Correction of a CADASIL point mutation using adenine base editors in hiPSCs and blood vessel organoids.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a monogenic small vessel disease caused by mutations in the NOTCH3 gene. However, the pathogenesis of CADASIL remains unclear, and patients have limited treatment options. Here, we use human induced pluripotent stem cells (hiPSCs) generated from the peripheral blood mononuclear cells of a patient with CADASIL carrying a heterozygous NOTCH3 mutation (c.1261C>T, p.R421C) to develop a disease model. The correction efficiency of different adenine base editors (ABEs) is tested using the HEK293T-NOTCH3 reporter cell line. ABEmax is selected based on its higher efficiency and minimization of predicted off-target effects. Vascular smooth muscle cells (VSMCs) differentiated from CADASIL hiPSCs show NOTCH3 deposition and abnormal actin cytoskeleton structure, and the abnormalities are recovered in corrected hiPSC-derived VSMCs. Furthermore, CADASIL blood vessel organoids generated for in vivo modeling show altered expression of genes related to disease phenotypes, including the downregulation of cell adhesion, extracellular matrix organization, and vessel development. The dual adeno-associated virus (AAV) split-ABEmax system is applied to the genome editing of vascular organoids with an average editing efficiency of 8.82%. Collectively, we present potential genetic therapeutic strategies for patients with CADASIL using blood vessel organoids and the dual AAV split-ABEmax system.

Current therapies for osteoarthritis and prospects of CRISPR-based genome, epigenome, and RNA editing in osteoarthritis treatment.

Osteoarthritis (OA) is one of the most common degenerative joint diseases worldwide, causing pain, disability, and decreased quality of life. The balance between regeneration and inflammation-induced degradation results in multiple etiologies and complex pathogenesis of OA. Currently, there is a lack of effective therapeutic strategies for OA treatment. With the development of CRISPR-based genome, epigenome, and RNA editing tools, OA treatment has been improved by targeting genetic risk factors, activating chondrogenic elements, and modulating inflammatory regulators. Supported by cell therapy and in vivo delivery vectors, genome, epigenome, and RNA editing tools may provide a promising approach for personalized OA therapy. This review summarizes CRISPR-based genome, epigenome, and RNA editing tools that can be applied to the treatment of OA and provides insights into the development of CRISPR-based therapeutics for OA treatment. Moreover, in-depth evaluations of the efficacy and safety of these tools in human OA treatment are needed.

CRISPR-assisted transcription activation by phase-separation proteins.

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system has been widely used for genome engineering and transcriptional regulation in many different organisms. Current CRISPR-activation (CRISPRa) platforms often require multiple components because of inefficient transcriptional activation. Here, we fused different phase-separation proteins to dCas9-VPR (dCas9-VP64-P65-RTA) and observed robust increases in transcriptional activation efficiency. Notably, human NUP98 (nucleoporin 98) and FUS (fused in sarcoma) IDR domains were best at enhancing dCas9-VPR activity, with dCas9-VPR-FUS IDR (VPRF) outperforming the other CRISPRa systems tested in this study in both activation efficiency and system simplicity. dCas9-VPRF overcomes the target strand bias and widens gRNA designing windows without affecting the off-target effect of dCas9-VPR. These findings demonstrate the feasibility of using phase-separation proteins to assist in the regulation of gene expression and support the broad appeal of the dCas9-VPRF system in basic and clinical applications.

Identification and Analysis of Small Molecule Inhibitors of CRISPR-Cas9 in Human Cells.

