Engineering APOBEC3A deaminase for highly accurate and efficient base editing.

Cytosine base editors (CBEs) are effective tools for introducing C-to-T base conversions, but their clinical applications are limited by off-target and bystander effects. Through structure-guided engineering of human APOBEC3A (A3A) deaminase, we developed highly accurate A3A-CBE (haA3A-CBE) variants that efficiently generate C-to-T conversion with a narrow editing window and near-background level of DNA and RNA off-target activity, irrespective of methylation status and sequence context. The engineered deaminase domains are compatible with PAM-relaxed SpCas9-NG variant, enabling accurate correction of pathogenic mutations in homopolymeric cytosine sites through flexible positioning of the single-guide RNAs. Dual adeno-associated virus delivery of one haA3A-CBE variant to a mouse model of tyrosinemia induced up to 58.1% editing in liver tissues with minimal bystander editing, which was further reduced through single dose of lipid nanoparticle-based messenger RNA delivery of haA3A-CBEs. These results highlight the tremendous promise of haA3A-CBEs for precise genome editing to treat human diseases.

Engineering a precise adenine base editor with minimal bystander editing.

Adenine base editors (ABEs) catalyze A-to-G transitions showing broad applications, but their bystander mutations and off-target editing effects raise safety concerns. Through structure-guided engineering, we found ABE8e with an N108Q mutation reduced both adenine and cytosine bystander editing, and introduction of an additional L145T mutation (ABE9), further refined the editing window to 1-2 nucleotides with eliminated cytosine editing. Importantly, ABE9 induced very minimal RNA and undetectable Cas9-independent DNA off-target effects, which mainly installed desired single A-to-G conversion in mouse and rat embryos to efficiently generate disease models. Moreover, ABE9 accurately edited the A5 position of the protospacer sequence in pathogenic homopolymeric adenosine sites (up to 342.5-fold precision over ABE8e) and was further confirmed through a library of guide RNA-target sequence pairs. Owing to the minimized editing window, ABE9 could further broaden the targeting scope for precise correction of pathogenic single-nucleotide variants when fused to Cas9 variants with expanded protospacer adjacent motif compatibility. bpNLS, bipartite nuclear localization signals.

Generation of genetically modified rat models via the CRISPR/Cas9 technology.

The RNA-guided CRISPR/Cas9 genomic editing system consists of a single guide RNA (sgRNA) and a Cas9 nuclease. The two components form a complex in cells and target the genomic loci complementary to the sgRNA. The Cas9 nuclease cleaves the target site creating a double stranded DNA break (DSB). In mammalian cells, DSBs are often repaired via error prone non-homologous end joining (NHEJ) or via homology directed repair (HDR) with the presence of donor DNA templates. Micro-injection of the CRISPR/Cas9 system into the rat embryos enables generation of genetically modified rat models. Here, we describe a detailed protocol for creating gene knockout or knockin rat models via the CRISPR/Cas9 technology.

Amelioration of an Inherited Metabolic Liver Disease through Creation of a De Novo Start Codon by Cytidine Base Editing.

Base editing technology efficiently generates nucleotide conversions without inducing excessive double-strand breaks (DSBs), which makes it a promising approach for genetic disease therapy. In this study, we generated a novel hereditary tyrosinemia type 1 (HT1) mouse model, which contains a start codon mutation in the fumarylacetoacetate hydrolase (Fah) gene by using an adenine base editor (ABE7.10). To investigate the feasibility of base editing for recombinant adeno-associated virus (rAAV)-mediated gene therapy, an intein-split cytosine base editor (BE4max) was developed. BE4max efficiently induced C-to-T conversion and restored the start codon to ameliorate HT1 in mice, but an undesired bystander mutation abolished the effect of on-target editing. To solve this problem, an upstream sequence was targeted to generate a de novo in-frame start codon to initiate the translation of FAH. After treatment, almost all C-to-T conversions created a start codon and restored Fah expression, which efficiently ameliorated the disease without inducing off-target mutations. Our study demonstrated that base editing-mediated creation of de novo functional elements would be an applicable new strategy for genetic disease therapy.

