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The adenine base editor (ABE), capable of catalyzing A•T to G•C conversions, is an important gene editing toolbox. Here, we systematically evaluate genome-wide off-target deamination by ABEs using the EndoV-seq platform we developed. EndoV-seq utilizes Endonuclease V to nick the inosine-containing DNA strand of genomic DNA deaminated by ABE in vitro. The treated DNA is then whole-genome sequenced to identify off-target sites. Of the eight gRNAs we tested with ABE, 2–19 (with an average of 8.0) off-target sites are found, significantly fewer than those found for canonical Cas9 nuclease (7–320, 160.7 on average). In vivo off-target deamination is further validated through target site deep sequencing. Moreover, we demonstrated that six different ABE-gRNA complexes could be examined in a single EndoV-seq assay. Our study presents the first detection method to evaluate genome-wide off-target effects of ABE, and reveals possible similarities and differences between ABE and canonical Cas9 nuclease. Adenine base editors are an important contribution to the genome editing toolbox. Here the authors present EndoV-seq, an endonuclease-based assay for evaluating genomewide off-target effects of base editing.

Class 2 CRISPR–Cas proteins have been widely developed as genome editing and transcriptional regulating tools. Class 1 type I CRISPR–Cas constitutes ~60% of all the CRISPR–Cas systems. However, only type I–B and I–E systems have been used to control mammalian gene expression and for genome editing. Here we demonstrate the feasibility of using type I–F system to regulate human gene expression. By fusing transcription activation domain to Pseudomonas aeruginosa type I–F Cas proteins, we activate gene transcription in human cells. In most cases, type I–F system is more efficient than other CRISPR-based systems. Transcription activation is enhanced by elongating the crRNA. In addition, we achieve multiplexed gene activation with a crRNA array. Furthermore, type I–F system activates target genes specifically without off-target transcription activation. These data demonstrate the robustness and programmability of type I–F CRISPR–Cas in human cells. Class 1 type I CRISPR–Cas systems have not been as extensively developed for genome engineering as Class 2 systems. Here the authors modify the Type I–F CRISPR–Cas system for transcriptional activation of gene expression.

Recent advances in genome editing, especially CRISPR-Cas nucleases, have revolutionized both laboratory research and clinical therapeutics. CRISPR-Cas nucleases, together with the DNA damage repair pathway in cells, enable both genetic diversification by classical non-homologous end joining (c-NHEJ) and precise genome modification by homology-based repair (HBR). Genome editing in zygotes is a convenient way to edit the germline, paving the way for animal disease model generation, as well as human embryo genome editing therapy for some life-threatening and incurable diseases. HBR efficiency is highly dependent on the DNA donor that is utilized as a repair template. Here, we review recent progress in improving CRISPR-Cas nuclease-induced HBR in mammalian embryos by designing a suitable DNA donor. Moreover, we want to provide a guide for producing animal disease models and correcting genetic mutations through CRISPR-Cas nuclease-induced HBR in mammalian embryos. Finally, we discuss recent developments in precise genome-modification technology based on the CRISPR-Cas system.

Macrophage polarization to proinflammatory M1-like or anti-inflammatory M2-like cells is critical to mount a host defense or repair tissue. The exact molecular mechanisms controlling this process are still elusive. Here, we report that ubiquitin-specific protease 19 (USP19) acts as an anti-inflammatory switch that inhibits inflammatory responses and promotes M2-like macrophage polarization. USP19 inhibited NLRP3 inflammasome activation by increasing autophagy flux and decreasing the generation of mitochondrial reactive oxygen species. In addition, USP19 inhibited the proteasomal degradation of inflammasome-independent NLRP3 by cleaving its polyubiquitin chains. USP19-stabilized NLRP3 promoted M2-like macrophage polarization by direct association with interferon regulatory factor 4, thereby preventing its p62-mediated selective autophagic degradation. Consistent with these observations, compared to wild-type mice, Usp19−/− mice had decreased M2-like macrophage polarization and increased interleukin-1β secretion, in response to alum and chitin injections. Thus, we have uncovered an unexpected mechanism by which USP19 switches the proinflammatory function of NLRP3 into an anti-inflammatory function, and suggest that USP19 is a potential therapeutic target for inflammatory interventions.

