RESUMEN
Mutations in SAMHD1, encoding SAM and HD domain-containing protein 1, cause Aicardi-Goutières syndrome (AGS) 5, an infancy-onset autoinflammatory disease characterized by neurodegeneration and chronic activation of type I interferon. Here, we report the generation and characterization of induced pluripotent stem cells (iPSCs) derived from fibroblasts and peripheral blood mononuclear cells from three AGS patients with biallelic SAMHD1 mutations. These cell lines provide a valuable source to study disease mechanisms and to assess therapeutic molecules.
Asunto(s)
Células Madre Pluripotentes Inducidas , Interferón Tipo I , Proteínas de Unión al GTP Monoméricas , Humanos , Proteína 1 que Contiene Dominios SAM y HD/genética , Células Madre Pluripotentes Inducidas/metabolismo , Leucocitos Mononucleares/metabolismo , Proteínas de Unión al GTP Monoméricas/genética , Proteínas de Unión al GTP Monoméricas/metabolismo , Mutación/genética , Interferón Tipo I/genética , Interferón Tipo I/metabolismoRESUMEN
Mutations in TREX1, encoding three prime repair exonuclease 1, cause Aicardi-Goutières syndrome (AGS) 1, an autoinflammatory disease characterized by neurodegeneration and constitutive activation of the antiviral cytokine type I interferon. Here, we report the generation and characterization of induced pluripotent stem cells (iPSCs) derived from fibroblasts from two AGS patients with biallelic TREX1 mutations. These cell lines offer a unique resource to investigate disease processes in a cell-type specific manner.
Asunto(s)
Células Madre Pluripotentes Inducidas , Interferón Tipo I , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Exodesoxirribonucleasas/genética , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Mutación/genética , Interferón Tipo I/genética , Citocinas , AntiviralesRESUMEN
In contrast to CRISPR/Cas9 nucleases, CRISPR base editors (BE) and prime editors (PE) enable predefined nucleotide exchanges in genomic sequences without generating DNA double strand breaks. Here, we employed BE and PE mRNAs in conjunction with chemically synthesized sgRNAs and pegRNAs for efficient editing of human induced pluripotent stem cells (iPSC). Whereas we were unable to correct a disease-causing mutation in patient derived iPSCs using a CRISPR/Cas9 nuclease approach, we corrected the mutation back to wild type with high efficiency utilizing an adenine BE. We also used adenine and cytosine BEs to introduce nine different cancer associated TP53 mutations into human iPSCs with up to 90% efficiency, generating a panel of cell lines to investigate the biology of these mutations in an isogenic background. Finally, we pioneered the use of prime editing in human iPSCs, opening this important cell type for the precise modification of nucleotides not addressable by BEs and to multiple nucleotide exchanges. These approaches eliminate the necessity of deriving disease specific iPSCs from human donors and allows the comparison of different disease-causing mutations in isogenic genetic backgrounds.
Asunto(s)
Adenina/química , Sistemas CRISPR-Cas , Citosina/química , Edición Génica/métodos , Células Madre Pluripotentes Inducidas/metabolismo , Mutagénesis , Mutación , ARN Mensajero/genética , Aminohidrolasas , Enfermedades Autoinmunes del Sistema Nervioso/genética , Secuencia de Bases , Proteína 9 Asociada a CRISPR , Línea Celular , Técnicas de Reprogramación Celular , Cuerpos Embrioides , Genes p53 , Células HEK293 , Humanos , Imidazoles/farmacología , Malformaciones del Sistema Nervioso/genética , Piperazinas/farmacología , ARN Guía de Kinetoplastida/genética , ARN Mensajero/metabolismo , Proteína 1 que Contiene Dominios SAM y HD/genética , TransfecciónRESUMEN
Immobilization of bioactive molecules (BAMs) on a nanometer scale is of great interest for functionalization of implant and scaffold surfaces in current biomaterials research. A system for immobilization of one or more compounds is described, which is based on nanomechanical fixation of single-stranded nucleic acids into an anodic titanium oxide layer and their subsequent hybridization with BAMs conjugated to the respective complementary strands. This paper focuses on further development and in depth understanding of the immobilization system, as some of the major findings established for common sensor applications for immobilization of single-stranded DNA onto gold surfaces cannot be transferred to the TiO2 surface. The first approach concerning the influence of the internal spacer sequence revealed the best performance for a polyadenine based sequence out of four homologous spacer sequences (A30, T30, C30, and G30). This overall best performance of the A30 spacer is attributed to an increased contour length by nucleotide staggering, which resulted in the best protection of the hybridization sequence from unfavorable interactions with the surface or damaging attacks by reactive oxygen species. The second approach comprises the implementation of a lateral spacer, also based on a homologous sequence of A30. Simultaneous as well as sequential adsorption of anchor strands and spacer strands were performed, and it could be shown that a preadsorption with high density of the spacer was most effective to increase hybridization efficiency.