Genome editing tools based on CRISPR-Cas systems can repair genetic mutations in situ; however, off-target effects and DNA damage lesions that result from genome editing remain major roadblocks to its full clinical implementation. Protein and chemical inhibitors of CRISPR-Cas systems may reduce off-target effects and DNA damage. Here we describe the identification of several lead chemical inhibitors that could specifically inhibit the activity of Streptococcus pyogenes Cas9 (SpCas9). In addition, we obtained derivatives of lead inhibitors that could penetrate the cell membrane and inhibit SpCas9 in cellulo. Two of these compounds, SP2 and SP24, were able to improve the specificity of SpCas9 in cellulo at low-micromolar concentration. Furthermore, microscale thermophoresis (MST) assays showed that SP24 might inhibit SpCas9 activity by interacting with both the SpCas9 protein and the SpCas9-gRNA ribonucleoprotein complex. Taken together, SP24 is a novel chemical inhibitor of SpCas9 which has the potential to enhance therapies that utilize SpCas9.

Generation of C-to-G transversion in mouse embryos via CG editors.

Base editors (BEs) are efficient and precise tools for generating single base conversions in living organisms. While most BE systems are limited in mediating C-to-T or A-to-G conversions, recently developed C-to-G base editors (CGBEs) could produce C-to-G transversions. CGBEs convert cytosine within the editing window to abasic intermediates, which would be replaced with any base after base excision repair (BER). By far, though the efficiency and editing scope of CGBEs have been investigated in cultured cells via gRNA library and machine-learning, the viability of CGBEs in generating mouse models has not been adequately tested. In this study, we tested the C-to-G transversion efficiency of the CGBE1 and CGBE-XRCC1 systems in mouse embryos. Our results showed that both of the CGBE systems were able to mediate C-to-G transversion on 2 out of 3 targets tested, with up to 20% frequency within the editing window. Notably, most of the groups showed over 40% of other base conversions, predominantly C-to-T. Lastly, we successfully acquired the F1 mouse carrying a disease-causing mutation. In all, our study suggested that CGBEs systems held great potential in generating mouse models and indicated that XRCC1 based system is applicable in mouse embryos.

Single AAV-mediated CRISPR-Nme2Cas9 efficiently reduces mutant hTTR expression in a transgenic mouse model of transthyretin amyloidosis.

Transthyretin (TTR) amyloidosis is a hereditary life-threatening disease characterized by deposition of amyloid fibrils. The main causes of TTR amyloidosis are mutations in the TTR gene that lead to the production of misfolded TTR protein. Reducing the production of toxic protein in the liver is a validated strategy to treat TTR amyloidosis. In this study, we established a humanized mouse model that expresses mutant human TTR (hTTR; V30M) protein in the liver to model TTR amyloidosis. Then, we compared the efficiency of reducing the expression of mutant hTTR by dual adeno-associated virus 8 (AAV8)-mediated split SpCas9 with that by single AAV8-mediated Nme2Cas9 in this model. With two gRNAs targeting different exons, dual AAV-mediated split SpCas9 system achieved efficiencies of 37% and 34% reduction of hTTR mRNA and reporter GFP expression, respectively, in the liver. Surprisingly, single AAV-mediated Nme2Cas9 treatment resulted in 65% and 71% reduction of hTTR mRNA and reporter GFP, respectively. No significant editing was identified in predicted off-target sites in the mouse and human genomes after Nme2Cas9 targeting. Thus, we provide proof of principle for using single AAV-mediated CRISPR-Nme2Cas9 to effectively reduce mutant hTTR expression in vivo, which may translate into gene therapy for TTR amyloidosis.

Homology-based repair induced by CRISPR-Cas nucleases in mammalian embryo genome editing.

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.

Dual-AAV delivering split prime editor system for in vivo genome editing.

Prime editor (PE), a new genome editing tool, can generate all 12 possible base-to-base conversions, insertion, and deletion of short fragment DNA. PE has the potential to correct the majority of known human genetic disease-related mutations. Adeno-associated viruses (AAVs), the safe vector widely used in clinics, are not capable of delivering PE (∼6.3 kb) in a single vector because of the limited loading capacity (∼4.8 kb). To accommodate the loading capacity of AAVs, we constructed four split-PE (split-PE994, split-PE1005, split-PE1024, and split-PE1032) using Rma intein (Rhodothermus marinus). With the use of a GFP-mutated reporter system, PE reconstituting activities were screened, and two efficient split-PEs (split-PE1005 and split-PE1024) were identified. We then demonstrated that split-PEs delivered by dual-AAV1, especially split-PE1024, could mediate base transversion and insertion at four endogenous sites in human cells. To test the performance of split-PE in vivo, split-PE1024 was then delivered into the adult mouse retina by dual-AAV8. We demonstrated successful editing of Dnmt1 in adult mouse retina. Our study provides a new method to deliver PE to adult tissue, paving the way for in vivo gene-editing therapy using PE.

