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To explore the editing specificity of CRISPR/Cpf1 system, effects of target mutation were systematically examined using a reporter activation assay, with a set of single-nucleotide mutated target site. Consistent with our previous study performed with CRISPR/Cas9, a “core” sequence region that is highly sensitive to target mutation was characterized. The region is of 4-nucleotide long, located from +4 to +7 position of the target site, and positioned within a positively charged central channel when assembled into Cpf1 endonuclease. Single-nucleotide mutation at the core sequence could abolish gene editing mediated by a however active sgRNA. With a great majority of the target sites, a kind of ‘super’ off-target gene editing was observed with both CRISPR/Cpf1 and CRISPR/Cas9. For a given target site, mutation at certain positions led to greatly enhanced off-target gene editing efficacy, even up to 10-fold of that of the fully-matched target. Study further found that these effects were determined by the identity of target nucleotide, rather than the nucleotide of crRNA. This likely suggests that the interactions between target nucleotide and the endonuclease are involved in this process.

Targeting specificity is an essential issue in the development of CRISPR-Cas technology. Using a luciferase activation assay, off-target cleavage activity of sgRNA was systematically investigated on single nucleotide-mismatched targets. In addition to confirming that PAM-proximal mismatches are less tolerated than PAM-distal mismatches, our study further identified a “core” sequence that is highly sensitive to target-mismatch. This sequence is of 4-nucleotide long, located at +4 to +7 position upstream of PAM, and positioned in a steric restriction region when assembled into Cas9 endonuclease. Our study also found that, single or multiple target mismatches at this region abolished off-target cleavage mediated by active sgRNAs, thus proposing a principle for gene-specific sgRNA design. Characterization of a mismatch sensitive “core” sequence not only enhances our understanding of how this elegant system functions, but also facilitates our efforts to improve targeting specificity of a sgRNA.

Systemic lupus erythematosus (SLE) is a complex autoimmune disease characterized by augmented type I interferon signaling. High-throughput technologies have identified plenty of SLE susceptibility single-nucleotide polymorphisms (SNPs) yet the exact roles of most of them are still unknown. Functional studies are principally focused on SNPs in the coding regions, with limited attention paid to the SNPs in non-coding regions. Long non-coding RNAs (lncRNAs) are important players in shaping the immune response and show relationship to autoimmune diseases. In order to reveal the role of SNPs located near SLE related lncRNAs, we performed a transcriptome profiling of SLE patients and identified linc00513 as a significantly over expressed lncRNA containing functional SLE susceptibility loci in the promoter region. The risk-associated G allele of rs205764 and A allele of rs547311 enhanced linc00513 promoter activity and related to increased expression of linc00513 in SLE. We also identified linc00513 to be a novel positive regulator of type I interferon pathway by promoting the phosphorylation of STAT1 and STAT2. Elevated linc00513 expression positively correlated with IFN score in SLE patients. Linc00513 expression was higher in active disease patients than those inactive ones. In conclusion, our data identify two functional promoter variants of linc00513 that contribute to increased level of linc00513 and confer susceptibility on SLE. The study provides new insights into the genetics of SLE and extends the role of lncRNAs in the pathogenesis of SLE.

To date, the non-viral vector Chimeric Antigen Receptor (CAR) T cell preparation platform, exemplified by transposons, has demonstrated significant potential in tumor immunotherapy and yielded positive results in multiple clinical trials. Nonetheless, non-methylated CpG sequences within plasmid DNA can elicit an inflammatory response via Toll-like receptor 9 (TLR9) during CAR-T cell preparation, adversely affecting transgene expression. Additionally, de novo DNA methylation programs promote T cell exhaustion, which poses a significant limitation for CAR-T cell therapy applications. High-throughput liquid protein chip and CBA analyses were utilized to determine the expression levels of inflammatory factors. Flow cytometry and luciferase reporter assays were employed for mutation screening. BALB/c mice and M-NSG mice were used to evaluate the inflammatory response and efficacy of LCG CAR-T , with TIL grouping detected via immunohistochemistry. In this study, we modified the newly discovered Passer (JL) transposon to construct a low-CpG content transposon for CAR-T cell (LCG CAR-T cell) preparation. experiments demonstrated that LCG CAR-T cells prepared using this new transposon exhibited stronger cytotoxicity. In animal models, LCG CAR-T cells significantly inhibited tumor growth and increased the populations of CD4+CAR-T cells and tumor-infiltrating lymphocytes. Furthermore, LCG CAR-T cells modulated pro-inflammatory cytokine release, thereby reducing inflammatory responses and surpassing the effects observed with unmodified CAR-T cells. Collectively, our results demonstrate the high safety and efficacy of non-viral, low CpG Passer transposon CAR-T cells, offering new avenues for improving CAR-T cell efficacy while minimizing inflammation.

