DNA repair protein RAD51 enhances the CRISPR/Cas9-mediated knock-in efficiency in brain neurons

Kurihara, Taiga; Kouyama-Suzuki, Emi; Satoga, Michiru; Li, Xue; Badawi, Moataz; Thiha,; Baig, Deeba Noreen; Yanagawa, Toru; Uemura, Takeshi; Mori, Tomohisa; Tabuchi, Katsuhiko

doi:10.1016/j.bbrc.2020.01.132

Cited by 27 publications

(15 citation statements)

References 23 publications

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“…Cas9 fused to a dominant-negative 53BP1 enhances HDR and inhibits NHEJ in a target-specific manner, without modifying cellular DNA repair mechanisms overall (Jayavaradhan et al, 2019). Efforts have also been made to improve HDR by fusing Cas9 to RecA (RAD51 in eukaryotes), which plays a key role in homologous recombination (Cai et al, 2019;Kurihara et al, 2020), or by altering the conformational checkpoints for Cas9 binding to DNA (Kato-Inui et al, 2018). No product for therapeutic gene editing has yet been approved, but the first clinical trials based on this technology have demonstrated the safety of this approach (Schacker and Seimetz, 2019).…”

Section: Gene Editingmentioning

confidence: 99%

Genome Editing for CNS Disorders

Duarte

Déglon

2020

Front. Neurosci.

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Central nervous system (CNS) disorders have a social and economic burden on modern societies, and the development of effective therapies is urgently required. Gene editing may prevent or cure a disease by inducing genetic changes at endogenous loci. Genome editing includes not only the insertion, deletion or replacement of nucleotides, but also the modulation of gene expression and epigenetic editing. Emerging technologies based on ZFs, TALEs, and CRISPR/Cas systems have extended the boundaries of genome manipulation and promoted genome editing approaches to the level of promising strategies for counteracting genetic diseases. The parallel development of efficient delivery systems has also increased our access to the CNS. In this review, we describe the various tools available for genome editing and summarize in vivo preclinical studies of CNS genome editing, whilst considering current limitations and alternative approaches to overcome some bottlenecks.

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Section: Gene Editingmentioning

confidence: 99%

Genome Editing for CNS Disorders

Duarte

Déglon

2020

Front. Neurosci.

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“…In the present study, RAD51 gene was overexpressed in MAC-T cells in order to assess whether RAD51 overexpression promoted knock-in efficiency. A recent study reported that the knock-in efficiency by CRISPR/Cas9-mediated HR was enhanced up to 2.5-fold by co-transfection of RAD51 in brain neurons [25]. RAD51 gene overexpression has not only revealed such positive results but also negative effects on initiation of HR.…”

Section: A C C E P T E D a R T I C L E Discussionmentioning

confidence: 99%

Relationship between DNA mismatch repair and CRISPR/Cas9-mediated knock-in in the bovine β-casein gene locus

Kim

You

et al. 2022

Anim Biosci

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Objective: Efficient gene editing technology is critical for successful knock-in in domestic animals.RAD51 gene plays an important role in strand invasion during homologous recombination (HR) in mammals, and is regulated by CHK1 and CHK2 genes, which are upstream elements of RAD51. In addition, mismatch repair (MMR) system is inextricably linked to HR-related pathways and regulates HR via heteroduplex rejection. Thus, the aim of this study was to investigate whether CRISPR/Cas9mediated knock-in efficiency of human lactoferrin (hLF) knock-in vector in the bovine β-casein gene locus can be increased by suppressing DNA mismatch repair (MMR)-related genes (MSH2, MSH3, MSH6, MLH1 and PMS2) and overexpressing DNA double-strand break (DSB) repair-related genes (RAD51, CHK1, CHK2). Methods: MAC-T cells were transfected with a knock-in vector, RAD51, CHK1 or CHK2 overexpression vector and CRISPR/sgRNA expression vector to target the bovine β-casein gene locus, followed by treatment of the cells with CdCl 2 for 24 hours. After 3 days of CdCl 2 treatment, the knock-in efficiency was confirmed by polymerase chain reaction (PCR). The mRNA expression levels of DNA MMR-related and DNA DSB repair-related genes were assessed by quantitative realtime PCR (RT-qPCR).Results: Treatment with CdCl 2 decreased the mRNA expression of RAD51 and MMR-related genes but did not increase the knock-in efficiency in MAC-T cells. Also, the overexpression of DNA DSB repair-related genes in MAC-T cells did not significantly affect the mRNA expression of MMRrelated genes and failed to increase the knock-in efficiency. Conclusion:Treatment with CdCl 2 inhibited the mRNA levels of RAD51 and DNA MMR-related genes in MAC-T cells. However, the function of MMR pathway in relation to HR may differ in A c c e p t e d A r t i c l e various cell types or species.

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“…When this project was initiated, co-transfection of CRISPR components and donor DNA was the only option available for precise genome editing. Methods for introducing discrete substitutions in eukaryotic genomes have vastly improved and diversified since and now include adeno-associated viral vector-mediated delivery of the donor DNA for HDR [78]; various pharmacological, physical or genetic methods to improve HDR rates [79][80][81][82]; and base editing, which does not require DNA cuts nor a donor DNA [83]. Taking advantage of these recent innovations, we consider it likely that it will soon be possible to achieve efficient biallelic editing of TRIM5, along with other restriction factor genes, in human cells, resulting in a profound disruption of HIV-1 infectivity.…”

Section: Discussionmentioning

confidence: 99%

Editing of the TRIM5 Gene Decreases the Permissiveness of Human T Lymphocytic Cells to HIV-1

Désaulniers

Ortiz

Dufour

et al. 2020

Viruses

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Tripartite-motif-containing protein 5 isoform α (TRIM5α) is a cytoplasmic antiretroviral effector upregulated by type I interferons (IFN-I). We previously showed that two points mutations, R332G/R335G, in the retroviral capsid-binding region confer human TRIM5α the capacity to target and strongly restrict HIV-1 upon overexpression of the mutated protein. Here, we used clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-mediated homology-directed repair (HDR) to introduce these two mutations in the endogenous human TRIM5 gene. We found 6 out of 47 isolated cell clones containing at least one HDR-edited allele. One clone (clone 6) had both alleles containing R332G, but only one of the two alleles containing R335G. Upon challenge with an HIV-1 vector, clone 6 was significantly less permissive compared to unmodified cells, whereas the cell clones with monoallelic modifications were only slightly less permissive. Following interferon (IFN)-β treatment, inhibition of HIV-1 infection in clone 6 was significantly enhanced (~40-fold inhibition). TRIM5α knockdown confirmed that HIV-1 was inhibited by the edited TRIM5 gene products. Quantification of HIV-1 reverse transcription products showed that inhibition occurred through the expected mechanism. In conclusion, we demonstrate the feasibility of potently inhibiting a viral infection through the editing of innate effector genes. Our results also emphasize the importance of biallelic modification in order to reach significant levels of inhibition by TRIM5α.

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DNA repair protein RAD51 enhances the CRISPR/Cas9-mediated knock-in efficiency in brain neurons

Cited by 27 publications

References 23 publications

Genome Editing for CNS Disorders

Genome Editing for CNS Disorders

Relationship between DNA mismatch repair and CRISPR/Cas9-mediated knock-in in the bovine β-casein gene locus

Editing of the TRIM5 Gene Decreases the Permissiveness of Human T Lymphocytic Cells to HIV-1

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