Conditional targeting of <i>Ispd</i> using paired Cas9 nickase and a single DNA template in mice

Lee, Angus Yiu-Fai; Lloyd, Kevin C K

doi:10.1016/j.fob.2014.06.007

Cited by 35 publications

(36 citation statements)

References 36 publications

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“…There are three previous studies reporting the generation of conditional mutant mice by introduction of two flox sites using the CRISPR/Cas9 system101112. However, no reported studies have used the one-step CRISPR/Cas9 system to generate floxed mice and subsequently tissue-specific knockout mice.…”

Section: Discussionmentioning

confidence: 99%

“…Because the binding of Cas9 is guided by simple base-pair complementarities between engineered single-guide RNA (sgRNA) and target genomic DNA sequences, it is possible to direct Cas9 to any genomic locus by providing engineered sgRNA. Previous reports have demonstrated that the generation of floxed mice in one step by the insertion of two loxP sites into the same allele of genes using the CRISPR/Cas9 system101112. However, the generation of tissue-specific knockout (KO) mice by crossing floxed mice developed using the methods outlined above with tissue-specific Cre Tg mice has yet to reported.…”

mentioning

confidence: 99%

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Hyperlipidemia and hepatitis in liver-specific CREB3L3 knockout mice generated using a one-step CRISPR/Cas9 system

Nakagawa

Oikawa

Mizuno

et al. 2016

Sci Rep

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cAMP responsive element binding protein 3-like 3 (CREB3L3), a transcription factor expressed in the liver and small intestine, governs fasting-response energy homeostasis. Tissue-specific CREB3L3 knockout mice have not been generated till date. To our knowledge, this is the first study using the one-step CRISPR/Cas9 system to generate CREB3L3 floxed mice and subsequently obtain liver- and small intestine-specific Creb3l3 knockout (LKO and IKO, respectively) mice. While LKO mice as well as global KO mice developed hypertriglyceridemia, LKO mice exhibited hypercholesterolemia in contrast to hypocholesterolemia in global KO mice. LKO mice demonstrated up-regulation of hepatic Srebf2 and its corresponding target genes. No phenotypic differences were observed between IKO and floxed mice. Severe liver injury was observed in LKO mice fed a methionine-choline deficient diet, a model for non-alcoholic steatohepatitis. These results provide new evidence regarding the hepatic CREB3L3 role in plasma triglyceride metabolism and hepatic and intestinal CREB3L3 contributions to cholesterol metabolism.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Hyperlipidemia and hepatitis in liver-specific CREB3L3 knockout mice generated using a one-step CRISPR/Cas9 system

Nakagawa

Oikawa

Mizuno

et al. 2016

Sci Rep

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“…Nevertheless, there are reports of DSD templates injected into the mouse zygote for the integration of reporter transgenes 70, 71 or a conditional allele. 72 These studies utilized conventional homology arms of variable lengths (3–9 kb) in a circular DSD template; linearization of the DSD donor is not done so as to reduce random integration in the mouse genome. Recently, DSD templates with shorter homology arms (0.3 kb) were used in mouse ESC to generate a reporter mouse line.…”

Section: Components Of Crispr Genome Editingmentioning

confidence: 99%

“…One method combines a sgRNA with Cas9n and a double-strand DNA donor template carrying both loxP sites. 72 A second method involves the synthesis of two SSO of length no more than 180 nucleotides with the 34 base pair loxP sequence placed at the center of the SSO within 10 base pairs of the DSB (Supplemental Figure IV). This approach is essentially identical to one previously published.…”

Section: Three Component Crisprmentioning

confidence: 99%

A CRISPR Path to Engineering New Genetic Mouse Models for Cardiovascular Research

Miano

Zhu

Lowenstein

2016

ATVB

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Previous efforts to target the mouse genome for the addition, subtraction, or substitution of biologically informative sequences required complex vector design and a series of arduous steps only a handful of labs could master. The facile and inexpensive clustered regularly interspaced short palindromic repeats (CRISPR) method has now superseded traditional means of genome modification such that virtually any lab can quickly assemble reagents for developing new mouse models for cardiovascular research. Here we briefly review the history of CRISPR in prokaryotes, highlighting major discoveries leading to its formulation for genome modification in the animal kingdom. Core components of CRISPR technology are reviewed and updated. Practical pointers for two-component and three-component CRISPR editing are summarized with a number of applications in mice including frameshift mutations, deletion of enhancers and non-coding genes, nucleotide substitution of protein-coding and gene regulatory sequences, incorporation of loxP sites for conditional gene inactivation, and epitope tag integration. Genotyping strategies are presented and topics of genetic mosaicism and inadvertent targeting discussed. Finally, clinical applications and ethical considerations are addressed as the biomedical community eagerly embraces this astonishing innovation in genome editing to tackle previously intractable questions.

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“…This has now been demonstrated both in cell lines and in murine models. 65,66 Again, CRISPR/Cas provides an advantage compared with ZFNs and TALENs here, because the flexibility of sgRNA-dependent targeting increases the likelihood that any particular mutation can be introduced. One of the most established methods for generating an allelic series in mammalian cells via HDR is that which uses adeno-associated virus (AAV).…”

Section: Crispr/cas and Other Editing Technologiesmentioning

confidence: 99%

High-Throughput Silencing Using the CRISPR-Cas9 System: A Review of the Benefits and Challenges

Wade

2015

SLAS Discovery

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The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system has been seized upon with a fervor enjoyed previously by small interfering RNA (siRNA) and short hairpin RNA (shRNA) technologies and has enormous potential for high-throughput functional genomics studies. The decision to use this approach must be balanced with respect to adoption of existing platforms versus awaiting the development of more "mature" next-generation systems. Here, experience from siRNA and shRNA screening plays an important role, as issues such as targeting efficiency, pooling strategies, and off-target effects with those technologies are already framing debates in the CRISPR field. CRISPR/Cas can be exploited not only to knockout genes but also to up-or down-regulate gene transcription-in some cases in a multiplex fashion. This provides a powerful tool for studying the interaction among multiple signaling cascades in the same genetic background. Furthermore, the documented success of CRISPR/Cas-mediated gene correction (or the corollary, introduction of disease-specific mutations) provides proof of concept for the rapid generation of isogenic cell lines for highthroughput screening. In this review, the advantages and limitations of CRISPR/Cas are discussed and current and future applications are highlighted. It is envisaged that complementarities between CRISPR, siRNA, and shRNA will ensure that all three technologies remain critical to the success of future functional genomics projects.

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Conditional targeting of Ispd using paired Cas9 nickase and a single DNA template in mice

Cited by 35 publications

References 36 publications

Hyperlipidemia and hepatitis in liver-specific CREB3L3 knockout mice generated using a one-step CRISPR/Cas9 system

Hyperlipidemia and hepatitis in liver-specific CREB3L3 knockout mice generated using a one-step CRISPR/Cas9 system

A CRISPR Path to Engineering New Genetic Mouse Models for Cardiovascular Research

High-Throughput Silencing Using the CRISPR-Cas9 System: A Review of the Benefits and Challenges

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