The CRISPR/Cas9 system has emerged as a revolutionary tool for gene editing, widely used in the biomedical field due to its simplicity, efficiency, and cost-effectiveness. However, evidence suggests that CRISPR/Cas9 can induce off-target effects, leading to unintended mutations that may compromise the precision of gene modifications. Consequently, predicting，detecting and evaluating these off-target effects is crucial for optimizing the accuracy and reliability of CRISPR/Cas9 system. This paper provides an overview of the various methodologies and strategies, used or to be used for identifying off-target effects in CRISPR/Cas9-based genome editing, offering insights to improve the precision and safety of CRISPR applications in research and therapeutics.

Objective:Individual differences were observed in the clinical efficacy of Botulinum toxin A (BoNT-A) in the treatment of the primary Meige syndrome. Our study aimed to explore the potential associations between the clinical efficacy of BoNT-A in the treatment of the primary Meige syndrome and variants of SNAP25, SV2C and ST3GAL2, which are involving in the translocation of the BoNT-A in vivo. Methods:Patients with the primary Meige syndrome treated with BoNT-A were enrolled. Clinical efficacy was evaluated by the maximum improvement rate of motor symptoms and the duration of efficacy. Variants of SNAP25, SV2C and ST3GAL2 were obtained by Sanger sequencing. Another cohort diagnosed with primary cervical dystonia was also enrolled in the replication stage. Results:Among the 104 primary Meige syndrome patients, 80 patients (76.9%) had a good efficacy (the maximum improvement rate of motor symptoms ≥30%) and 24 (23. 1%) had a poor (the maximum improvement rate of motor symptoms <30%). As to the duration of efficacy, 52 patients (50.0%) had a long duration of efficacy (≥4 months), and 52 (50.0%) had a short (<4 months). In terms of primary Meige syndrome, SNAP25 rs6104571 was found associating with the maximum improvement rate of motor symptoms (Genotype: P = 0.02, OR = 0.26; Allele: P = 0.013, OR = 0.29), and SV2C rs31244 was found associating with the duration of efficacy (Genotype: P = 0.024, OR = 0.13; Allele: P = 0.012, OR = 0.13). Besides, we also conducted the association analyses between the variants and BoNT-A-related adverse reactions. Although, there was no statistical difference between the allele of SV2C rs31244 and BoNT-A-related adverse reactions, there was a trend (P = 0.077, OR = 2.56). In the replication stage, we included 39 patients with primary cervical dystonia to further expanding the samples' size. Among the 39 primary cervical dystonia patients, 25 patients (64.1%) had a good efficacy (the maximum improvement rate of motor symptoms ≥50%) and 14 (35.9%) had a poor (the maximum improvement rate of motor symptoms <50%). As to the duration of efficacy, 32 patients (82.1%) had a long duration of efficacy (≥6 months), and 7 (17.9%) had a short (<6 months). Integrating primary Meige syndrome and primary cervical dystonia, SV2C rs31244 was still found associating with the duration of efficacy (Genotype: P = 0.002, OR = 0. 23; Allele: P = 0.001, OR = 0. 25). Conclusion:In our study, SNAP25 rs6104571 was associated with the maximum improvement rate of motor symptoms in patients with primary Meige syndrome treated with BoNT-A, and patients carrying this variant had a lower improvement rate of motor symptoms. SV2C rs31244 was associated with duration of treatment in patients with primary Meige syndrome treated with BoNT-A and patients carrying this variant had a shorter duration of treatment. Patients with primary Meige syndrome carrying SV2C rs31244 G allele have an increase likelihood of BoNT-A-related adverse reactions. Involving 39 patients with primary cervical dystonia, the results further verify that SV2C rs31244 was associated with duration of treatment and patients carrying this variant had a shorter duration of treatment.

