CD34+ cells from dental pulp stem cells with a ZFN-mediated and homology-driven repair-mediated locus-specific knock-in of an artificial β-globin gene

Chattong, Supreecha; Ruangwattanasuk, O; Yindeedej, W; Setpakdee, Anant; Manotham, Krissanapong

doi:10.1038/gt.2017.42

“…Various studies targeted HBB gene with different gene editing methodologies in various types of cell and cell lines (Antony et al, 2018;Cai et al, 2018;Chattong et al, 2017;Dever et al, 2016;Hoban et al, 2016a;Vakulskas et al, 2018;Wattanapanitch et al, 2018;Ye et al, 2016). However, our study is the first study testing long gRNA including SCD specific mutation and its effects in HBB gene editing.…”

Section: Discussionmentioning

confidence: 99%

Development of Gene Editing Strategies for Humanβ-Globin (HBB) Gene Mutations

Kalkan

¹

,

Kala

²

,

Yuce

³

et al. 2020

Preprint

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Recent developments in gene editing technology have enabled scientists to modify DNA sequence by using engineered endonucleases. These gene editing tools are promising candidates for clinical applications, especially for treatment of inherited disorders like sickle cell disease (SCD). SCD is caused by a point mutation in human β -globin gene (HBB). Clinical strategies have demonstrated substantial success, however there is not any permanent cure for SCD available. CRISPR/Cas9 platform uses a single endonuclease and a single guide RNA (gRNA) to induce sequence-specific DNA double strand break (DSB). When this accompanies a repair template, it allows repairing the mutated gene. In this study, it was aimed to target HBB gene via CRISPR/Cas9 genome editing tool to introduce nucleotide alterations for efficient genome editing and correction of point mutations causing SCD in 2 human cell line, by Homology Directed Repair (HDR). We have achieved to induce target specific nucleotide changes on HBB gene in the locus of mutation causing SCD. The effect of on-target activity of bone fide standard gRNA and newly developed longer gRNA were examined. It is observed that longer gRNA has higher affinity to target DNA while having the same performance for targeting and Cas9 induced DSBs. HDR mechanism was triggered by co-delivery of donor DNA repair templates in circular plasmid form. In conclusion, we have suggested methodological pipeline for efficient targeting with higher affinity to target DNA and generating desired modifications on HBB gene. Graphical abstract Highlights• HBB gene were targeted by spCas9 in close proximity to the SCD mutation • Long gRNA, which is designed to target SCD mutation, is sickle cell disease specific and exhibits indistinguishable level of cleavage activity on target locus.• Functional HBB HDR repair templates with 1 Kb and 2 Kb size were generated to cover all known mutations in the HBB gene.• Replacement of PAM sequence in HDR template with HindIII recognition sequence allowed a quick assessment of the HDR efficiency.• HDR template: Cas9-GFP vector 2:1 ratio yielded the highest HDR events/GFP+ cells. 5

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“…Various studies targeted HBB gene with different gene editing methodologies in various types of cell and cell lines (Antony et al, 2018;Cai et al, 2018;Chattong et al, 2017;Dever et al, 2016;Hoban et al, 2016a;Vakulskas et al, 2018;Wattanapanitch et al, 2018;Ye et al, 2016). However, our study is the first study testing long gRNA including SCD specific mutation and its effects in HBB gene editing.…”

Section: Discussionmentioning

confidence: 99%

Development of gene editing strategies for human β-globin (HBB) gene mutations

Kalkan

¹

,

Kala

²

,

Yuce

³

et al. 2020

Gene

View full text Add to dashboard Cite

Recent developments in gene editing technology have enabled scientists to modify DNA sequence by using engineered endonucleases. These gene editing tools are promising candidates for clinical applications, especially for treatment of inherited disorders like sickle cell disease (SCD). SCD is caused by a point mutation in human β -globin gene (HBB). Clinical strategies have demonstrated substantial success, however there is not any permanent cure for SCD available. CRISPR/Cas9 platform uses a single endonuclease and a single guide RNA (gRNA) to induce sequence-specific DNA double strand break (DSB). When this accompanies a repair template, it allows repairing the mutated gene. In this study, it was aimed to target HBB gene via CRISPR/Cas9 genome editing tool to introduce nucleotide alterations for efficient genome editing and correction of point mutations causing SCD in .

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“…In addition, the role of SCAPs during root formation is observed in many studies [5]. Interestingly, in vivo studies showed that root development can be halted when cells were isolated from tooth buds at an early stage [6]. Taken together, the easy accessibility and superior properties established them as an attractive choice for tooth and bone tissue engineering.…”

Section: Introductionmentioning

confidence: 99%

Intermittent Administration of Parathyroid Hormone Enhances Odonto/Osteogenic Differentiation of Stem Cells from the Apical Papilla via JNK and P38 MAPK Pathways

Pang

¹

,

Zhuang

²

,

Li

³

et al. 2020

Stem Cells International

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Objective. Parathyroid hormone (PTH) is considered to be essential during the tooth development. Stem cells from the apical papilla (SCAPs) are responsible for dentine formation. However, the interaction between PTH and SCAPs remains unclear. This study was aimed at investigating the effects of PTH on odonto/osteogenic differentiation capacity of SCAPs and elucidating the underlying molecular mechanisms. Materials and Methods. Here, SCAPs were isolated and identified in vitro. Effects of PTH on the proliferation of SCAPs were determined by Cell Counting Kit-8 (CCK-8), flow cytometry (FCM), and EdU. Alkaline phosphatase (ALP) activity, alizarin red staining, Western blot, and RT-PCR were carried out to detect the odonto/osteogenic differentiation of PTH-treated SCAPs as well as the participation of the MAPK signaling pathway. Results. An ALP activity assay determined that 10-8 mol/L PTH was the optimal concentration for the induction of SCAPs with no significant influence on the proliferation of SCAPs as indicated by CCK-8, FCM, and EdU. The expression of odonto/osteogenic markers was significantly upregulated in mRNA levels and protein levels. Moreover, intermittent treatment of PTH also increased phosphorylation of JNK and P38, and the differentiation was suppressed following the inhibition of JNK and P38 MAPK pathways. Conclusion. PTH can regulate the odonto/osteogenic differentiation of SCAPs via JNK and P38 MAPK pathways.

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CD34+ cells from dental pulp stem cells with a ZFN-mediated and homology-driven repair-mediated locus-specific knock-in of an artificial β-globin gene

Cited by 9 publications

References 50 publications

Development of Gene Editing Strategies for Humanβ-Globin (HBB) Gene Mutations

Development of Gene Editing Strategies for Humanβ-Globin (HBB) Gene Mutations

Development of gene editing strategies for human β-globin (HBB) gene mutations

Intermittent Administration of Parathyroid Hormone Enhances Odonto/Osteogenic Differentiation of Stem Cells from the Apical Papilla via JNK and P38 MAPK Pathways

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