mediated genome engineering

Kimura, Yukiko; Hisano, Yu; Kawahara, Atsuo; Higashijima, Shin‐ichi

doi:10.1038/srep06545

Cited by 304 publications

(312 citation statements)

References 26 publications

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“…Published reports of CRISPR-Cas9 deployment in zebrafish vary widely in terms of the reported concentrations for sgRNA and Cas9 mRNA or protein, mutagenesis efficiency, and the assessment of resulting mutant alleles (Auer et al, 2014a,b;Chang et al, 2013;Gagnon et al, 2014;Hruscha et al, 2013;Hwang et al, 2013;Irion et al, 2014;Jao et al, 2013;Kimura et al, 2014;Moreno-Mateos et al, 2015;Sung et al, 2014). In our hands, some of this stems from experimental variability in the delicate preparation and handling of in vitro transcribed RNA components and in the mechanical process of individual microinjections.…”

Section: Discussionmentioning

confidence: 99%

Maximizing mutagenesis with solubilized CRISPR-Cas9 ribonucleoprotein complexes.

et al. 2016

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CRISPR-Cas9 enables efficient sequence-specific mutagenesis for creating somatic or germline mutants of model organisms. Key constraints in vivo remain the expression and delivery of active Cas9-sgRNA ribonucleoprotein complexes (RNPs) with minimal toxicity, variable mutagenesis efficiencies depending on targeting sequence, and high mutation mosaicism. Here, we apply in vitro assembled, fluorescent Cas9-sgRNA RNPs in solubilizing salt solution to achieve maximal mutagenesis efficiency in zebrafish embryos. MiSeq-based sequence analysis of targeted loci in individual embryos using CrispRVariants, a customized software tool for mutagenesis quantification and visualization, reveals efficient biallelic mutagenesis that reaches saturation at several tested gene loci. Such virtually complete mutagenesis exposes loss-of-function phenotypes for candidate genes in somatic mutant embryos for subsequent generation of stable germline mutants. We further show that targeting of non-coding elements in gene regulatory regions using saturating mutagenesis uncovers functional control elements in transgenic reporters and endogenous genes in injected embryos. Our results establish that optimally solubilized, in vitro assembled fluorescent Cas9-sgRNA RNPs provide a reproducible reagent for direct and scalable loss-of-function studies and applications beyond zebrafish experiments that require maximal DNA cutting efficiency in vivo.

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Section: Discussionmentioning

confidence: 99%

Maximizing mutagenesis with solubilized CRISPR-Cas9 ribonucleoprotein complexes.

et al. 2016

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“…S1) followed the strategy described by Kimura et al (2014), albeit with our original modifications as will be described elsewhere in detail. pSpCas9 (BB)-2A-Puro (Addgene plasmid, #48139) (Ran et al, 2013) was used as the vector to generate a single guide RNA (sgRNA).…”

Section: Establishment Of Ko Cell Lines Using the Crispr/cas9 Systemmentioning

confidence: 99%

Regulation of ciliary retrograde protein trafficking by the Joubert syndrome proteins ARL13B and INPP5E

Nozaki

Katoh

Terada

et al. 2017

Journal of Cell Science

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ARL13B (a small GTPase) and INPP5E (a phosphoinositide 5-phosphatase) are ciliary proteins encoded by causative genes of Joubert syndrome. We here showed, by taking advantage of a visible immunoprecipitation assay, that ARL13B interacts with the IFT46-IFT56 (IFT56 is also known as TTC26) dimer of the intraflagellar transport (IFT)-B complex, which mediates anterograde ciliary protein trafficking. However, the ciliary localization of ARL13B was found to be independent of its interaction with IFT-B, but dependent on the ciliary-targeting sequence RVEP in its C-terminal region. ARL13B-knockout cells had shorter cilia than control cells and exhibited aberrant localization of ciliary proteins, including INPP5E. In particular, in ARL13B-knockout cells, the IFT-A and IFT-B complexes accumulated at ciliary tips, and GPR161 (a negative regulator of Hedgehog signaling) could not exit cilia in response to stimulation with Smoothened agonist. This abnormal phenotype was rescued by the exogenous expression of wild-type ARL13B, as well as by its mutant defective in the interaction with IFT-B, but not by its mutants defective in INPP5E binding or in ciliary localization. Thus, ARL13B regulates IFT-A-mediated retrograde protein trafficking within cilia through its interaction with INPP5E.

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“…CRISPR/Cas9 genome modification is a powerful approach for generating both targeted indel mutations by non-homologous endjoining (NHEJ) repair and more precise gene editing by the introduction of specific sequences through homology-directed repair (HDR) (Auer et al, 2014;Bassett et al, 2014;Bhattacharya et al, 2015;Blitz et al, 2013;Chen et al, 2013;Friedland et al, 2013;Guo et al, 2014;Hisano et al, 2015;Kimura et al, 2014;Kotani et al, 2015;Li et al, 2015;Nakayama et al, 2013Nakayama et al, , 2014Ran et al, 2013;Shi et al, 2015;Wang et al, 2015;Yang et al, 2014). While NHEJ-mediated indel mutations have proven to be effective in animal models including mouse, zebrafish and Xenopus (Blitz et al, 2013;Kotani et al, 2015;Nakayama et al, 2013;Wang et al, 2015;Xue et al, 2014), HDR-mediated targeted DNA insertion has proven more challenging.…”

Section: Introductionmentioning

confidence: 99%

High-efficiency non-mosaic CRISPR-mediated knock-in and indel mutation in F0 Xenopus

et al. 2017

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The revolution in CRISPR-mediated genome editing has enabled the mutation and insertion of virtually any DNA sequence, particularly in cell culture where selection can be used to recover relatively rare homologous recombination events. The efficient use of this technology in animal models still presents a number of challenges, including the time to establish mutant lines, mosaic gene editing in founder animals, and low homologous recombination rates. Here we report a method for CRISPR-mediated genome editing in Xenopus oocytes with homology-directed repair (HDR) that provides efficient non-mosaic targeted insertion of small DNA fragments (40-50 nucleotides) in 4.4-25.7% of F0 tadpoles, with germline transmission. For both CRISPR/Cas9-mediated HDR gene editing and indel mutation, the gene-edited F0 embryos are uniformly heterozygous, consistent with a mutation in only the maternal genome. In addition to efficient tagging of proteins in vivo, this HDR methodology will allow researchers to create patient-specific mutations for human disease modeling in Xenopus.

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Efficient generation of knock-in transgenic zebrafish carrying reporter/driver genes by CRISPR/Cas9-mediated genome engineering

Cited by 304 publications

References 26 publications

Maximizing mutagenesis with solubilized CRISPR-Cas9 ribonucleoprotein complexes.

Maximizing mutagenesis with solubilized CRISPR-Cas9 ribonucleoprotein complexes.

Regulation of ciliary retrograde protein trafficking by the Joubert syndrome proteins ARL13B and INPP5E

High-efficiency non-mosaic CRISPR-mediated knock-in and indel mutation in F0 Xenopus

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