Use of CRISPR/Cas9 for the Modification of the Mouse Genome

Klimke, Ansgar; Güttler, Steffen; Kuballa, Petric; Janzen, Simone; Ortmann, Sonja; Flora, Adriano

doi:10.1007/978-1-4939-9145-7_13

Cited by 3 publications

(2 citation statements)

References 24 publications

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“…A variety of sequencing-based approaches are used to detect and quantify off-target effects caused by CRISPR ( Manghwar et al, 2020 ). To comprehensively assess the risk of off-target effects, genome-wide sequencing is a popular choice ( Klimke et al, 2019 ; Yu and Wu, 2019 ). Akcakaya et al (2018) described verification of in vivo off-targets (VIVO), a highly sensitive strategy that can identify the genome-wide off-target effects of CRISPR/Cas nucleases in vivo .…”

Section: Discussionmentioning

confidence: 99%

Inhibition of Hepatitis B Virus by AAV8-Derived CRISPR/SaCas9 Expressed From Liver-Specific Promoters

et al. 2021

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Curative therapies for chronic hepatitis B virus (HBV) infection remain a distant goal, and the persistence of stable covalently closed circular DNA (cccDNA) during HBV replication is a key barrier that is hard to break through using the drugs currently approved for HBV treatment. Due to the accuracy, efficiency, and cost-effectiveness of genome editing, CRISPR/Cas technologies are being widely used for gene therapy and in antiviral strategies. Although CRISPR/Cas could possibly clear cccDNA, ensuring its safety is requirement for application. In our study, we analyzed the liver specificity of several promoters and constructed candidate promoters in the CRISPR/Staphylococcus aureus Cas9 (SaCas9) system combined with hepatotropic AAV8 (whereby AAV refers to adeno-associated virus) to verify the efficacy against HBV. The results revealed that the reconstructed CRISPR/SaCas9 system in which the original promoter replaced with a liver-specific promoter could still inhibit HBV replication both in vitro and in vivo. Three functional guide RNAs (gRNAs), T2, T3, and T6, which target the conserved regions of different HBV genotypes, demonstrated consistently better anti-HBV effects with different liver-specific promoters. Moreover, the three gRNAs inhibited the replication of HBV genotypes A, B, and C to varying degrees. Under the action of the EnhII-Pa1AT promoter and AAV8, the expression of SaCas9 was further decreased in other organs or tissues in comparison to liver. These results are helpful for clinical applications in liver by ensuring the effects of the CRISPR/Cas9 system remain restricted to liver and, thereby, reducing the probability of undesired and harmful effects through nonspecific targeting in other organs.

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Section: Discussionmentioning

confidence: 99%

Inhibition of Hepatitis B Virus by AAV8-Derived CRISPR/SaCas9 Expressed From Liver-Specific Promoters

et al. 2021

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“…However, this conventional method takes up to months to a year to generate knockout mice for experimental studies of gene function. Recently, advancements in genetic engineering have led to the development of the CRISPR-Cas9 technology that in addition to producing genetically modified mice in a shorter time, can be applied to both cell lines and whole organisms (Klimke et al, 2019). In the present review, we will give an update of the most used gene knockout models in the field of psychiatric disorders including depression, anxiety and obsessive-compulsive disorders.…”

Section: Introductionmentioning

confidence: 99%

Gene knockout animal models of depression, anxiety and obsessive compulsive disorders

Scherma

Giunti

Fratta

et al. 2019

Psychiatric Genetics

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In the past decades, the improving knowledge of genes implicated in the pathogenesis of psychiatric disorders together with the advancements in genetic engineering has led to the creation of mice in which one or more genes are inactivated or 'knocked out'. Knockout mice are extensively used to better investigate the molecular and cellular mechanisms underlying these diseases as well as the biological role of specific genes. Moreover, they are also useful tools for developing new therapeutic strategies. The success of using knockout mice is possible due to availability of several models used to mimic some clinical manifestations reported in psychiatric patients. In the present review, we will give an update of the most used gene knockout models in the field of psychiatric disorders including depression, anxiety, and obsessive-compulsive disorder.

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