Background: Right ventricle failure (RVF) is a clinical syndrome with high mortality that is frequently secondary to cardiovascular and pulmonary vascular diseases. There is growing evidence that the outcome of RVF is closely related to the immune response. This study is to explore SPP1 as a potential diagnostic marker related to the infiltration of immune cells in RVF. Methods: The gene expression profile of GSE161473 was downloaded from Gene Expression Omnibus and analyzed by R. Protein-protein interaction (PPI) networks which were established with STRING. Gene Ontology and KEGG enrichment analysis were performed using R-package “Clusterprofiler.” Differentially expressed immune-related genes (DEIRG) were identified using ImmPort. The infiltration of immune cells in RVF was evaluated using CIBERSORT, and the correlation between key hub genes and immune cells was analyzed by R. Rats model of RVF which was established by monocrotaline (MCT) (60 mg/kg, IP). The immune status and differentially expressed immune-related genes (IRGs) in the right heart tissue of rats with RVF in PAH were verified by immunofluorescence, immunohistochemistry, Real-time PCR, and western blotting. In addition, the serum of right heart-failing patients was used to verify the possibility of SPP1 as a biomarker. Result: A total of 107 differentially expressed genes (DEGs) were screened, from which 20 DEIRGs were screened by ImmPort, PPI, and functional enrichment analysis. 4 Hub genes (ADM, FGF12, AREG and SPP1) were confirmed by RT-PCR. In addition, SPP1 was identified as the most potent biomarker. ImmuCellAI immune infiltration analysis showed that a higher proportion of monocytes, M2 macrophages, memory B cells, resting NK cells, and resting mast cells were elavated in the RVF group. Finally, SPP1 was positively correlated with monocytes and M2 macrophages and negatively correlated with memory B cells and CD8 + T cells. Conclusion: A variety of immune cells such as monocytes, macrophages, T cells, B cells, and mast cells are involved in the development of RVF. Dysregulated immune responses were found in the right cardiac tissue of patients with RVF. SPP1 was considered a key hub gene to discriminate between RVF and normal groups. Parts of IRGs, especially SPP1, may be used as biomarkers to assist in the diagnosis and evaluation of RVF. This work was supported by the Shandong Provincial Natural Science Foundation of China (ZR2022MH052), China Postdoctoral Science Foundation (No. 2021M691944) to Xiaomei Yang. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Background:The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has triggered a global health crisis, with genetic mutations and evolution further creating uncertainty about epidemic risk. It is imperative to rapidly determine the nucleic acid sequence of SARS-CoV-2 and its variants to combat the coronavirus pandemic. Our goal was to develop a rapid, room-temperature, point-of-care (POC) detection system to determine the nucleic acid sequences of SARS-CoV-2 isolates, especially omicron variants. Methods:Based on the conserved nucleotide sequence of SARS-CoV-2, bioinformatics software was used to analyze, design, and screen optimal enzymatic isothermal amplification primers and efficient CRISPR RNAs (crRNAs) of CRISPR/Cas13a to the target sequences. Reverse transcription-recombinase polymerase amplification (RT-RPA) was used to amplify the virus, and CRISPR/Cas13a-crRNA was used to cleave the SARS-CoV-2 target sequence. The sensitivity of nucleic acid detection was assessed by serial dilution of plasmid templates. All reactions were performed at room temperature. Results:RT-RPA, combined with CRISPR/Cas13a, can detect the SARS-CoV-2 with a minimum content of 102 copies/μL, and can effectively distinguish between the original strain and the Omicron variant with a minimum limit of detection (LOD) of 103 copies/μL. Conclusions:The method developed in this study has potential application in clinical detection of SARS-CoV-2 and its omicron variants.

