Creating a monomeric endonuclease TALE-I-SceI with high specificity and low genotoxicity in human cells

Background: Premature ovarian insufficiency (POI) is a severe disorder of female infertility, characterized by 4-6 months of amenorrhea before the age of 40 years, with elevated follicle stimulating hormone (FSH) levels (> 25 IU/ L). Although several genes have been reported to contribute to the genetic basis of POI, the molecular mechanism of POI remains unclear. Methods: Whole-exome sequencing (WES) was performed. Sanger sequencing was carried out to validate the variant in the proband and her mother. In silico algorithms were used to analyze the mutational effect of the variant. Protein 3D structural modeling was used for predicting mutated protein structures. Vector construction and plasmids transfection were performed, and subsequently RNA-sequencing (RNA-seq) was carried out in each group to dissect the differentially expressed genes in wild-type (WT) and D1853H NOTCH2 mutant expressing groups. Gene Ontology analysis was also used to analyze the enriched biological processes or pathways among the differentially expressed genes. Results: We report two non-syndromic POI patients from a Chinese pedigree. The FSH level of the proband (the daughter) was 46 IU/L at the age of 22. Her menarche was at the age of 12, but she was amenorrhea at the age of 20. By WES, a rare heterozygous variant (c.5557G > C;p.D1853H) in the NOTCH2 gene was identified. In silico analysis suggested that p.D1853H was a pathogenic allele. Protein 3D structural modeling suggested that D1853H may enhance or weaken the electrostatic surface potential. By molecular analysis, we found that cells expressing the D1853H NOTCH2 mutant had similar effect in activating the NOTCH signaling pathway downstream target genes. However, 106 protein-coding genes were differentially expressed between D1853H expressing cells and WT NOTCH2 expressing cells, and these genes were enriched for collagen degradation, NCAM1 interactions and HDACs deacetylate histones, revealing a unknown underlying mechanism of the pathology that leads to POI.

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“…The procedures of culture, passage and plasmid transfection of 293FT cells were performed as previously described [2,27].…”

Section: Cell Culture and Plasmid Transfectionmentioning

confidence: 99%

NOTCH2 variant D1853H is mutated in two non-syndromic premature ovarian insufficiency patients from a Chinese pedigree

Feng

Minying

et al. 2020

J Ovarian Res

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“…This overlap in form and function make their repurposing challenging, and limits their utility for more routine applications of genome editing. More recently megaTALsfusions of a rare-cleaving homing endonuclease to a TALE-binding domain-have been reported to induce highly specific gene modifications (Boissel et al 2014;Lin et al 2015a). These enzymes have enabled integration of antitumor and anti-HIV factors into the human CCR5 gene in both primary T cells and hematopoietic stem/progenitor cells (Sather et al 2015), as well as disruption of endogenous T-cell receptor elements in T cells (Osborn et al 2016), indicating their potential for enabling and enhancing immunotherapies.…”

Section: Homing Endonucleasesmentioning

confidence: 99%

Genome-Editing Technologies: Principles and Applications

Gaj

Sirk

Shui

et al. 2016

Cold Spring Harb Perspect Biol

297

142

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Targeted nucleases have provided researchers with the ability to manipulate virtually any genomic sequence, enabling the facile creation of isogenic cell lines and animal models for the study of human disease, and promoting exciting new possibilities for human gene therapy. Here we review three foundational technologies-clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9), transcription activator-like effector nucleases (TALENs), and zinc-finger nucleases (ZFNs). We discuss the engineering advances that facilitated their development and highlight several achievements in genome engineering that were made possible by these tools. We also consider artificial transcription factors, illustrating how this technology can complement targeted nucleases for synthetic biology and gene therapy.

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“…Preference of NH or NK instead of NN for guanine recognition could be another way of improving specificity (Sun and Zhao 2013). In a different approach to increase specificity of TALENs, Lin et al fused a TALEN monomer to an engineered homing endonuclease I-SceI (Lin et al 2015). Although they have very high specificity, homing endonucleases are not modular as programmable nucleases and it is difficult to change their targeting specificity.…”

Section: Major Obstacles In Translation Of Gene Editing Tools For mentioning

confidence: 99%

Use of genome-editing tools to treat sickle cell disease

2016

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Recent advances in genome editing techniques have made it possible to modify any desired DNA sequence by employing programmable nucleases. These next generation genome-modifying tools are the ideal candidates for therapeutic applications, especially for the treatment of genetic disorders like sickle cell disease (SCD). SCD is an inheritable monogenic disorder which is caused by a point mutation in the β-globin gene. Substantial success has been achieved in the development of supportive therapeutic strategies for SCD but unfortunately there is still a lack of long-term universal cure. The only existing curative treatment is based on allogeneic stem cell transplantation from healthy donors; however, this treatment is applicable to a limited number of patients only. Hence, a universally applicable therapy is highly desirable. In this review we will discuss the three programmable nucleases that are commonly used for genome editing purposes: zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR/Cas9). We will continue by exemplifying uses of these methods to correct the sickle cell mutation. Additionally, we will present induction of fetal globin expression as an alternative approach to cure sickle cell disease. We will conclude by comparing the three methods and explaining the concerns about their use in therapy.

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Creating a monomeric endonuclease TALE-I-SceI with high specificity and low genotoxicity in human cells

Cited by 24 publications

References 31 publications

NOTCH2 variant D1853H is mutated in two non-syndromic premature ovarian insufficiency patients from a Chinese pedigree

NOTCH2 variant D1853H is mutated in two non-syndromic premature ovarian insufficiency patients from a Chinese pedigree

Genome-Editing Technologies: Principles and Applications

Use of genome-editing tools to treat sickle cell disease

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