CAS9 is a genome mutator by directly disrupting DNA-PK dependent DNA repair pathway

With its high efficiency for site-specific genome editing and easy manipulation, the clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR associated protein 9 (CAS9) system has become the most widely used gene editing technology in biomedical research. In addition, significant progress has been made for the clinical development of CRISPR/CAS9 based gene therapies of human diseases, several of which are entering clinical trials. Here we report that CAS9 protein can function as a genome mutator independent of any exogenous guide RNA (gRNA) in human cells, promoting genomic DNA double-stranded break (DSB) damage and genomic instability. CAS9 interacts with the KU86 subunit of the DNA-dependent protein kinase (DNA-PK) complex and disrupts the interaction between KU86 and its kinase subunit, leading to defective DNA-PK-dependent repair of DNA DSB damage via non-homologous end-joining (NHEJ) pathway. XCAS9 is a CAS9 variant with potentially higher fidelity and broader compatibility, and dCAS9 is a CAS9 variant without nuclease activity. We show that XCAS9 and dCAS9 also interact with KU86 and disrupt DNA DSB repair. Considering the critical roles of DNA-PK in maintaining genomic stability and the pleiotropic impact of DNA DSB damage responses on cellular proliferation and survival, our findings caution the interpretation of data involving CRISPR/CAS9-based gene editing and raise serious safety concerns of CRISPR/CAS9 system in clinical application.

CAS9 is a genome mutator by directly disrupting DNA-PK dependent DNA repair pathway

With its high efficiency for site-specific genome editing and easy manipulation, the clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR associated protein 9 (CAS9) system has become the most widely used gene editing technology in biomedical research. In addition, significant progress has been made for the clinical development of CRISPR/CAS9 based gene therapies of human diseases, several of which are entering clinical trials. Here we report that CAS9 protein can function as a genome mutator independent of any exogenous guide RNA (gRNA) in human cells, promoting genomic DNA double-stranded break (DSB) damage and genomic instability. CAS9 interacts with the KU86 subunit of the DNA-dependent protein kinase (DNA-PK) complex and disrupts the interaction between KU86 and its kinase subunit, leading to defective DNA-PK-dependent repair of DNA DSB damage via non-homologous end-joining (NHEJ) pathway. XCAS9 is a CAS9 variant with potentially higher fidelity and broader compatibility, and dCAS9 is a CAS9 variant without nuclease activity. We show that XCAS9 and dCAS9 also interact with KU86 and disrupt DNA DSB repair. Considering the critical roles of DNA-PK in maintaining genomic stability and the pleiotropic impact of DNA DSB damage responses on cellular proliferation and survival, our findings caution the interpretation of data involving CRISPR/CAS9-based gene editing and raise serious safety concerns of CRISPR/CAS9 system in clinical application.

Core pluripotency factors promote glycolysis of human embryonic stem cells by activating GLUT1 enhancer

Human embryonic stem cells (hESCs) depend on glycolysis for energy and substrates for biosynthesis. To understand the mechanisms governing the metabolism of hESCs, we investigated the transcriptional regulation of glucose transporter 1 (GLUT1, SLC2A1), a key glycolytic gene to maintain pluripotency. By combining the genome-wide data of binding sites of the core pluripotency factors (SOX2, OCT4, NANOG, denoted SON), chromosomal interaction and histone modification in hESCs, we identified a potential enhancer of the GLUT1 gene in hESCs, denoted GLUT1 enhancer (GE) element. GE interacts with the promoter of GLUT1, and the deletion of GE significantly reduces the expression of GLUT1, glucose uptake and glycolysis of hESCs, confirming that GE is an enhancer of GLUT1 in hESCs. In addition, the mutation of SON binding motifs within GE reduced the expression of GLUT1 as well as the interaction between GE and GLUT1 promoter, indicating that the binding of SON to GE is important for its activity. Therefore, SON promotes glucose uptake and glycolysis in hESCs by inducing GLUT1 expression through directly activating the enhancer of GLUT1.

Expression of voltagE-gated sodium channel SCN5A/Nav1.5 in human ovarian cancer and its significance / 肿瘤

Objective:The different subtypes of voltagE-gated sodium channels (VGSCs) are known to correlate with the migration of many malignant cancers. This study was to investigate the significance of functional expression of SCN5A/Nav1.5 in human ovarian cancer and its effects on migration capability of ovarian cancer cells in vitro. Methods: Sodium indicator SBFI and immunofluorescence method were used to detect the distribution of intracellular Na+. Real-time PCR, Western blotting, and immunohistochemistry were used to detect the mRNA and protein expression of SCN5A/Nav1.5. The effect of specific voltagE-gated sodium channels inhibitor tetrodotoxin (TTX) on cell viability was measured by CCK-8 kit. The migration and invasion of ovarian cancer cell lines SKOV-3 were tested by Transwell chamber assay. Results:SCN5A/Nav1.5 were over-expressed in ovarian cancer cell lines SKOV-3 and ovarian cancer specimens at mRNA and protein levels. TTX 30 μmol/L inhibited the intracellular Na+ concentration by (41.51±0.41)%. TTX also suppressed the invasion and migration capacities of SKOV-3 cells by (33.80±1.6)% and (43.60±2.9)%, respectively. The difference was significant (P<0.05). Conclusion:SCN5A/Nav1.5 is involved in the metastasis progression of ovarian cancer in vitro and plays an important role in the initiation and progression of ovarian cancer. It may become a target for ovarian cancer therapy.

INVESTIGATION AND ANALYSIS OF STEREOSCOPIC VISION OF MILITARY PERSONNEL / 解放军医学杂志

An examination was conducted with holographic stereopsis test instrument on the stereoscopic vision function of 341 military personnel in different services. 94.7 percent of the total examined enjoyed normal stereopsis acuity. The pilots obtained the far most satisfactory result. One hundred percent of them were up to normal standard in terms of both far and near visions and stereopsis acuity. Meanwhile, 94.6 percent of the vehicle drivers examined also had normal stereopsis acuity. This percentage was higher compared with 83.78 percent of the civilian personnel of the same occupation. The above results indicate that the stereopsis function of personnel in military services is in good condition, and such examination is very effective and necessary as part of the physical examinations in conscription to assure the standard of the service men.