Prevalence, risk factors, and clinical correlations of underweight in Chinese newly diagnosed and drug-naïve patients with Parkinson's disease.

OBJECTIVE: Studies have shown that patients with Parkinson's disease (PD) will experience weight loss during the progression of the illness, which suggests an increased rate of underweight. However, few studies have addressed underweight in early de novo population. This study aimed to examine the prevalence, risk factors, and clinical correlations of underweight in Chinese newly diagnosed and drug-naïve patients with PD. METHODS: A total of 245 inpatients with newly diagnosed PD and 213 age-, sex-, and education-matched healthy controls were enrolled in Ningbo. BMI, demographics, supine and upright blood pressure, Montreal Cognitive Assessment (MoCA), Hamilton Anxiety Scale (HAMA), Hamilton Depression Scale (HAMD) together with fasting blood glucose, low-density lipoprotein, total cholesterol, uric acid (UA), and homocysteine were collected in all subjects. Hoehn and Yahr (HY) rating and Unified Parkinson's Disease Rating Scale (UPDRS) motor scores were also measured in patients. RESULTS: Overall, 35 (14.3%) patients were underweight and 14 (6.6%) controls were underweight. Binary regression analyses showed that low MoCA (p = 0.035), ΔSBP and ΔDBP values (both p < 0.001) were risk factors for underweight. Furthermore, correlation analysis indicated that BMI was associated with HY grade, UPDRS motor, HAMA, HAMD, MoCA, ΔSBP, ΔDBP, and UA values, stepwise multiple regression revealed significant correlations between BMI and ΔSBP (p < 0.001), ΔDBP (p = 0.001), MoCA (p = 0.002), UPDRS motor (p = 0.005), and HAMD scores (p = 0.014). CONCLUSIONS: Our study showed that the prevalence of underweight was significantly higher in Chinese newly diagnosed and drug-naïve patients with PD than in the healthy population, and several clinical variables were risk factors for underweight.

Lipidomic characteristics and clinical findings of epileptic patients treated with valproic acid.

Our early study has found valproic acid (VPA)-induced lipid dysmetabolism in animal model, however, the details of lipid profiling of VPA-treated epileptic patients remain unknown. Therefore, in this study, the blood samples of VPA-treated epileptic patients and VPA-free controls were collected for lipidomic and biochemical assays. As results, clinical data showed the changes of some blood lipid molecules in VPA-treated epileptic patients. In lipidomic assays, all 3797 annotated positive ions were identified prior to the data validation. In addition, the number of differentially expressed lipids were identified. And the 133 lipid molecules in VPA-treated cases were significantly up-regulated when compared to those in controls, while other 250 lipid metabolites were down-regulated. Further, these lipid metabolites were mainly constituted with glycerolipids, glycerophopholipids, fatty acyls, sterol lipids. In addition, the most significant elevations of metabolite molecules of triglyceride, sphingomyelin, phosphorylcholine, ceramides, phenolic phthiocerol, as well as topped reductions of phosphoethanolamines, diradylglycerols, 1α,25-dihydroxy-24-oxo-22-oxavitamin D3, 2-deoxy-20-hydroxy-5alpha-ecdysone 3-acetate, dolichyl-4 phosphate were identified respectively. Taken together, these clinical findings demonstrate that negative impacts of exposure to VPA on expression of lipid mediators, progressively disrupting the functions of lipid molecules. Interestingly, these differentially expressed metabolites may be potential biomarkers for screening VPA-induced dyslipidemia.

Adverse impact of carbon tetrachloride on metabolic function in mice.