β-Thalassemia is a global health issue, caused by mutations in the HBB gene. Among these mutations, HBB -28 （A〉G） mutations is one of the three most common mutations in China and Southeast Asia patients with β-thalassemia. Correcting this mutation in human embryos may prevent the disease being passed onto future generations and cure anemia. Here we report the first study using base editor （BE） system to correct disease mutant in human embryos. Firstly, we produced a 293T cell line with an exogenous HBB -28 （A〉G） mutant fragment for gRNAs and targeting efficiency evaluation. Then we collected primary skin fibroblast cells from a β-thalassemia patient with HBB -28 （A〉G） homozygous mutation. Data showed that base editor could precisely correct HBB -28 （A〉G） mutation in the patient＇s primary cells. To model homozygous mutation disease embryos, we consb＇ucted nuclear transfer embryos by fusing the lymphocyte or skin fibroblast cells with enucleated in vitro matured （IVM） oocytes.Notably, the gene correction efficiency was over 23.0% in these embryos by base editor. Although these embryos were still mosaic, the percentage of repaired blastomeres was over 20.0%. In addition, we found that base editor variants, with narrowed deamination window, could promote G-to-A conversion at HBB -28 site precisely in human embryos. Collectively, this study demonstrated the feasibility of curing genetic disease in human somatic cells and embryos by base editor system.

Targeted point mutagenesis through homologous recombination has been widely used in genetic studies and holds considerable promise for repairing disease- causing mutations in patients. However, problems such as mosaicism and low mutagenesis efficiency continue to pose challenges to clinical applicaUon of such approaches. Recently, a base editor （BE） system built on cytidine （C） deaminase and CRISPR/Cas9 technology was developed as an alternative method for targeted point mutagenesis in plant, yeast, and human cells. Base editors convert C in the deamination window to thymidine （T） efficiently, however, it remains unclear whether targeted base editing in mouse embryos is feasible. In this report, we generated a modified high- fidelity version of base editor 2 （HF2-BE2）, and investigated its base editing efficacy in mouse embryos. We found that HF2-BE2 could convert C to T efficiently, with up to 100% biallelic mutation efficiency in mouse embryos. Unlike BE3, HF2-BE2 could convert C to T on both the target and non-target strand, expanding the editing scope of base editors. Surprisingly, we found HF2-BE2 could also deaminate C that was proximal to the gRNA-binding region. Taken together, our work demonstrates the feasibility of generating point mutations in mouse by base editing, and underscores the need to carefully optimize base editing systems in order to eliminate proximal-site deamination.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Background This study seeks to elucidate the clinical and biochemical features of Ornithine transcarbamylase deficiency (OTCD), a pleomorphic congenital hyperammonemia disorder with a non-specific clinical phenotype. Additionally, the research aims to analyze the mutation spectrum of the OTC gene and its potential association with phenotype, as well as to perform an in silico analysis of novel OTC variants to elucidate their structure-function relationship. Methods In this study, we conducted a retrospective analysis of the clinical and biochemical features of 12 patients with OTCD and examined their metabolite profiles. Additionally, we reviewed existing literature to explore the range of mutations in the OTC gene and their possible associations with phenotypic outcomes. Furthermore, we employed the high ambiguity-driven protein-protein docking (HADDOCK) algorithm and protein-ligand interaction profiler (PLIP) to predict the pathogenicity of these mutations and elucidate the underlying mechanisms of pathogenesis in novel variants of the OTC gene. Results Nine cases, all of which were male, presented with early onset, while two cases, all of which were female, exhibited late onset. Additionally, one male case was asymptomatic. The ages of the patients at the time of diagnosis ranged from 1 day to 12 years. Peak plasma ammonia levels were found to be higher in patients with early onset compared to those with late onset. Molecular analyses identified a total of 12 different mutations, including two novel mutations (V323G and R320P). In silico analysis indicated a potential difference in affinity between wild-type and mutant OTCase, with V323G and R320P mutations leading to a decreased binding ability of OTCase to the substrate, potentially disrupting its function. Conclusion This study broadened the genetic variation spectrum of OTCD and provided substantial evidence for genetic counselling to affected families. Additionally, we elucidated variant data of OTC in Chinese patients through comprehensive literature review. Given the ongoing uncertainty surrounding the genotype-phenotype correlation of OTCD, the results of our in silico analysis can contribute to a deeper understanding of this complex, rare, and severe genetic disorder.