USP19 suppresses inflammation and promotes M2-like macrophage polarization by manipulating NLRP3 function via autophagy.

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-1ß 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.

USP38 Couples Histone Ubiquitination and Methylation via KDM5B to Resolve Inflammation.

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.

Author Correction: Repurposing type I-F CRISPR-Cas system as a transcriptional activation tool in human cells.

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

Melatonin ameliorates necrotizing enterocolitis by preventing Th17/Treg imbalance through activation of the AMPK/SIRT1 pathway.

Necrotizing enterocolitis (NEC) is a severe gastrointestinal disease affecting premature infants. Mounting evidence supports the therapeutic effect of melatonin on NEC, although the underlying mechanisms remain unclear. Methods: NEC was induced in 10-day-old C57BL/6 pups via hypoxia and gavage feeding of formula containing enteric bacteria, and then, mice received melatonin, melatonin + recombinant IL-17, melatonin + anti-CD25 monoclonal antibody, melatonin + Ex-527, or melatonin + Compound C treatment. Control mice were left with their dams to breastfeed and vehicle-treated NEC pups were used as controls for treatment. Ileal tissues were collected from mice and analyzed by histopathology, immunoblotting, and flow cytometry. FITC-labeled dextran was administered to all surviving pups to evaluate gut barrier function by fluorometry. We used molecular biology and cell culture approaches to study the related mechanisms in CD4+ T cells from umbilical cord blood. Results: We demonstrated that melatonin treatment ameliorates disease in an NEC mouse model in a manner dependent on improved intestinal Th17/Treg balance. We also showed that melatonin blocks the differentiation of pathogenic Th17 cells and augments the generation of protective Treg cells in vitro. We further demonstrated that the Th17/Treg balance is influenced by melatonin through activation of AMPK in the intestine, in turn promoting SIRT1 activation and stabilization. Conclusions: These results demonstrate that melatonin-induced activation of AMPK/SIRT1 signaling regulates the balance between Th17 and Treg cells and that therapeutic strategies targeting the Th17/Treg balance via the AMPK/SIRT1 pathway might be beneficial for the treatment of NEC.

Single AAV-Mediated CRISPR-SaCas9 Inhibits HSV-1 Replication by Editing ICP4 in Trigeminal Ganglion Neurons.

Herpes simplex keratitis (HSK) is the most common cause of corneal blindness in developed nations, caused by primary or recurrent herpes simplex virus 1 (HSV-1) infection of the cornea. Latent infection of HSV-1, especially in the trigeminal ganglion (TG), causes recurrence of HSV-1 infection. As antiviral treatment is not effective on latent HSV-1, to test the possibility of inhibiting HSV-1 by SpCas9 (Streptococcus pyogenes Cas9) or SaCas9 (Staphylococcus aureus Cas9), ICP0 and ICP4, two important genes required for HSV-1 replication and reactivation, were chosen as targets. In Vero cells, SpCas9 and SaCas9 targeting ICP0 or ICP4 can effectively inhibit the proliferation of HSV-1 without affecting cell viability. No significant guide RNA (gRNA)-dependent off-targets were observed in the human genome by digenome sequencing and deep sequencing verification. Adeno-associated virus 1 (AAV1)-mediated delivery of SaCas9 inhibits HSV-1 replication by targeting ICP4 in mouse primary TG neuronal cells. SpCas9 and SaCas9 are able to inhibit HSV-1 infection in Vero cells and mouse TG neuronal cultures with high efficiency and good biosafety. AAV1-mediated delivery of SaCas9 shows great potential in treating HSK and inhibiting HSV-1 in TG neurons. Further investigations may be needed to test the inhibition of latent infections, which may result in the development of novel methods for treating viral diseases.