Carbon tetrachloride (CCl4 ), a potent hepatotoxin, is linked to the histopathological outcomes of inflammatory or oxidative stress, and cell death. However, further study of additional dysmetabolism induced by CCl 4 toxicant has not yet been investigated. In current study, chronical and acute exposures of CCl 4 in mice were used to unmask the biological molecular mechanism responsible for insulin-dependent metabolic disorder. In experimental methods, a number of biochemical assays were used in assessment of biological impacts on insulin-produced pancreas and insulin-responsive hepatocyte after long- and short-term exposures of CCl 4 toxicant, respectively. As a result, data from oral glucose tolerance test showed that CCl 4 exposures induced glucose tolerance and disrupted blood insulin and glucagon levels time-dependently. Meanwhile, biochemical and histocytological analyses further indicated that CCl 4 exposures significantly resulted in liver cell damage, induced abnormal changes of hepatic and skeletal glycogen synthesis. In addition, acute CCl 4 -exposed mice showed reduced functional proteins of glucose transporter 2 (GLUT2), insulin receptor ß, insulin receptor substrate 1, glycogen synthase kinase 3ß (GSK3ß), p-AKT Ser473 associated with AKT signaling pathway in liver cells, whereas acute CCl 4 exposure downregulated the endogenous expressions of the insulin and glucagon hormonal proteins in the pancreas. Taken together, the current findings highlight that CCl 4 impaired insulin-dependent glucose homeostasis through modulating hepatocellular AKT signaling pathway in acute CCl 4 exposure and GLUT2/GSK3ß pathway in chronic CCl 4 -exposed liver cells.

Phylogeny and ecological processes influence grass coexistence at different spatial scales within the steppe biome.

Phylogenetic analyses are essential for disentangling how environmental filtering and competition determine species coexistence across spatial scales. Inner Mongolia steppe has strong environmental gradients, but how the phylogenetic relatedness of co-occurring species and phylogenetic signals of functional traits change across spatial scales remains unclear. We investigated the phylogenetic structure of grass assemblages along environmental gradients from regional to local scales, and measured functional traits within assemblages. We compared phylogenetic signals of plant traits between the same numbers of species randomly selected from the regional pool and species observed at the local scale, did phylogenetic principal component analysis to infer the main factors driving species coexistence, and examined the key plant trait-environment relationships across the phylogeny to reveal ecological adaptation mechanisms. Regionally, grass species were phylogenetically clustered with contrasting climate preferences. With decreasing spatial scales, species richness declined, changing from phylogenetically clustered to overdispersed, and phylogenetic signals of plant traits became weaker. At the local scale, grass assemblages were structured by soil water content and neighbor density, and the trait-environment relationships were less clear than those at the regional scale. This study demonstrated that at smaller scales, co-occurring grass species in the steppe tended to be more phylogenetically overdispersed, and that phylogenetic signals of plant functional traits became weaker with increasing abiotic and biotic interactions. Our findings contributed evidence for understanding species coexistence and maintenance at scales spanning regional to local communities in the East Asia steppe biome.

Immunophenotypes of Ductal Epithelial Cells in Advanced Pancreatic Ductal Adenocarcinoma.

BACKGROUND/AIMS: In this report, we aimed to investigate the distribution and characterization of biomarker-immunolabeled ductal epithelium in advanced pancreatic ductal adenocarcinoma (PDAC). DESIGN: Eighteen patients with PDAC were medically diagnosed prior to being treated periodically. All clinical records were collected and assayed. The PDAC samples were subjected to routine biochemical tests and immuno-stains of immunohistochemistry and immunofluorescence. RESULTS: As results, immunohistochemical findings indicated pancreatic ductal cells in PDAC showed plenty of Ki-67, P53, RP, SAM positive cells expressed in nuclei. In addition, ductal epithelial cells exhibited numerous positive cells of CK7, 18, 19, 20 biomarkers, respectively. Interestingly, compared to non-PDAC controls, immunostaining results showed that endocrine hormones were positively expressed in pancreatic ducts of PDAC, characterized with increased insulin-, Ngn3-, PDX1-fluorescence-labeled cells, and reduced F-box and WD-40 domain protein 7 (Fbxw7)-labeled cells in ducts. CONCLUSION: These clinicopathologic findings preliminarily disclose that there may be a potential for insulin-producing cells in PDAC, possibly through negatively inducing Fbxw7 ubiquitination in pancreatic ducts.