The noncanonical NF‐κB signaling pathway plays a critical role in a variety of biological functions including chronic inflammation and tumorigenesis. Activation of noncanonical NF‐κB signaling largely relies on the abundance as well as the processing of the NF‐κB family member p100/p52. Here, TRIM14 is identified as a novel positive regulator of the noncanonical NF‐κB signaling pathway. TRIM14 promotes noncanonical NF‐κB activation by targeting p100/p52 in vitro and in vivo. Furthermore, a mechanistic study shows that TRIM14 recruits deubiquitinase USP14 to cleave the K63‐linked ubiquitin chains of p100/p52 at multiple sites, thereby preventing p100/p52 from cargo receptor p62‐mediated autophagic degradation. TRIM14 deficiency in mice significantly impairs noncanonical NF‐κB‐mediated inflammatory responses as well as acute colitis and colitis‐associated colon cancer development. Taken together, these findings establish the TRIM14‐USP14 axis as a crucial checkpoint that controls noncanonical NF‐κB signaling and highlight the crosstalk between autophagy and innate immunity. TRIM14 is a novel positive regulator of the noncanonical NF‐κB signaling pathway. It recruits deubiquitinase USP14 to cleave the K63‐linked ubiquitin chains of p100/p52 at multiple sites, thereby preventing p100/p52 from cargo receptor p62‐mediated autophagic degradation. TRIM14 deficiency in mice significantly impairs noncanonical NF‐κB‐mediated inflammatory responses as well as acute colitis and colitis‐associated colon cancer development.

Chromatin modifications, such as histone acetylation, ubiquitination, and methylation, play fundamental roles in maintaining chromatin architecture and regulating gene transcription. Although their crosstalk in chromatin remodeling has been gradually uncovered, the functional relationship between histone ubiquitination and methylation in regulating immunity and inflammation remains unclear. Here, it is reported that USP38 is a novel histone deubiquitinase that works together with the histone H3K4 modifier KDM5B to orchestrate inflammatory responses. USP38 specifically removes the monoubiquitin on H2B at lysine 120, which functions as a prerequisite for the subsequent recruitment of demethylase KDM5B to the promoters of proinflammatory cytokines Il6 and Il23a during LPS stimulation. KDM5B in turn inhibits the binding of NF‐κB transcription factors to the Il6 and Il23a promoters by reducing H3K4 trimethylation. Furthermore, USP38 can bind to KDM5B and prevent it from proteasomal degradation, which further enhances the function of KDM5B in the regulation of inflammation‐related genes. Loss of Usp38 in mice markedly enhances susceptibility to endotoxin shock and acute colitis, and these mice display a more severe inflammatory phenotype compared to wild‐type mice. The studies identify USP38‐KDM5B as a distinct chromatin modification complex that restrains inflammatory responses through manipulating the crosstalk of histone ubiquitination and methylation. USP38 is a novel histone deubiquitinase of H2B that couples the regulation of histone ubiquitination (H2Bub) and methylation (H3K4me3) with KDM5B to selectively inhibit the transcription of the proinflammatory cytokines and prevent excessive inflammation. USP38 deficiency in mice enhances the inflammatory response, and renders animals more susceptible to acute inflammation and dextran sulfate sodium‐induced acute colitis.