The BCR-ABL fusion gene, formed by the fusion of the breakpoint cluster region protein ( BCR) and the Abl Oncogene 1, Receptor Tyrosine Kinase ( ABL) genes, encodes the BCR-ABL oncoprotein, which plays a crucial role in leukemogenesis. Current therapies have limited efficacy in patients with chronic myeloid leukemia (CML) because of drug resistance or disease relapse. Identification of novel strategies to treat CML is essential. This study aims to explore the efficiency of novel CRISPR-associated protein 9 (Cas9)/dual-single guide RNA (sgRNA)-mediated disruption of the BCR-ABL fusion gene by targeting BCR and cABL introns. A co-expression vector for Cas9 green fluorescent protein (GFP)/dual-BA-sgRNA targeting BCR and cABL introns is constructed to produce lentivirus to affect BCR-ABL expression in CML cells. The effects of dual-sgRNA virus-mediated disruption of BCR-ABL are analyzed via the use of a genomic sequence and at the protein expression level. Cell proliferation, cell clonogenic ability, and cell apoptosis are assessed after dual sgRNA virus infection, and phosphorylated BCR-ABL and its downstream signaling molecules are detected. These effects are further confirmed in a CML mouse model via tail vein injection of Cas9-GFP/dual-BA-sgRNA virus-infected cells and in primary cells isolated from patients with CML. Cas9-GFP/dual-BA-sgRNA efficiently disrupts BCR-ABL at the genomic sequence and gene expression levels in leukemia cells, leading to blockade of the BCR-ABL tyrosine kinase signaling pathway and disruption of its downstream molecules, followed by cell proliferation inhibition and cell apoptosis induction. This method prolongs the lifespan of CML model mice. Furthermore, the effect is confirmed in primary cells derived from patients with CML.

The PIK3CA mutation is considered a potential target for treatment of colorectal cancer. We evaluated a PIK3CA mutation assay on plasma cell-free DNA (cfDNA) using a newly developed PCR with restriction digestion integrated and followed by Sanger’s sequencing. We analyzed PIK3CA mutation in plasma with our newly developed assays and in matching tumor tissues by routine methods. We detected the PIK3CA gene mutation status by both methods in samples from 40 colorectal cancer patients. Three H1047R mutations of PIK3CA gene were detected in the cfDNA of the 40 patients by restriction digestion PCR. Neither E545K nor H1047R mutations were detected in the cfDNA by routine PCR/sequencing. The PIK3CA H1047R and E545K mutations in cfDNA can be sensitively detected with our newly developed assays. The colorectal cancer has been used as a clinical example in testing our new assays, which indicates that the new assays may have wider applications in detecting mutations in precision oncology. Trial registration: Current Controlled Trials ChiCTR-DDT-12002848, 8 October 2012.

In our recent effort of developing methylation-dependent endonuclease mediated methylation sequencing technology, efficiency and reproducibility were restrained by adapters’ self-ligation. In the present study, three strategies to prevent self-ligation of adapters used in enzyme mediated methylation sequencing have been developed. Our data have demonstrated that these strategies can either inhibit or eliminate the adapters’ self-ligation. These strategies are crucial in enzyme-mediated methylation sequencing and may be useful in some other genomic sequencing technologies.

In recent years, small intestine as a key target in the treatment of Inflammatory bowel disease caused by NSAIDs has become a hot topic. Sanguinarine (SA) is one of the main alkaloids in the Macleaya cordata extracts with strong pharmacological activity of anti-tumor, anti-inflammation and anti-oxidant. SA is reported to inhibit acetic acid-induced colitis, but it is unknown whether SA can relieve NSAIDs-induced small intestinal inflammation. Herein, we report that SA effectively reversed the inflammatory lesions induced by indomethacin (Indo) in rat small intestine and IEC-6 cells in culture. Our results showed that SA significantly relieved the symptoms and reversed the inflammatory lesions of Indo as shown in alleviation of inflammation and improvement of colon macroscopic damage index (CMDI) and tissue damage index (TDI) scores. SA decreased the levels of TNF-α, IL-6, IL-1β, MDA and LDH in small intestinal tissues and IEC-6 cells, but increased SOD activity and ZO-1 expression. Mechanistically, SA dose-dependently promoted the expression of Nrf2 and HO-1 by decreasing Keap-1 level, but inhibited p65 phosphorylation and nuclear translocation in Indo-treated rat small intestine and IEC-6 cells. Furthermore, in SA treated cells, the colocalization between p-p65 and CBP in the nucleus was decreased, while the colocalization between Nrf2 and CBP was increased, leading to the movement of gene expression in the nucleus to the direction of anti-inflammation and anti-oxidation. Nrf2 silencing blocked the effects of SA. Together our results suggest that SA can significantly prevent intestinal inflammatory lesions induced by Indo in rats and IEC-6 cells through regulation of the Nrf2 pathway and NF-κBp65 pathway.