Background Systemic lupus erythematosus (SLE) is a complex systemic autoimmune disease characterized by the presence of numerous autoantibodies. The interaction of infectious agents (viruses, bacteria and parasites) and a genetically susceptible host may be a key mechanism for SLE. Toxoplasma gondii is a widespread intracellular parasite that has been implicated in the pathogenesis of autoimmune diseases. However, the relationship between T. gondii infection and the increased risk of SLE in Chinese populations remains unclear. Methods The seroprevalence of T. gondii infection was assessed in 1771 serum samples collected from Chinese individuals (908 healthy controls and 863 SLE patients) from different regions of China using an enzyme-linked immunosorbent assay. Serum autoantibodies and clinical information were obtained and analysed. Results Our observations revealed a higher prevalence of anti- T. gondii antibodies (ATxA) immunoglobulin G (IgG) in serum samples from SLE patients (144/863, 16.7%) than in those from the healthy controls (53/917, 5.8%; P  < 0.0001), indicating a 2.48-fold increased risk of SLE in the ATxA-IgG + population, after adjustment for age and sex (95% confidence interval [CI] 1.70–3.62, P  < 0.0001). ATxA-IgG + SLE patients also showed a 1.75-fold higher risk of developing moderate and severe lupus symptoms (95% CI 1.14–2.70, P  = 0.011) compared to ATxA-IgG − patients. Relative to ATxA-IgG − patients, ATxA-IgG + patients were more likely to develop specific clinical symptoms, including discoid rash, oral ulcer, myalgia and alopecia. Seven antibodies, namely anti-ribosomal RNA protein (rRNP), anti-double stranded DNA (dsDNA), anti-cell membrane DNA (cmDNA), anti-scleroderma-70 (Scl-70), anti-cardiolipin (CL), anti-beta2-glycoprotein-I (B2GPI) and rheumatoid factor (RF), occurred more frequently in ATxA-IgG + patients. When combined with anti-dsDNA and RF/anti-rRNP/anti-cmDNA/ESR, ATxA-IgG significantly increased the risk for severe lupus. Conclusions Our results suggest that ATxA-IgG may be a significant risk factor for SLE prevalence and severity in Chinese populations. Graphical Abstract

The problems of tuberculosis (TB) and its drug resistance are very severe in China. New therapeutic agents or regimens to treat multi-drug-resistant tuberculosis (MDR-TB) are urgently needed. In this study, the effects of Ag85A DNA or ESAT6/Ag85A chimeric DNA vaccines alone or in combination with rifampin (RFP) were studied for the treatment of mice with MDR-TB. Eighty female BALB/c mice infected with Mycobacterium tuberculosis clinical isolate HB361, which was resistant to high level of RFP, and low level of isoniazid (INH), were treated with the saline, plasmid vector pVAX1, RFP, HSP65 DNA, Ag85A DNA, Ag85A DNA combined with RFP, chimeric ESAT6/Ag85A DNA, chimeric ESAT6/Ag85A DNA combined with RFP, respectively. Different effects of DNA vaccines for the treatment of MDR-TB were demonstrated in this study. Compared with saline group, Ag85A DNA vaccine alone or Ag85A DNA in combination with rifampin group reduced the pulmonary and splenic bacterial loads by 0.58, 0.82 and 0.51, 0.69 logs, respectively. The pathological changes of lungs were also slight and the lesions were limited in comparison with that of the control mice in which the lesions were extensive and more necrotic changes were observed. Interestingly, the chimeric Ag85A/ESAT6 DNA vaccine showed the lower effect for the treatment of MDR-TB. Ag85A DNA vaccine played a main role for the treatment of TB and MDR-TB. We believe that this is the first report of the use of DNA vaccine in the treatment of MDR-TB, and that these data suggest that DNA vaccine was effective for the treatment of MDR-TB which might have the potential contribution for resolving this problem in developing countries.

Both ZeBrafish (ZB), a recently identified DNA transposon in the zebrafish genome, and SB, a reconstructed transposon originally discovered in several fish species, are known to exhibit high transposition activity in vertebrate cells. Although a similar structural organization was observed for ZB and SB transposons, the evolutionary profiles of their homologs in various species remain unknown. In the present study, we compared their taxonomic ranges, structural arrangements, sequence identities, evolution dynamics, and horizontal transfer occurrences in vertebrates. In total, 629 ZB and 366 SB homologs were obtained and classified into four distinct clades, named ZB, ZB-like, SB, and SB-like. They displayed narrow taxonomic distributions in eukaryotes, and were mostly found in vertebrates, Actinopterygii in particular tended to be the major reservoir hosts of these transposons. Similar structural features and high sequence identities were observed for transposons and transposase, notably homologous to the SB and ZB elements. The genomic sequences that flank the ZB and SB transposons in the genomes revealed highly conserved integration profiles with strong preferential integration into AT repeats. Both SB and ZB transposons experienced horizontal transfer (HT) events, which were most common in Actinopterygii. Our current study helps to increase our understanding of the evolutionary properties and histories of SB and ZB transposon families in animals.

