Rapid generation of mouse models with defined point mutations by the CRISPR/Cas9 system

Inui, Masafumi; Miyado, Mami; Igarashi, Maki; Tamano, Moe; Kubo, Atsushi; Yamashita, Satoshi; Asahara, Hiroshi; Fukami, Maki; Takada, Shuji

doi:10.1038/srep05396

Cited by 206 publications

(200 citation statements)

References 13 publications

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“…Wang et al [8] reported the efficient production of mice with nucleotide substitutions, in which two nucleotides were converted at Tet1 (GC>AT substitution) and Tet2 (TA>AT substitution) loci by microinjection of ssODN, sgRNAs, and Cas9 mRNA in fertilized eggs. Inui et al [9] also showed induction of point mutations in the Sf1 (C>T substitution) gene by this strategy. Reports showing the production of animals with nucleotide substitutions are summarized in Table 1.…”

Section: Hdr-mediated Production Of Nucleotide Substitution In Micementioning

confidence: 99%

Genome editing for the reproduction and remedy of human diseases in mice

Hara

Takada

2017

J Hum Genet

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With the recent progress in genome-editing technologies, such as the CRISPR/Cas9 system, genetically modified animals carrying nucleotide substitutions or large chromosomal rearrangements can be produced rapidly and at low cost. Such genome-editing techniques have been applied in the generation of animal models, especially mice, for reproducing human disease mutations, such as single-nucleotide polymorphisms (SNPs) or large chromosomal rearrangements identified by genome-wide screening analyses. While application methods are under development for various complex mutations involving genome editing for mimicking human disease-causing mutations in mice, functional studies of mouse models carrying replicated human mutations are gradually being published. In this review, we discuss the recent progress in application methods of the CRISPR/Cas9 system, focusing on the production of mouse models of diseases.

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Section: Hdr-mediated Production Of Nucleotide Substitution In Micementioning

confidence: 99%

Genome editing for the reproduction and remedy of human diseases in mice

Hara

Takada

2017

J Hum Genet

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“…CRISPR/Cas9-induced genome editing was recently performed in ␤-Thal iPSCs using a piggyBac or donor vector as the repair template (15,16). Previous studies demonstrated that single strand oligodeoxynucleotides (ssODNs) could be used as templates to generate point mutations and short sequence insertions in human cells and animal models (21)(22)(23); however, no studies have been reported showing the efficacy of using ssODNs as a template to correct the HBB mutation in ␤-Thal patient-specific iPSCs.…”

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confidence: 99%

Combining Single Strand Oligodeoxynucleotides and CRISPR/Cas9 to Correct Gene Mutations in β-Thalassemia-induced Pluripotent Stem Cells

Niu

Song

et al. 2016

Journal of Biological Chemistry

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␤-Thalassemia (␤-Thal) is one of the most common genetic diseases in the world. The generation of patient-specific ␤-Thalinduced pluripotent stem cells (iPSCs), correction of the disease-causing mutations in those cells, and then differentiation into hematopoietic stem cells offers a new therapeutic strategy for this disease. Here, we designed a CRISPR/Cas9 to specifically target the Homo sapiens hemoglobin ␤ (HBB) gene CD41/ 42(؊CTTT) mutation. We demonstrated that the combination of single strand oligodeoxynucleotides with CRISPR/Cas9 was capable of correcting the HBB gene CD41/42 mutation in ␤-Thal iPSCs. After applying a correction-specific PCR assay to purify the corrected clones followed by sequencing to confirm mutation correction, we verified that the purified clones retained full pluripotency and exhibited normal karyotyping. Additionally, whole-exome sequencing showed that the mutation load to the exomes was minimal after CRISPR/Cas9 targeting. Furthermore, the corrected iPSCs were selected for erythroblast differentiation and restored the expression of HBB protein compared with the parental iPSCs. This method provides an efficient and safe strategy to correct the HBB gene mutation in ␤-Thal iPSCs.

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“…With a CRISPR approach, injection of an sgRNA and Cas9 mRNA or protein into the one-cell embryo creates germline knockout mice in one generation-4 weeks. 31 Moreover, multiple genes can be targeted at once, which is impossible using standard approaches. 32 One-step generation of mouse knockins is also possible, but it is less efficient than creating knockouts.…”

Section: Genome Engineering In Model Organismsmentioning

confidence: 99%

Gene Editing: Powerful New Tools for Nephrology Research and Therapy

Miyagi

Humphreys

2016

JASN

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Biologic research is experiencing a transformation brought about by the ability of programmable nucleases to manipulate the genome. In the recently developed CRISPR/ Cas system, short RNA sequences guide the endonuclease Cas9 to any location in the genome, causing a DNA double-strand break (DSB). Repair of DSBs allows the introduction of targeted genetic manipulations with high precision. Cas9-mediated gene editing is simple, scalable, and rapid, and it can be applied to virtually any organism. Here, we summarize the development of modern gene editing techniques and the biology of DSB repair on which these techniques are based. We discuss technical points in applying this technology and review its use in model organisms. Finally, we describe prospects for the use of gene editing to treat human genetic diseases. This technology offers tremendous promise for equipping the nephrology research community to better model and ultimately, treat kidney diseases.

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Rapid generation of mouse models with defined point mutations by the CRISPR/Cas9 system

Cited by 206 publications

References 13 publications

Genome editing for the reproduction and remedy of human diseases in mice

Genome editing for the reproduction and remedy of human diseases in mice

Combining Single Strand Oligodeoxynucleotides and CRISPR/Cas9 to Correct Gene Mutations in β-Thalassemia-induced Pluripotent Stem Cells

Gene Editing: Powerful New Tools for Nephrology Research and Therapy

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