Clusters of regularly interspaced short palindromic repeats (CRISPR)/Cas systems have a powerful ability to edit DNA and RNA targets. However, the need for a specific recognition site, protospacer adjacent motif (PAM), of the CRISPR/Cas system limits its application in gene editing. Some Argonaute (Ago) proteins have endonuclease functions under the guidance of 5′ phosphorylated or hydroxylated guide DNA (gDNA). The NgAgo protein might perform RNA gene editing at 37 °C, suggesting its application in mammalian cells; however, its mechanisms are unclear. In the present study, the target of NgAgo in RNA was confirmed in vitro and in vivo. Then, an in vitro RNA cleavage system was designed and the cleavage site was verified by sequencing. Furthermore, NgAgo and gDNA were transfected into cells to cleave an intracellular target sequence. We demonstrated targeted degradation of GFP, HCV, and AKR1B10 RNAs in a gDNA-dependent manner by NgAgo both in vitro and in vivo, but no effect on DNA was observed. Sequencing demonstrated that the cleavage sites are located at the 3′ of the target RNA which is recognized by 5′ sequence of the gDNA. These results confirmed that NgAgo–gDNA cleaves RNA not DNA. We observed that the cleavage site is located at the 3′ of the target RNA, which is a new finding that has not been reported in the past.

During the outbreak of coronavirus disease 2019 (COVID-19), the identification of a SARS-CoV-2 infected case with untraceable epidemic origin has three values: (1) the region has community transmission of the virus; (2) acertain portion of population in the community is getting immunized and more individuals have already been immunized; (3) an unpredictable future risk exists for regions where there are no infected cases with untraceable epidemic origin. Minimizing or avoiding the aggregation infection through individuals with no clinical symptoms is crucial and possible as its occurrence is mainly attributed to the local environment as opposed to thesuper spreader with or without clinical symptoms. As infected cases are not necessarily positive with gene test by definition, proper application of gene test is crucial in the identification of asymptomatic cases. In the early stage of an outbreak of infectious disease, gene test can be used to identify asymptomatic cases but it should not be used to exclude cases with typical clinical symptoms and signs. In the middle or late stages of an outbreak gene test should be applied in evaluation of infection rate of the population of a region, in addition to be used in spreader identification and isolation. Although asymptomatic cases are attributed to the overwhelming effect of personalized defense against pathogens, they are still a source of infection. Asymptomatic cases are considered to be Qi deficiency and with lingering toxicity in traditional Chinese medicine (TCM). Treatment in such cases, typically, involves therapy focused on replenishing the Qi, tonifying the lung, clearing the fever, and detoxification in order to return to a normal health condition. The recommended Chinese medicines include Qing Fei Pai Du Decoction, Yi Qi Qu Du Decoction, Xuan Fei Bai Du Granule, Lian Hua Qing Wen Capsule (Granule), and Jin Hua Qing Gan Granule, etc.

BACKGROUND:The present study sought to identify a panel of DNA markers for noninvasive diagnosis using cell-free DNA (cfDNA) from urine supernatant or cellular DNA from urine sediments of hematuria patients. A panel of 48 bladder cancer-specific genes was selected. A next-generation sequencing-based assay with a cfDNA barcode-enabled single-molecule test was employed. Mutation profiles of blood, urine, and tumor sample from 16 bladder cancer patients were compared. Next, urinary cellular DNA and cfDNA were prospectively collected from 125 patients (92 bladder cancer cases and 33 controls) and analyzed using the 48-gene panel. The individual gene markers and combinations of markers were validated according to the pathology results. The mean areas under the receiver operating characteristic (ROC) curves (AUCs) obtained with the various modeling approaches were calculated and compared.RESULTS:This pilot study of 16 bladder cancer patients demonstrated that gene mutations in urine supernatant and sediments had better concordance with cancer tissue as compared with plasma. Logistic analyses suggested two powerful combinations of genes for genetic diagnostic modeling: five genes for urine supernatant (TERT, FGFR3, TP53, PIK3CA, and KRAS) and seven genes for urine sediments (TERT, FGFR3, TP53, HRAS, PIK3CA, KRAS, and ERBB2). The accuracy of the five-gene panel and the seven-gene panel in the validation cohort yielded AUCs of 0.94 [95% confidence interval (CI) 0.91-0.97] and 0.91 (95% CI 0.86-0.96), respectively. With the addition of age and gender, the diagnostic power of the urine supernatant five-gene model and the urine sediment seven-gene model improved as the revised AUCs were 0.9656 (95% CI 0.9368-0.9944) and 0.9587 (95% CI 0.9291-0.9883).CONCLUSIONS:cfDNA from urine bears great diagnostic potential. A five-gene panel for urine supernatant and a seven-gene panel for urine sediments are promising options for identifying bladder cancer in hematuria patients.

