Simple, Efficient CRISPR-Cas9-Mediated Gene Editing in Mice: Strategies and Methods

Low, Benjamin E.; Kutny, Peter M.; Wiles, Michael V.

doi:10.1007/978-1-4939-3661-8_2

Cited by 36 publications

(41 citation statements)

References 73 publications

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“…To determine whether Acer2 has a role in the regulation of blood S1P and dhS1P levels, we generated Acer2 mutant mouse strains by using CRISPR/Cas9 technology (36, 40, 41) as depicted in Fig. 2A.…”

Section: Resultsmentioning

confidence: 99%

“…A mouse strain that is deficient in Acer1 (33) or Acer3 (34) was generated previously in our laboratory. To generate an Acer2 knockout mouse strain, female C57BL/6J mice were superovulated and mated, and zygotes were collected and microinjected with ~2–5 pl of Cas9 mRNA (100 ng/μl) and 2 single‐guide RNAs (sgRNAs; 50 ng/μl each) as previously described (35, 36). Cas9 mRNA was synthesized from the DNA template—the Cas9 coding sequence preceded with T7 promoter—by using mMESSAGE mMCHINE T7 ULTRA Transcription Kit (Ambion, Austin, TX, USA) according to manufacturer instructions.…”

Section: Methodsmentioning

confidence: 99%

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Alkaline ceramidase 2 is essential for the homeostasis of plasma sphingoid bases and their phosphates

Low

et al. 2018

FASEB j.

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Sphingosine-1-phosphate (S1P) plays important roles in cardiovascular development and immunity. S1P is abundant in plasma because erythrocytes-the major source of S1P-lack any S1P-degrading activity; however, much remains unclear about the source of the plasma S1P precursor, sphingosine (SPH), derived mainly from the hydrolysis of ceramides by the action of ceramidases that are encoded by 5 distinct genes, acid ceramidase 1 ( ASAH1)/ Asah1, ASAH2/ Asah2, alkaline ceramidase 1 ( ACER1)/ Acer1, ACER2/ Acer2, and ACER3/ Acer3, in humans/mice. Previous studies have reported that knocking out Asah1 or Asah2 failed to reduce plasma SPH and S1P levels in mice. In this study, we show that knocking out Acer1 or Acer3 also failed to reduce the blood levels of SPH or S1P in mice. In contrast, knocking out Acer2 from either whole-body or the hematopoietic lineage markedly decreased the blood levels of SPH and S1P in mice. Of interest, knocking out Acer2 from whole-body or the hematopoietic lineage also markedly decreased the levels of dihydrosphingosine (dhSPH) and dihydrosphingosine-1-phosphate (dhS1P) in blood. Taken together, these results suggest that ACER2 plays a key role in the maintenance of high plasma levels of sphingoid base-1-phosphates-S1P and dhS1P-by controlling the generation of sphingoid bases-SPH and dhSPH-in hematopoietic cells.-Li, F., Xu, R., Low, B. E., Lin, C.-L., Garcia-Barros, M., Schrandt, J., Mileva, I., Snider, A., Luo, C. K., Jiang, X.-C., Li, M.-S., Hannun, Y. A., Obeid, L. M., Wiles, M. V., Mao, C. Alkaline ceramidase 2 is essential for the homeostasis of plasma sphingoid bases and their phosphates.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Alkaline ceramidase 2 is essential for the homeostasis of plasma sphingoid bases and their phosphates

Low

et al. 2018

FASEB j.

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“…The founder animals are backcrossed to the appropriate strain, and offspring are genotyped for the required events. Subsequently, heterozygous animals are bred to generate homozygous animals (for our detailed methodology see Low et al [2016]). a The high degree of variation in efficiency is linked we believe to microinjection conditions and the targeting of different chromosomal loci.…”

Section: Discussionmentioning

confidence: 99%

Development of Humanized Mice in the Age of Genome Editing

Hosur

Low

Avery

et al. 2017

J of Cellular Biochemistry

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Mice are the most commonly used model organisms to study human disease. Many genetic human diseases can be recapitulated by modifying the mouse genome, which permits testing of existing and novel therapeutics, including combinatorial therapeutics, without putting humans at risk. Specifically, the development of “humanized” mice, i.e., severely immunodeficient mice engrafted with functional human hematopoietic and immune cells and tissues, has revolutionized our ability to study and model human diseases in preclinical in vivo systems. Until recently it has been challenging to develop strains of humanized mice with targeted mutations or that transgenically express human genes with site-specific mutations, permitting optimal growth of functional human cells and tissues. However, recent advances in targeted nuclease-based genetic engineering have enabled precise modification and development of humanized mouse models at an unprecedented pace. These modifications permit optimal growth of functional human cells and tissues and can replicate human genetically determined diseases.

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“…The CRISPR approach is also useful for the creation of genetically modified animal models for the study of human diseases [Low et al, 2016], especially by the modification of the mouse genome since mice have a short generation time and are comparatively easy and inexpensive to produce and maintain. Thus, mice provide an ideal mammal in which to examine genome functions and complexities.…”

Section: Recent Applications Of Crisprmentioning

confidence: 99%

CRISPR Editing Technology in Biological and Biomedical Investigation

White

Kaminski

Young

et al. 2017

J of Cellular Biochemistry

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The CRISPR or clustered regularly interspaced short palindromic repeats system is currently the most advanced approach to genome editing and is notable for providing an unprecedented degree of specificity, effectiveness and versatility in genetic manipulation. CRISPR evolved as a prokaryotic immune system to confer provide an acquired immunity and resistance to foreign genetic elements such as bacteriophages. It has recently been developed into a tool for the specific targeting of nucleotide sequences within complex eukaryotic genomes for the purpose of genetic manipulation. The power of CRISPR lies in its simplicity and ease of use, its flexibility to be targeted to any given nucleotide sequence by the choice of an easily synthesized guide RNA, and its ready ability to continue to undergo technical improvements. Applications for CRISPR are numerous including creation of novel transgenic cell animals for research, high-throughput screening of gene function, potential clinical gene therapy and nongene-editing approaches such as modulating gene activity and fluorescent tagging. In this prospect article, we will describe the salient features of the CRISPR system with an emphasis on important drawbacks and considerations with respect to eliminating off-target events and obtaining efficient CRISPR delivery. We will discuss recent technical developments to the system and we will illustrate some of the most recent applications with an emphasis on approaches to eliminate human viruses including HIV-1, JCV and HSV-1 and prospects for the future.

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Simple, Efficient CRISPR-Cas9-Mediated Gene Editing in Mice: Strategies and Methods

Cited by 36 publications

References 73 publications

Alkaline ceramidase 2 is essential for the homeostasis of plasma sphingoid bases and their phosphates

Alkaline ceramidase 2 is essential for the homeostasis of plasma sphingoid bases and their phosphates

Development of Humanized Mice in the Age of Genome Editing

CRISPR Editing Technology in Biological and Biomedical Investigation

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