A modified glycosylase base editor without predictable DNA off-target effects.

Glycosylase base editor (GBE) can induce C-to-G transversion in mammalian cells, showing great promise for the treatment of human genetic disorders. However, the limited efficiency of transversion and the possibility of off-target effects caused by Cas9 restrict its potential clinical applications. In our recent study, we have successfully developed TaC9-CBE and TaC9-ABE by separating nCas9 and deaminase, which eliminates the Cas9-dependent DNA off-target effects without compromising editing efficiency. We developed a novel GBE called TaC9-GBEYE1, which utilizes the deaminase and UNG-nCas9 guided by TALE and sgRNA, respectively. TaC9-GBEYE1 showed comparable levels of on-target editing efficiency to traditional GBE at 19 target sites, without any off-target effects caused by Cas9 or TALE. The TaC9-GBEYE1 is a safe tool for gene therapy.

Improvement of TaC9-ABE mediated correction of human SMN2 gene.

Spinal muscular atrophy (SMA) is a devastating neuromuscular disease caused by mutations in the survival motor neuron 1 (SMN1) gene. Gene editing technology repairs the conversion of the 6th base T to C in exon 7 of the paralogous SMN2 gene, compensating for the SMN protein expression and promoting the survival and function of motor neurons. However, low editing efficiency and unintended off-target effects limit the application of this technology. Here, we optimized a TaC9-adenine base editor (ABE) system by combining Cas9 nickase with the transcription activator-like effector (TALE)-adenosine deaminase fusion protein to effectively and precisely edit SMN2 without detectable Cas9 dependent off-target effects in human cell lines. We also generated human SMA-induced pluripotent stem cells (SMA-iPSCs) through the mutation of the splice acceptor or deletion of the exon 7 of SMN1. TaC9-R10 induced 45% SMN2 T6 > C conversion in the SMA-iPSCs. The SMN2 T6 > C splice-corrected SMA-iPSCs were directionally differentiated into motor neurons, exhibiting SMN protein recovery and antiapoptosis ability. Therefore, the TaC9-ABE system with dual guides from the combination of Cas9 with TALE could be a potential therapeutic strategy for SMA with high efficacy and safety.

Establishment and characterization of Lesch-Nyhan syndrome rabbit model.

Lesch-Nyhan syndrome (LNS) is a congenital defect disease that results in defective purine metabolism. It is caused by pathogenic variants of the HPRT gene. Its clinical symptoms mainly include high uric acid levels, gout, and kidney stones and damage. The mechanism of LNS has not been fully elucidated, and no cure exists. Animal models have always played an important role in exploring causative mechanisms and new therapies. This study combined CRISPR/Cas9 and microinjection to knock out the HPRT gene to create an LNS rabbit model. A sgRNA targeting exon 3 of HPRT gene was designed. Subsequently, Cas9 mRNA and sgRNA were injected into rabbit zygotes, and injected embryos were transferred to the uterus. The genotype and phenotype of rabbits were analyzed after birth. Four infant rabbits (named R1, R2, R3 and R4), which showed varying levels of gene modification, were born. The gene-editing efficiency was 100%. No wild-type sequences at the target HPRT gene were detected in R4 rabbit. Next, 6-thioguanine drug testing confirmed that HPRT enzymatic activity was deficient in R4 infant rabbit. HE staining revealed kidney abnormalities in all infant rabbits. Overall, an sgRNA capable of knocking out the HPRT gene in rabbits was successfully designed, and HPRT gene-modified rabbits were successfully constructed by using CRISPR/Cas9 technology and microinjection. This study provides a new nonrodent animal model for studying LNS syndrome.

Stability of African swine fever virus genome under different environmental conditions.

Background and Aim: African swine fever (ASF), a globally transmitted viral disease caused by ASF virus (ASFV), can severely damage the global trade economy. Laboratory diagnostic methods, including pathogen and serological detection techniques, are currently used to monitor and control ASF. Because the large double-stranded DNA genome of the mature virus particle is wrapped in a membrane, the stability of ASFV and its genome is maintained in most natural environments. This study aimed to investigate the stability of ASFV under different environmental conditions from both genomic and antibody perspectives, and to provide a theoretical basis for the prevention and elimination of ASFV. Materials and Methods: In this study, we used quantitative real-time polymerase chain reaction for pathogen assays and enzyme-linked immunosorbent assay for serological assays to examine the stability of the ASFV genome and antibody, respectively, under different environmental conditions. Results: The stability of the ASFV genome and antibody under high-temperature conditions depended on the treatment time. In the pH test, the ASFV genome and antibody remained stable in both acidic and alkaline environments. Disinfection tests revealed that the ASFV genome and antibody were susceptible to standard disinfection methods. Conclusion: Collectively, the results demonstrated that the ASFV genome is highly stable in favorable environments but are also susceptible to standard disinfection methods. This study focuses on the stability of the ASFV genome under different conditions and provides various standard disinfection methods for the prevention and control of ASF.

Fibroblast growth factor 13 regulates apoptosis of A549 cells through the ROS/ Caspase-3 pathway / 中国肿瘤生物治疗杂志

@#目的：探讨成纤维细胞生长因子 13（fibroblast growth factor 13，FGF13）对非小细胞肺癌 A549 细胞活性氧（reactive oxygen species，ROS）的生成和凋亡的影响及其调控机制。方法：WB 法检测 FGF13 在人正常肺上皮细胞 BEAS-2B 和肺癌 A549、H460 细胞中的本底表达量。采用 FGF13 过表达载体转染 BEAS-2B 和 A549 细胞；设计两组靶向 FGF13 的 shRNA 序 列，构建慢病毒干扰载体，包装病毒后侵染 A549 细胞，采用 qPCR 和 WB 法检测干扰效果，DCFH-DA 探针结合荧光酶标仪分 析敲减 FGF13 对 A549 细胞内 ROS 水平的影响，MitoSOX 与 WB 法检测对线粒体 ROS 水平及烟酰胺腺嘌呤二核苷酸磷酸氧 化酶 4（nicotinamide adenine dinucleotide phosphate oxidase 4，NOX4）蛋白表达量的影响，Annexin V-FITC-PI 双染法检测对细胞 凋亡和 Caspase-3 及 Cleaved Caspase-3 蛋白表达的影响。结果：与 BEAS-2B 细胞相比，FGF13 蛋白在两种肺癌细胞中均高表 达（均 P<0.05）。成功构建 FGF13 过表达、低表达的 A549 细胞系。过表达 FGF13 后，BEAS-2B 和 A549 细胞内 ROS 水平显著 降低（P<0.05）；敲减 FGF13 表达后，A549 细胞内 ROS 水平显著升高（P<0.05）；然而过表达及干扰 FGF13 对 A549 细胞内线粒 体 ROS 水平无显著影响，但 NOX4 蛋白表达量显著下调（P<0.05）及显著上调（P<0.05）。FGF13 干扰后 A549 细胞凋亡率显著 升高（P<0.01），Caspase-3 及 Cleaved Caspase-3 蛋白表达量显著上调（P<0.05）。结论：FGF13 可能通过 NOX 家族途径调控 ROS 的生成，并通过 ROS/Caspase-3 通路调控 A549 细胞凋亡。