Both ZeBrafish (ZB), a recently identified DNA transposon in the zebrafish genome, and SB, a reconstructed transposon originally discovered in several fish species, are known to exhibit high transposition activity in vertebrate cells. Although a similar structural organization was observed for ZB and SB transposons, the evolutionary profiles of their homologs in various species remain unknown. In the present study, we compared their taxonomic ranges, structural arrangements, sequence identities, evolution dynamics, and horizontal transfer occurrences in vertebrates. In total, 629 ZB and 366 SB homologs were obtained and classified into four distinct clades, named ZB, ZB-like, SB, and SB-like. They displayed narrow taxonomic distributions in eukaryotes, and were mostly found in vertebrates, Actinopterygii in particular tended to be the major reservoir hosts of these transposons. Similar structural features and high sequence identities were observed for transposons and transposase, notably homologous to the SB and ZB elements. The genomic sequences that flank the ZB and SB transposons in the genomes revealed highly conserved integration profiles with strong preferential integration into AT repeats. Both SB and ZB transposons experienced horizontal transfer (HT) events, which were most common in Actinopterygii. Our current study helps to increase our understanding of the evolutionary properties and histories of SB and ZB transposon families in animals.

Highly pathogenic avian influenza viruses (HPAIV) have historically caused disastrous damage to the poultry industry, and recently they have shown their zoonotic potential by causing human infections and deaths. Control and prevention of HPAIV are therefore important issues for both veterinary and human public health. In this study, we constructed a plasmid, pCAGGoptiH7, encoding a codon-optimized HA gene of the H7N1 avian influenza virus A/FPV/Rostock/34 (RK/34). To evaluate the vaccine efficacy of pCAGGoptiH7, groups of specific-pathogen-free (SPF) chickens were intramuscularly inoculated with one or two doses of 100 µg, 50 µg, or 10 µg of the plasmid in 3-wk intervals. Four weeks after the single vaccination or 2 wk after the second dose, all chickens were challenged with 100CLD50 (chicken lethal dose) of highly pathogenic RK/34. After the single dose vaccination, only 90% of chickens were protected in all of the pCAGGoptiH7-immunized groups, although all of the chickens immunized generated detectable HI antibodies. After the second dose of vaccination, HI antibodies increased sharply, and chickens in the 100-µg and 50-µg pCAGGoptiH7-immunized groups were completely protected from virus challenge (no disease signs, no virus shedding, and no deaths). Low titers of virus shedding were detected in two out of ten chickens inoculated with two doses of 10-µg pCAGGoptiH7, although no disease or death was observed. These results provide a strong argument for the continued evaluation of this vaccine in field trials.

The problems of tuberculosis (TB) and its drug resistance are very severe in China. New therapeutic agents or regimens to treat multi-drug-resistant tuberculosis (MDR-TB) are urgently needed. In this study, the effects of Ag85A DNA or ESAT6/Ag85A chimeric DNA vaccines alone or in combination with rifampin (RFP) were studied for the treatment of mice with MDR-TB. Eighty female BALB/c mice infected withMycobacterium tuberculosisclinical isolate HB361, which was resistant to high level of RFP, and low level of isoniazid (INH), were treated with the saline, plasmid vector pVAX1, RFP, HSP65 DNA, Ag85A DNA, Ag85A DNA combined with RFP, chimeric ESAT6/Ag85A DNA, chimeric ESAT6/Ag85A DNA combined with RFP, respectively. Different effects of DNA vaccines for the treatment of MDR-TB were demonstrated in this study. Compared with saline group, Ag85A DNA vaccine alone or Ag85A DNA in combination with rifampin group reduced the pulmonary and splenic bacterial loads by 0.58, 0.82 and 0.51, 0.69 logs, respectively. The pathological changes of lungs were also slight and the lesions were limited in comparison with that of the control mice in which the lesions were extensive and more necrotic changes were observed. Interestingly, the chimeric Ag85A/ESAT6 DNA vaccine showed the lower effect for the treatment of MDR-TB. Ag85A DNA vaccine played a main role for the treatment of TB and MDR-TB. We believe that this is the first report of the use of DNA vaccine in the treatment of MDR-TB, and that these data suggest that DNA vaccine was effective for the treatment of MDR-TB which might have the potential contribution for resolving this problem in developing countries.