The detection of circulating tumor DNA is important in cancer research and clinical practice. In the present study, we aimed to improve the sensitivity of downstream mutation detection of next-generation sequencing using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system to selectively target wild-type fragments but with low or no cleavage activity to mutant fragments, followed by amplification using polymerase chain reaction. We selected different mutant sites of epidermal growth factor receptor gene (EGFR)-exon19 deletions in patients with lung cancer and constructed mixed templates of mutant and wild-type DNA comprising ratios of 10% to 0.01% to test the effectiveness of the enrichment method. The results showed that after CRISPR/Cas9 enrichment, a low concentration of mutant DNA fragments (0.01%) could be detected by Sanger sequencing, which represented a 1000-fold increase compared with the untreated samples. We further verified the feasibility of the introduced method and obtained similar results in clinical samples from patients with non-small cell lung cancer, indicating that this method has the potential to detect low copy number mutations at the early stage.

A primerless amplification suitable for enrichment of particular genotype cfDNA which is a one-dimensional material has been developed. This primerless amplification coordinated by two thermostable enzymes of endonuclease and proofreading polymerase, functions as a genotype switch in analyzing cfDNA. The endonuclease digests the wild-typed fragments into mega-primer and discriminately destroys the wild-type DNA alleles. The DNA polymerase proofreads the megaprimer and then extends the mega-primer using the mutant DNA as the template. The prototypes of this technology were applied to two hotspot mutations of APC and EGFR with confirmed by DNA sequencing analysis. Genotype switch was then employed to clinical cfDNA assay targeting PIK3CA. Data from the clinical application suggest its potential in early cancer diagnosis.

The combination of phosphorothioate-modified primer and high-fidelity DNA polymerase constitute a mutation sensitive on/off switch. Herein, a modified mutation-sensitive on/off switch was designed with an extra point mutation at 5′ far away from the 3′ of the phosphorothioate modified primer to test the power of the new enzymes: Phusion, Q5 and Phanta UC polymerases. A modified on/off switch with the introduction of an extra point mismatched nucleotide at –8 from the 3′ terminus, and the use of Phanta UC Super-fidelity DNA polymerase has been confirmed to be highly sensitive and specific as compared to those without the 5′ artificial mismatched nucleotide, when applied in circulating tumor DNA (ctDNA) analysis of BRAF hotspot mutation.

Overlap extension polymerase chain reaction (PCR) is a powerful technology for DNA assembly. Based on this technology, we synthesized DNA templates, which were transcribed into sgRNA in vitro, and further detected their efficiency of purified sgRNAs with Cas9 nuclease. The sgRNAs synthesized by this approach can effectively cleave the DNA fragments of interest in vitro and in vivo. Compared with the conventional method for generating sgRNA, it does not require construction of recombinant plasmids and design of primers to amplify sgRNA core fragment. Only several short primers with overlapped sequences are needed to assemble a DNA fragment as the template of sgRNA. This modified and simplified method is highly applicable and less time-consuming.