Benzo[a]pyrene promotes progression in tongue squamous cell carcinoma.

OBJECTIVES: Benzo[a]pyrene (B[a]P) is a member of the polycyclic aromatic hydrocarbon (PAH) family. Although the potent carcinogenicity of high-dose B[a]P has been extensively reported, the effects of long-term exposure to B[a]P on the progression of tongue squamous cell carcinoma (TSCC) are poorly understood. METHODS: In the present study, TSCC cells were treated with 5 or 50 nM of B[a]P for three months. The proliferation and chemoresistance of B[a]P-treated cells to 5-fluorouracil or cisplatin were detected by CCK8. The motility of the B[a]P-treated cells was evaluated with wound healing analysis, invasion assay, and three-dimensional culture in decellularized mouse tongue matrix. Xenograft assay was used to investigate the aggressiveness of B[a]P-treated cells. Immunofluorescence staining, terminal restriction fragment assay, and whole-genome sequence were used to determine the mutation spectrums. RESULTS: Long-term 50 nM B[a]P-treated cells exhibited increased aggressiveness and chemoresistance to 5-fluorouracil or cisplatin. In addition, data from whole-genome sequencing demonstrated that C:T to A:T transitions were the predominant nucleotide substitutions occurred in 50 nM B[a]P-treated CAL27 cells. Furthermore, 102 non-synonymous or stop-gain mutations were enriched in the extracellular-matrix-receptor interactive pathway. CONCLUSIONS: B[a]P exposure may contribute to genomic instability, and therefore, B[a]P may promote the progression of TSCC.

Repurposing type I-F CRISPR-Cas system as a transcriptional activation tool in human cells.

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.

Effective generation of maternal genome point mutated porcine embryos by injection of cytosine base editor into germinal vesicle oocytes.

Cytosine and adenine base editors are promising new tools for introducing precise genetic modifications that are required to generate disease models and to improve traits in pigs. Base editors can catalyze the conversion of C→T (C>T) or A→G (A>G) in the target site through a single guide RNA. Injection of base editors into the zygote cytoplasm can result in the production of offspring with precise point mutations, but most F0 are mosaic, and breeding of F1 heterozygous pigs is time-intensive. Here, we developed a method called germinal vesicle oocyte base editing (GVBE) to produce point mutant F0 porcine embryos by editing the maternal alleles during the GV to MII transition. Injection of cytosine base editor 3 (BE3) mRNA and X-linked Dmd-specific guide RNAs into GVoocytes efficiently edited maternal Dmd during in vitro maturation and did not affect the maturation potential of the oocytes. The edited MII oocytes developed into blastocysts after parthenogenetic activation (PA) or in vitro fertilization (IVF). However, BE3 may reduce the developmental potential of IVF blastocysts from 31.5%±0.8% to 20.4% ±2.1%. There 40%-78.3% diploid PA blastocysts had no more than two different alleles, including up to 10% embryos that had only C>T mutation alleles. Genotyping of IVF blastocysts indicated that over 70% of the edited embryos had one allele or two different alleles of Dmd. Since the male embryos had only a copy of Dmd allele, all five (5/19) F0 male embryos are homozygous and three of them were Dmd precise C>T mutation. Nine (9/19) female IVF embryos had two different alleles including a WT and a C>T mutation. DNA sequencing showed that some of them might be heterozygous embryos. In conclusion, the GVBE method is a valuable method for generating F0 embryos with maternal point mutated alleles in a single step.

TRIM14 Promotes Noncanonical NF-κB Activation by Modulating p100/p52 Stability via Selective Autophagy.

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.