High-efficiency and heritable gene targeting in mouse by transcription activator-like effector nucleases.

Transcription activator-like effector nucleases (TALENs) are a powerful new approach for targeted gene disruption in various animal models, but little is known about their activities in Mus musculus, the widely used mammalian model organism. Here, we report that direct injection of in vitro transcribed messenger RNA of TALEN pairs into mouse zygotes induced somatic mutations, which were stably passed to the next generation through germ-line transmission. With one TALEN pair constructed for each of 10 target genes, mutant F0 mice for each gene were obtained with the mutation rate ranged from 13 to 67% and an average of ∼40% of total healthy newborns with no significant differences between C57BL/6 and FVB/N genetic background. One TALEN pair with single mismatch to their intended target sequence in each side failed to yield any mutation. Furthermore, highly efficient germ-line transmission was obtained, as all the F0 founders tested transmitted the mutations to F1 mice. In addition, we also observed that one bi-allele mutant founder of Lepr gene, encoding Leptin receptor, had similar diabetic phenotype as db/db mouse. Together, our results suggest that TALENs are an effective genetic tool for rapid gene disruption with high efficiency and heritability in mouse with distinct genetic background.

Prevalence of asymptomatic Bordetella pertussis and Bordetella parapertussis infections among school children in China as determined by pooled real-time PCR: a cross-sectional study.

BACKGROUND: Studies have documented that older children and adolescents act as a reservoir of Bordetella pertussis infection for young infants who have not yet completed their primary immunization schedule. Asymptomatic pertussis infection has been reported during outbreaks. This cross-sectional study aimed to investigate whether B. pertussis and Bordetella parapertussis can colonize the nasopharynx of healthy school children, using culture and pooled real-time PCR with targets for insertion sequences IS481 and IS1001. METHODS: Nasopharyngeal (NP) swabs were taken from 629 asymptomatic school children aged 7 to 15 y in 4 counties of China during the period July-September 2011. The number of subjects included in each county ranged from 153 to 165. The 4 counties selected are located in the north, south, east, and southwest regions of China. NP swabs were inoculated onto Regan-Lowe agar for isolation of suspected Bordetella organisms. Pooled real-time PCRs were used to detect B. pertussis and B. parapertussis based on the IS481 and IS1001 targets separately. RESULTS: Of the 629 subjects, 2 (0.3%) and 30 (4.8%) were confirmed to be culture-positive and PCR-positive, respectively, for B. pertussis, and 1 (0.2%) and 13 (2.1%) were confirmed to be culture-positive and PCR-positive, respectively, for B. parapertussis. All culture-positive samples were also PCR-positive. Furthermore, positive B. pertussis and B. parapertussis samples were found in all counties. CONCLUSIONS: Our results indicate that asymptomatic B. pertussis infections are common in school children in China, and asymptomatic B. parapertussis infections are more prevalent than previously documented.

Differential freezing resistance and photoprotection in C3 and C4 eudicots and grasses.