The target lengths of gene-editing enzymes vary from approximately 20 to 80 nucleotides. The ideal reporter genes should be able to tolerate the insertion of target sequences to differentiate the phenotypic changes between in-frame and frame shift mutations for evaluating the enzymatic activities and off-target effects of gene-editing enzymes such as zinc finger nucleases, transcription activator-like effector nucleases, clustered regularly interspaced short palindromic repeats-associated system. This study demonstrated that the zeocin resistance gene (ZeoR) tolerated the insertion of long targets for a variety of tests using a strategy of inserting tandem repeats of NNT. Four genes such as CCR5, AR, HPV 16E7 early gene, and EGFR applications of ZeoR in quantitatively analyzing the off-targets of CRISPR were successful. The data indicated that evaluating the enzymatic activities and off-target effects with assays employing ZeoR had great potential in facilitating the application of gene editing to human gene therapy.

Development of sensitive assay for detection of hotspot mutations of cancer driving gene is crucial for circulating tumor DNA analysis. This study tested the possibilities of applying restriction enzyme digestion and dephosphorylation coupled with blue/white screening technology for analyzing a hotspot point mutation in codon 13 of KRAS gene. The present study has documented that the combination of PCR with restriction digestion, dephosphorylation, blue/white screening and Sanger's sequencing can identify rare mutations with sensitivities at 0.003%. This novel assay with high sensitivity may have application in the diagnosis of early cancer targeting ctDNAs.

Objective We found seminal B7-H3 was associated with human sperm concentration. However, the mechanism is unclear. The purpose of this study was to investigate the expression of B7-H3 in mouse testis and determine the effects of B7-H3 on the proliferation of mouse spermatogonial stem cells (SSCs) and the underlying mechanisms. Methods B7-H3 expression in the testis of mice at different ages (3 weeks, 8 weeks, 4 months and 9 months) was detected by western blot and immunohistochemistry. CCK-8 were used to measure mouse SSCs proliferation after incubation with different concentrations of B7-H3 for 1-72 h in vitro. Flow cytometry was used to analyze the cell cycle of mouse SSCs after incubation with different concentrations of B7-H3 for 48 and 72 h. The signaling pathways involved were assessed by western blot. Results Four-month-old mice had the highest expression of B7-H3 in the testis, while 3-week-old mice had the lowest expression of B7-H3. B7-H3 was predominantly detected on the membrane and in the cytoplasm of Sertoli cells. Furthermore, B7-H3 promoted mouse SSCs proliferation and increased the percentage of cells in S+G2/M phase in a time- and dose-dependent manner in vitro. These effects were inhibited by LY294002, indicating the involvement of the phosphoinositide 3-kinase signaling pathway. Conclusions The expression of B7-H3 in mouse testis, especially Sertoli cells, was associated with mouse age. In vitro, B7-H3 promoted the proliferation and accelerated the cell cycle of mouse SSCs via the PI3K pathway, indicating a critical role of B7-H3 expressed by Sertoli cells in mouse spermatogenesis.

The present study was designed to test a new strategy for comparing the off-target effects of CRISPR/cas9 employing a blue/white colony based assay. Eight types of AmpR plasmids with matched, one base mismatched, two bases mismatched and three bases mismatched targets were constructed. The wild typed alpha peptide of the beta-glactosidase gene and some with mutations, chosen as the targets, were successfully subcloned into the plasmids in an inframe pattern. The relevant gRNA and cas9 were subcloned into the ChloR plasmid. Transformation of the plasmid harboring the target gene yielded blue colonies, while white colonies formed in the plates following the co-transformation of target gene plasmid and the CRISPR/cas9 plasmid. Colonies in different levels of blue color were formed from the plasmids having completely matched target and mismatched targets. The appearance of different blue levels following the co-transformation of the different target plasmid and CRISPR/cas9 plasmid qualitatively indicated the effectiveness of the gRNA, while the ratio of the AMP/CL colonies can be used for relatively quantitative evaluation. The target and off-target effects among different targets of gRNA were differentially identifiable and these effects in turn demonstrated the effectiveness of the blue to white assay. Either the qualitative or the quantitative analysis of the blue to white depends on the final interactions among the target, the cas9 and the gRNA. Clearly, when co-transformation is replaced by single transformation using a specialized nanoparticle containing these three components, the target and off-target effect will be more accurately evaluated. Thus, this study provided a model for application of nanoparticle in gene editing studies.