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Detection of cytosolic DNA by the cyclic GMP-AMP (cGAMP) synthase (cGAS)-stimulator of interferon genes (STING) pathway provides immune defense against pathogens and cancer but can also cause autoimmunity when overactivated. The exonuclease three prime repair exonuclease 1 (TREX1) degrades DNA in the cytosol and prevents cGAS activation by self-DNA. Loss-of-function mutations of the TREX1 gene are linked to autoimmune diseases such as Aicardi-Goutières syndrome, and mice deficient in TREX1 develop lethal inflammation in a cGAS-dependent manner. In order to determine the type of cells in which cGAS activation drives autoinflammation, we generated conditional cGAS knockout mice on the Trex1-/- background. Here, we show that genetic ablation of the cGAS gene in classical dendritic cells (cDCs), but not in macrophages, was sufficient to rescue Trex1-/- mice from all observed disease phenotypes including lethality, T cell activation, tissue inflammation, and production of antinuclear antibodies and interferon-stimulated genes. These results show that cGAS activation in cDC causes autoinflammation in response to self-DNA accumulated in the absence of TREX1. [ABSTRACT FROM AUTHOR]

Huntington's disease (HD) is an autosomal dominant progressive neurodegenerative disorder, caused by a CAG/polyglutamine (polyQ) repeat expansion in the Huntingtin (HTT) gene. 2e polyQ tract is located in and transcribed from N-terminal HTT of exon 1. HTT is a large multifaceted protein, which participates in a range of cellular functions. Previous studies have shown that truncated HTT, which lacks N-terminus, retains specific functions that can produce neuroprotective benefits. It gives an insight that it is possible to repair HD by removing deleterious N-terminal HTT with CRISPR/Cas9, without compromising functions of remaining HTTpeptides. To successfully generate functional truncated HTTproteins, an alternative downstream ATG start codon that is capable of initiating truncated HTT expression is required. In this study, we searched all possible in-frame ATGs before exon 7 and demonstrated that one of them can indeed initiate the downstream GFP expression in plasmids. We then tried to remove endogenous N-terminal HTT with an optimized dual-sgRNA strategy by CRISPR/Cas9; however, we cannot detect obvious traits of truncated HTTexpression. Our results suggest that noncanonical ATGs of N-terminal HTTmay not be effective in the genomic context, as in the construct context. Nevertheless, our study examined the therapeutic efficacy of downstream noncanonical ATGs for protein translation and also provided an optimized dual-sgRNA strategy for further genome manipulation of the HTT gene. [ABSTRACT FROM AUTHOR]

Oculocutaneous albinism type 1 (OCA1), resulting from pathogenic variants in the tyrosinase (TYR) gene, refers to a group of phenotypically heterogeneous autosomal recessive disorders characterized by a partial or a complete absence of pigment in the skin/hair and is also associated with common developmental eye defects. In this study, we identified two novel compound heterozygous TYR variants from a Chinese hypopigmentary patient by whole-exome sequencing. Specifically, the two variants were c.-89T>G, located at the core of the initiator E-box (Inr E-box) of the TYR promoter, and p.S16Y (c.47C>A), located within the signal sequence. We performed both in silico analysis and experimental validation and verified these mutations as OCA1 variants that caused either impaired or complete loss of function of TYR. Mechanistically, the Inr E-box variant dampened TYR binding to microphthalmia-associated transcription factor, a master transcriptional regulator of the melanocyte development, whereas the S16Y variant contributed to endoplasmic reticulum retention, a common and principal cause of impaired TYR activity. Interestingly, we found that the Inr E-box variant creates novel protospacer adjacent motif sites, recognized by nucleases SpCas9 and SaCas9-KKH, respectively, without compromising the functional TYR coding sequence. We further used allele-specific genomic editing by CRISPR activation to specifically target the variant promoter and successfully activated its downstream gene expression, which could lead to potential therapeutic benefits. In conclusion, this study expands the spectrum of TYR variants, especially those within the promoter and noncoding regions, which can facilitate genetic counseling and clinical diagnosis of OCA1. [ABSTRACT FROM AUTHOR]