Globally, C4 plants dominate hot, open environments, but this general pattern is underpinned by important differences in the biogeography of C4 lineages. In particular, the species richness of C4 Poaceae (grasses) increases strongly with increasing temperature, whereas that of the major C4 eudicot group Chenopodiaceae correlates positively with aridity. Freezing tolerance is a crucial determinant of biogeographical relationships with temperature and is mediated by photodamage and cellular disruption by desiccation, but little is known about differences between C4 families. This study hypothesized that there is a greater risk of freezing damage via these mechanisms in C4 Poaceae than Chenopodiaceae, that freezing protection differs between the taxonomic groups, and that freezing tolerance of species is linked to arid habitat preference. Chlorophyll fluorescence, water relations, and freezing injury were compared in four C3 and six C4 species of Poaceae and Chenopodiaceae from the same Mongolian flora. Contrary to expectations, freezing-induced leaf mortality and photodamage were lower in Poaceae than Chenopodiaceae species, and unrelated to photosynthetic pathway. The freezing resistance of Poaceae species resulted from constitutive protection and cold acclimation and an ability to protect the photosynthetic apparatus from photodamage. Freezing protection was associated with low osmotic potential and low tissue elasticity, and freezing damage was accompanied by electrolyte leakage, consistent with cell-membrane disruption by ice. Both Chenopodiaceae and Poaceae had the potential to develop cold acclimation and withstand freezing during the growing season, which conflicted with the hypothesis. Instead, freezing tolerance was more closely associated with life history and ecological preference in these Mongolian species.

Bioinformatics approach and experimental validation reveal the hepatoprotective effect of pachyman against acetaminophen-associated liver injury.

Pachyman, known as Poria cocos polysaccharides, refers to the bioactive compounds isolated from Poria cocos. Pachyman is thought to exert cytoprotective action. However, the detailed mechanisms of pachyman action for hepatoprotection remain unknown. In this study, we aimed to assess the therapeutic actions, molecular mechanisms, and key target proteins of pachyman in the treatment of liver injury through network pharmacology and molecular docking assays. Furthermore, these bioinformatic findings were validated by an acetaminophen (APAP)-induced liver injury in vivo. Primarily using bioinformatic analysis, we screened and characterized 12 genes that act as potential therapeutic targets of pachyman against APAP-induced liver injury, in which all core targets were obtained. By using enrichment analysis, these core target genes of pachyman were characterized to reveal the pharmacological functions and molecular mechanisms of anti-liver injury induced by APAP. A molecular docking simulation was further performed to certain anti-liver injury target proteins of pachyman, including cytochrome P450 3A4 enzyme (CYP3A4) and inducible nitric oxide synthase (NOS2). In animal experiments, pachyman exerted potent hepatoprotective activities in prenatal APAP-exposed offspring livers, characterized by activated hepatocellular CYP3A4 and NOS2 expressions. These current findings have thus indicated that pachyman exerts hepatoprotective effects and may be the promising nutraceuticals for the treatment of APAP-induced liver injury.

The seroepidemiology of immunoglobulin G antibodies against pertussis toxin in China: a cross sectional study.

BACKGROUND: Pertussis is a reported vaccine-preventable respiratory disease in China. Because the routine laboratory methods for diagnosis are not in use, the reported cases are mainly in infants with classical paroxysmal cough and the true incidence related to pertussis is most likely under estimated. In China, however, few studies have attempted to address this issue. The purpose of this cross sectional study was to estimate the incidence rates using the method of sero-epidemiology of immunoglobulin (Ig) G antibodies against pertussis toxin (PT) among healthy populations in China. METHODS: Blood samples were obtained from 1313 healthy individuals aged 0 to 95 years in Guangdong province of China throughout September 2010. Serum IgG antibodies against PT were determined by commercial ELISA kits. Subjects with concentration of anti-PT IgG higher than 30 IU/mL were indicated to have recent Bordetella pertussis infection, if they have not received a booster dose of pertussis vaccine within one year. RESULTS: Of the 1313 study subjects, 117 (8.91%) were found to have anti-PT antibodies higher than 30 IU/mL. The estimated incidence of recent infection was thus 9395 per 100,000 for individuals older than 7 years. Peaks of the estimated incidence rate of recent infection were found to be 11561 per 100,000 in age group of 41-50 years and 11428 per 100,000 in the group aged 13-19 years. CONCLUSIONS: Our study indicated that B.pertussis infections are considerablely common, particularly in adolescents and adults in China. The study also stresses the importance of laboratory diagnosis for pertussis and employment of booster dose of pertussis vaccine in adolescents and adults in this country.

[Surveillance and analysis on the pathogenic features of Salmonella in Guangdong province in 2010].

