Cloning-free CRISPR/Cas system facilitates functional cassette knock-in in mice

Aida, Tomomi; Chiyo, Keiho; Usami, Takako; Ishikubo, Harumi; Imahashi, Risa; Wada, Yusaku; Tanaka, Kenji F.; Sakuma, Tetsushi; Yamamoto, Takashi; Tanaka, Kohichi

doi:10.1186/s13059-015-0653-x

Cited by 257 publications

(251 citation statements)

References 47 publications

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“…As direct introduction of Cas9 protein is more efficient than using a plasmid 9,19 , recombinant Cas9 protein microinjection was adopted, injecting a mixture of sgRNA, Cas9 protein and ssODN DNA into the cytoplasm of pronuclear stage zygotes 18 h after fertilization. Injected zygotes and intact controls were cultured for 3 days before each embryonic blastomere was isolated and individually analysed by sequencing (Fig.…”

Section: Hdr Efficiency In Heterozygous Mybpc3 ∆Gagt Zygotesmentioning

confidence: 99%

Correction of a pathogenic gene mutation in human embryos

Martí-Gutiérrez

Park

et al. 2017

Nature

788

512

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More than 10,000 monogenic inherited disorders have been identified, affecting millions of people worldwide. Among these are autosomal dominant mutations, where inheritance of a single copy of a defective gene can result in clinical symptoms. Genes in which dominant mutations manifest as late-onset adult disorders include BRCA1 and BRCA2, which are associated with a high risk of breast and ovarian cancers 1 , and MYBPC3, mutation of which causes hypertrophic cardiomyopathy (HCM) 2 . Because of their delayed manifestation, these mutations escape natural selection and are often transmitted to the next generation. Consequently, the frequency of some of these founder mutations in particular human populations is very high. For example, the MYBPC3 mutation is found at frequencies ranging from 2% to 8% 3 in major Indian populations, and the estimated frequency of both BRCA1 and BRCA2 mutations among Ashkenazi Jews exceeds 2% 4 .HCM is a myocardial disease characterized by left ventricular hypertrophy, myofibrillar disarray and myocardial stiffness; it has an estimated prevalence of 1:500 in adults 5 and manifests clinically with heart failure. HCM is the commonest cause of sudden death in otherwise healthy young athletes. HCM, while not a uniformly fatal condition, has a tremendous impact on the lives of individuals, including physiological (heart failure and arrhythmias), psychological (limited activity and fear of sudden death), and genealogical concerns. MYBPC3 mutations account for approximately 40% of all genetic defects causing HCM and are also responsible for a large fraction of other inherited cardiomyopathies, including dilated cardiomyopathy and left ventricular non-compaction 6 . MYBPC3 encodes the thick filament-associated cardiac myosin-binding protein C (cMyBP-C), a signalling node in cardiac myocytes that contributes to the maintenance of sarcomeric structure and regulation of both contraction and relaxation 2 .Current treatment options for HCM provide mostly symptomatic relief without addressing the genetic cause of the disease. Thus, the development of novel strategies to prevent germline transmission of founder mutations is desirable. One approach for preventing second-generation transmission is preimplantation genetic diagnosis (PGD) followed by selection of non-mutant embryos for transfer in the context of an in vitro fertilization (IVF) cycle. When only one parent carries a heterozygous mutation, 50% of the embryos should be mutationfree and available for transfer, while the remaining carrier embryos are discarded. Gene correction would rescue mutant embryos, increase the number of embryos available for transfer and ultimately improve pregnancy rates.Recent developments in precise genome-editing techniques and their successful applications in animal models have provided an option for correcting human germline mutations. In particular, CRISPR-Cas9 is a versatile tool for recognizing specific genomic sequences and inducing DSBs 7-10 . DSBs are then resolved by endogenous DNA repair mechanisms, prefer...

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Section: Hdr Efficiency In Heterozygous Mybpc3 ∆Gagt Zygotesmentioning

confidence: 99%

Correction of a pathogenic gene mutation in human embryos

Martí-Gutiérrez

Park

et al. 2017

Nature

788

512

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“…Aida et al [10] have demonstrated that the efficiency was improved by microinjection of recombinant Cas9 protein and crRNA/ tracrRNA, instead of Cas9 mRNA and sgRNA targeting Actb, with a targeting vector that contained an EGFP cassette with homologous arms at the Actb locus, as a donor for HDR-mediated editing, in fertilized eggs. They With silent mutations in ssODN to avoid re-cutting the target locus reported that the microinjection of RNA-protein complexes into pronuclei enables delivery of the complex at earlier stages than that of Cas9 mRNA/sgRNA in the cytoplasm of zygotes.…”

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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“…This recombinant protein is mixed with sgRNA in vitro before microinjection. Preparing sgRNAs by direct chemical synthesis (either as sgRNA or as two RNA molecules that will assemble into a guide RNA) goes one step further toward the simplification of the genome editing process, avoiding the need of molecular cloning (Aida et al 2015, Hendel et al 2015. Finally, the Cas9 protein can be encoded from a transgene and already present in the fertilized oocytes, in which case a single sgRNA microinjection is sufficient (Sakurai et al 2016).…”

Section: Delivery Of Cas9 and Sgrna To Mouse Oocytes And The Problem mentioning

confidence: 99%

“…Although this approach is more efficient than plasmid microinjection (Horii et al 2014), it does not eliminate mosaicism (Yen et al 2014). Cas9 protein produced by bacteria can also be purchased from several sources and is efficient in cultured cells ), brain stem cells (Kalebic et al 2016), and mouse oocytes (Aida et al 2015). This recombinant protein is mixed with sgRNA in vitro before microinjection.…”

Section: Delivery Of Cas9 and Sgrna To Mouse Oocytes And The Problem mentioning

confidence: 99%

CRISPR/Cas9: a breakthrough in generating mouse models for endocrinologists

Markossian¹,

Flamant²

2016

Journal of Molecular Endocrinology

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CRISPR/Cas9 is a recent development in genome editing which is becoming an indispensable element of the genetic toolbox in mice. It provides outstanding possibilities for targeted modification of the genome, and is often extremely efficient. There are currently two main limitations to in ovo genome editing in mice: the first is mosaicism, which is frequent in founder mice. The second is the difficulty to evaluate the advent of off-target mutations, which often imposes to wait for germline transmission to ensure genetic segregation between wanted and unwanted genetic mutations. However rapid progresses are made, suggesting that these difficulties can be overcome in the near future.

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Cloning-free CRISPR/Cas system facilitates functional cassette knock-in in mice

Cited by 257 publications

References 47 publications

Correction of a pathogenic gene mutation in human embryos

Correction of a pathogenic gene mutation in human embryos

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

CRISPR/Cas9: a breakthrough in generating mouse models for endocrinologists

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