Genome editing methods in animal models

Lee, Hyunji; Yoon, Da Eun; Kim, Kyoungmi

doi:10.1080/19768354.2020.1726462

Cited by 44 publications

(28 citation statements)

References 62 publications

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“…More precisely, traditional CRISPR-based approaches are limited in their ability to generate animal models of disease that arise as a consequence of single nucleotide mutations [ 46 , 47 ]. While base editors are able to generate such animal models, such base-editing-driven approaches suffer from low efficiency, similar to when employed for in vitro applications [ 46 , 47 ]. In this vein, XMAS-TREE can greatly facilitate the use of base editors in generating animal models of human disease.…”

Section: Discussionmentioning

confidence: 99%

A Cas9-mediated adenosine transient reporter enables enrichment of ABE-targeted cells

et al. 2020

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Background Adenine base editors (ABE) enable single nucleotide modifications without the need for double-stranded DNA breaks (DSBs) induced by conventional CRIPSR/Cas9-based approaches. However, most approaches that employ ABEs require inefficient downstream technologies to identify desired targeted mutations within large populations of manipulated cells. In this study, we developed a fluorescence-based method, named “Cas9-mediated adenosine transient reporter for editing enrichment” (CasMAs-TREE; herein abbreviated XMAS-TREE), to facilitate the real-time identification of base-edited cell populations. Results To establish a fluorescent-based assay able to detect ABE activity within a cell in real time, we designed a construct encoding a mCherry fluorescent protein followed by a stop codon (TGA) preceding the coding sequence for a green fluorescent protein (GFP), allowing translational readthrough and expression of GFP after A-to-G conversion of the codon to “TGG.” At several independent loci, we demonstrate that XMAS-TREE can be used for the highly efficient purification of targeted cells. Moreover, we demonstrate that XMAS-TREE can be employed in the context of multiplexed editing strategies to simultaneous modify several genomic loci. In addition, we employ XMAS-TREE to efficiently edit human pluripotent stem cells (hPSCs), a cell type traditionally resistant to genetic modification. Furthermore, we utilize XMAS-TREE to generate clonal isogenic hPSCs at target sites not editable using well-established reporter of transfection (RoT)-based strategies. Conclusion We established a method to detect adenosine base-editing activity within a cell, which increases the efficiency of editing at multiple genomic locations through an enrichment of edited cells. In the future, XMAS-TREE will greatly accelerate the application of ABEs in biomedical research.

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Section: Discussionmentioning

confidence: 99%

A Cas9-mediated adenosine transient reporter enables enrichment of ABE-targeted cells

et al. 2020

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“…Yet surprisingly, current technologies are still unable to generate porcine ESCs for gene editing. Somatic cell nuclear transfer (SCNT) technology, which was first used to clone the sheep dolly, has been employed to generate genetic PD pigs [227][228][229][230]. Zhou et al applied Cas9/sgRNAs to obtain heterozygous mutant porcine fetal fibroblasts to serve as nucleus donors and further generated homozygous gene-targeted pigs through a single round of SCNT in that 15 tyrosinase (TYR) biallelic mutant pigs (TYR -/-) and 20 PARK2 and PINK1 doublegene KO pigs (PARK2 -/-, PINK1 -/-) were successfully generated without detecting any off-targets [229].…”

Section: Domestic Pigsmentioning

confidence: 99%

Comprehensive Perspectives on Experimental Models for Parkinson’s Disease

Chong

Zhu

et al. 2021

Aging and disease

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“…One of the major applications of the CRISPR-Cas system is the induction of disease models in animals become faster and cheaper than conventional methods. The creation of these models is intended to simulate human diseases in mouse; due to its genomic similarity to the human genome, the low distance between generations, and its cost-effectiveness [62][63][64][65]. However, problem with CRISPR-mediated creation of animal models is the generation of mosaic animals [66].…”

Section: Applications Of Crispr-cas9 In Biomedical Engineeringmentioning

confidence: 99%

An overview of applications of CRISPR-Cas technologies in biomedical engineering

Jamehdor¹,

Kaboli

Naserian

et al. 2020

Folia Histochem Cytobiol.

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Clustered Regulatory Interspaced Short Palindromic Repeats (CRISPR) is one of the major genome editing systems and allows changing DNA levels of an organism. Among several CRISPR categories, the CRISPR-Cas9 system has shown a remarkable progression rate over its lifetime. Recently, other tools including CRISPR-Cas12 and CRISPR-Cas13 have been introduced. CRISPR-Cas9 system has played a key role in the industrial cell factory's production and improved our understanding of genome function. Additionally, this system has been used as one of the major genome editing systems for the diagnosis and treatment of several infectious and non-infectious diseases. In this review, we discuss CRISPR biology, its versatility, and its application in biomedical engineering.

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Genome editing methods in animal models

Cited by 44 publications

References 62 publications

A Cas9-mediated adenosine transient reporter enables enrichment of ABE-targeted cells

A Cas9-mediated adenosine transient reporter enables enrichment of ABE-targeted cells

Comprehensive Perspectives on Experimental Models for Parkinson’s Disease

An overview of applications of CRISPR-Cas technologies in biomedical engineering

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