OBJECTIVE: In order to better understand the nature of Salmonella infection in diarrheal patients in Guangdong province, the study analyzed the serum types, antibiotic resistance and molecular determinants of the isolated Salmonella strains. METHODS: In year 2010, 8405 diarrhea patients from 16 surveillant hospital in Guangzhou, Zhongshan, Dongguan, Zhuhai, Maoming, Yangjiang and Jiangmen cities in Guangdong province, were recruited in the study. A total of 8405 fecal specimen were collected and subjected to Salmonella isolation and culture. The isolated Salmonella strains were further analyzed via serotyping, antimicrobial susceptibility testing, and PFGE. The χ(2) test was applied to compare the differences between the isolated Salmonella strains in different seasons and districts. BioNumerics software was used to analyze the PFGE results in order to determine the correlation between different Salmonella strains. RESULTS: The positive rate of the surveillant Salmonella in Guangdong province was 3.58% (301/8405) in 2010; with the gender ratio at 1.34:1 (166/124). Salmonella infection was found in all age groups, and most in infants, accounting for 57.48% (173/301). The isolated rates of Salmonella were separately 3.48% (61/1751), 4.97% (134/2695), 3.08% (73/2370) and 2.08% (33/1589) in the four seasons; and the difference was statistically significant (χ(2) = 27.29, P < 0.01). The isolated rates of Salmonella in different regions were as follows: Zhuhai 15.43% (25/162), Maoming 7.53% (18/239), Dongguan 6.51% (39/599), Yangjiang 3.64% (14/385), Zhongshan 3.03% (70/2309), Guangzhou 2.90% (126/4349) and Jiangmen 2.49% (9/362). The difference between regions was statistically significant (χ(2) = 100.75, P < 0.01). Except one strain of the isolated Salmonella cannot be serotyped, the other 300 strains were divided into 42 serotypes, of which Salmonella typhimurium and Salmonella enteritidis were dominant, account for 45.18% (136/301) and 10.96% (33/301) respectively. Although over 85% of Salmonella were sensitive to cephalosporin, ACSSuT resistance patterns (defined as resistance to at least ampicillin, chloramphenicol, streptomycin, sulfamethoxazole and tetracycline) reached 34.88% (105/301), the highest resistant rate was found in serotype Salmonella typhimurium, as high as 65.44% (89/136). 136 strains of Salmonella typhimurium were divided into 51 PFGE types, showed great genetic diversity. 33 strains of Salmonella enteritidis were divided into 18 PFGE types. The strains with same PFGE pattern may have different drug-resistant patterns, and vice versa. CONCLUSION: Salmonella typhimurium and Salmonella enteritidis were the dominant serotypes causing infectious diarrhea in Guangdong province. Cephalosporin was the primary choice in clinical medicine. However, Salmonella typhimurium was resistant to drug most seriously in Guangdong province. There was no significant correlation between Salmonella resistance patterns and PFGE type.

Innovatively treat rural food waste through producing organic grains.

Food waste (FW) in a whole country contains a large amount of nitrogen which could be used to replace chemical fertilizers to produce organic grains, thus mitigating environmental pollution from the source. A 2-year field experiment was carried out using rural FW to grow organic grains in Shandong Province, China. Different proportions of FW and cattle manure were designed: FM0, 100% cattle manure compost (CMC); FM1, 75% CMC + 25% FW; FM2, 50% CMC + 50% FW; FM3, 25% CMC + 75% FW; FM4, 100% FW; CF, 100% chemical fertilizer; CK, without any fertilizers. Compared with CK and FM0, the application of FW significantly increased the total nitrogen, total phosphorus, and total potassium content of the soil. Simultaneously, all the three indicators increased with the increase of the proportion of FW. FW did not cause increase of contents of heavy metals such as cuprum, zinc, and chromium in the soils, nor did it increase the heavy metals in the grains. Using FW to replace all cattle manure, the total organic yield of grains reached to an average of 18,163 kg ha-1. We found that 1 kg dry FW could produce 1.64 kg organic grains under organic conditions, with the average net income being 5.42 times that of chemical mode. Our findings may provide an innovative solution for treating rural food wastes, ensuring food safety, and conservating the agriculture ecosystem.

Immunogenicity of meningococcal polysaccharide conjugate vaccine as a replacement for meningococcal polysaccharide vaccine in children in Guangzhou, China.

To assess the durability of antibody persistence after substitution of the MPCV vaccine for the MPSV-A vaccine in children, an observational study was conducted in children who voluntarily received two doses of MPCV-AC instead of MPSV-A between March 2017 and March 2018 in Guangzhou, China. In total, 131 and 47 participants were enrolled in the 3-year-old and 6-year-old groups, respectively. In the 3-year-old group, the seroprotection rate and GMT values for Men A and Men C were raised significantly after 1-month post- dose 1 MPSV booster vaccination. All immune indicators were significantly lower in pre- dose 1 MPSV booster vaccination in the 3-year-old group than after pre- dose 2 MPSV booster vaccination in the 6-year-old group. While no significant differences were found in most immune indicators between the 1-month post- dose 1 MPSV booster vaccination in the 3-year-old group and pre- dose 2 MPSV booster vaccination in 6-year-old group. The substitute meningococcal immunization schedule showed a good immunogenicity in young children, and good sequential immunogenicity with MPSV booster immunization.

Rescue of mis-splicing of a common SLC26A4 mutant associated with sensorineural hearing loss by antisense oligonucleotides.

A wide spectrum of SLC26A4 mutations causes Pendred syndrome and enlarged vestibular aqueduct, both associated with sensorineural hearing loss (SNHL). A splice-site mutation, c.919-2A>G (A-2G), which is common in Asian populations, impairs the 3' splice site of intron 7, resulting in exon 8 skipping during pre-mRNA splicing and a subsequent frameshift that creates a premature termination codon in the following exon. Currently, there is no effective drug treatment for SHNL. For A-2G-triggered SNHL, molecules that correct mis-splicing of the mutant hold promise to treat the disease. Antisense oligonucleotides (ASOs) can promote exon inclusion when targeting specific splicing silencers. Here, we systematically screened a large number of ASOs in a minigene system and identified a few that markedly repressed exon 8 skipping. A lead ASO, which targets a heterogeneous nuclear ribonucleoprotein (hnRNP) A1/A2 intronic splicing silencer (ISS) in intron 8, promoted efficient exon 8 inclusion in cultured peripheral blood mononuclear cells derived from two homozygous patients. In a partially humanized Slc26a4 A-2G mouse model, two subcutaneous injections of the ASO at 160 mg/kg significantly rescued exon 8 splicing in the liver. Our results demonstrate that the ISS-targeting ASO has therapeutic potential to treat genetic hearing loss caused by the A-2G mutation in SLC26A4.

Long-term correction of hemophilia B through CRISPR/Cas9 induced homology-independent targeted integration.

CRISPR/Cas9-mediated site-specific insertion of exogenous genes holds potential for clinical applications. However, it is still infeasible because homologous recombination (HR) is inefficient, especially for non-dividing cells. To overcome the challenge, we report that a homology-independent targeted integration (HITI) strategy is used for permanent integration of high-specificity-activity Factor IX variant (F9 Padua, R338L) at the albumin (Alb) locus in a novel hemophilia B (HB) rat model. The knock-in efficiency reaches 3.66%, as determined by droplet digital PCR (ddPCR). The clotting time is reduced to a normal level four weeks after treatment, and the circulating factor IX (FIX) level is gradually increased up to 52% of the normal level over nine months even after partial hepatectomy, demonstrating the amelioration of hemophilia. Through primer-extension-mediated sequencing (PEM-seq), no significant off-target effect is detected. This study not only provides a novel model for HB but also identifies a promising therapeutic approach for rare inherited diseases.