Direct Injection of CRISPR/Cas9-Related mRNA into Cytoplasm of Parthenogenetically Activated Porcine Oocytes Causes Frequent Mosaicism for Indel Mutations

Some reports demonstrated successful genome editing in pigs by one-step zygote microinjection of mRNA of CRISPR/Cas9-related components. Given the relatively long gestation periods and the high cost of housing, the establishment of a single blastocyst-based assay for rapid optimization of the above system is required. As a proof-of-concept, we attempted to disrupt a gene (GGTA1) encoding the α-1,3-galactosyltransferase that synthesizes the α-Gal epitope using parthenogenetically activated porcine oocytes. The lack of α-Gal epitope expression can be monitored by staining with fluorescently labeled isolectin BS-I-B₄ (IB4), which binds specifically to the α-Gal epitope. When oocytes were injected with guide RNA specific to GGTA1 together with enhanced green fluorescent protein (EGFP) and human Cas9 mRNAs, 65% (24/37) of the developing blastocysts exhibited green fluorescence, although almost all (96%, 23/24) showed a mosaic fluorescent pattern. Staining with IB4 revealed that the green fluorescent area often had a reduced binding activity to IB4. Of the 16 samples tested, six (five fluorescent and one non-fluorescent blastocysts) had indel mutations, suggesting a correlation between EGFP expression and mutation induction. Furthermore, it is suggested that zygote microinjection of mRNAs might lead to the production of piglets with cells harboring various mutation types.     

One-Step Generation of Multiple Gene-Edited Pigs by Electroporation of the CRISPR/Cas9 System into Zygotes to Reduce Xenoantigen Biosynthesis

Xenoantigens cause hyperacute rejection and limit the success of interspecific xenografts. Therefore, genes involved in xenoantigen biosynthesis, such as GGTA1, CMAH, and B4GALNT2, are key targets to improve the outcomes of xenotransplantation. In this study, we introduced a CRISPR/Cas9 system simultaneously targeting GGTA1, CMAH, and B4GALNT2 into in vitro-fertilized zygotes using electroporation for the one-step generation of multiple gene-edited pigs without xenoantigens. First, we optimized the combination of guide RNAs (gRNAs) targeting GGTA1 and CMAH with respect to gene editing efficiency in zygotes, and transferred electroporated embryos with the optimized gRNAs and Cas9 into recipient gilts. Next, we optimized the Cas9 protein concentration with respect to the gene editing efficiency when GGTA1, CMAH, and B4GALNT2 were targeted simultaneously, and generated gene-edited pigs using the optimized conditions. We achieved the one-step generation of GGTA1/CMAH double-edited pigs and GGTA1/CMAH/B4GALNT2 triple-edited pigs. Immunohistological analyses demonstrated the downregulation of xenoantigens; however, these multiple gene-edited pigs were genetic mosaics that failed to knock out some xenoantigens. Although mosaicism should be resolved, the electroporation technique could become a primary method for the one-step generation of multiple gene modifications in pigs aimed at improving pig-to-human xenotransplantation.     

An Engineered sgsh Mutant Zebrafish Recapitulates Molecular and Behavioural Pathobiology of Sanfilippo Syndrome A/MPS IIIA

Mucopolysaccharidosis IIIA (MPS IIIA, Sanfilippo syndrome type A), a paediatric neurological lysosomal storage disease, is caused by impaired function of the enzyme N-sulfoglucosamine sulfohydrolase (SGSH) resulting in impaired catabolism of heparan sulfate glycosaminoglycan (HS GAG) and its accumulation in tissues. MPS IIIA represents a significant proportion of childhood dementias. This condition generally leads to patient death in the teenage years, yet no effective therapy exists for MPS IIIA and a complete understanding of the mechanisms of MPS IIIA pathogenesis is lacking. Here, we employ targeted CRISPR/Cas9 mutagenesis to generate a model of MPS IIIA in the zebrafish, a model organism with strong genetic tractability and amenity for high-throughput screening. The sgsh^Δex5−6 zebrafish mutant exhibits a complete absence of Sgsh enzymatic activity, leading to progressive accumulation of HS degradation products with age. sgsh^Δex5−6 zebrafish faithfully recapitulate diverse CNS-specific features of MPS IIIA, including neuronal lysosomal overabundance, complex behavioural phenotypes, and profound, lifelong neuroinflammation. We further demonstrate that neuroinflammation in sgsh^Δex5−6 zebrafish is largely dependent on interleukin-1β and can be attenuated via the pharmacological inhibition of Caspase-1, which partially rescues behavioural abnormalities in sgsh^Δex5−6 mutant larvae in a context-dependent manner. We expect the sgsh^Δex5−6 zebrafish mutant to be a valuable resource in gaining a better understanding of MPS IIIA pathobiology towards the development of timely and effective therapeutic interventions.     

锥面共形阵列极化-DOA估计的降维MUSIC算法

针对传统MUSIC(multiple signal classification)算法在锥面共形阵列极化-DOA(direction of arrival)参数联合估计过程中计算复杂度较高的问题,利用单极化阵元构造极化敏感锥面共形阵列,并建立阵列接收信号模型.通过构造同极化接收子阵,将导向矢量中空域信息和极化域信息去"耦合",在考虑阵元遮挡效应的条件下,结合秩损原理实现了基于降维MUSIC算法的极化-DOA多参数估计,减小了极化-DOA参数估计的计算量.通过计算机仿真证明了方法的有效性.     

基于CRISPR-Cas13的单增李斯特菌RNA快速检测方法的建立

为实现食品中单增李斯特菌污染的快速检测,本研究构建了一种基于CRISPR-Cas系统和Broccoli适配体的RNA均相检测技术。利用Cas 13与cr RNA锚定序列结合形成识别元件cr RNA-Cas13复合物,靶标RNA存在时可激活Cas 13的非特异性RNase活性,并利用点亮型RNA适配体Broccoli作为信号探针,监测cr RNA此-Cas13的活化状态。荧光值的变化与单增李斯特菌浓度存在线性关系,利用来检测单增李斯特菌。本研究所构建的检测可在30min内完成对于单增李斯特菌的的识别与检测,检出限为148CFU/m L,对细菌具有良好的检测特异性,可区分大肠杆菌、鼠伤寒沙门氏菌和蜡样芽孢杆菌。在牛奶模型中单增李斯特菌的加标回收率为95.15%～97.99%。该方法具有较好的灵敏度、特异性,可直接靶向检测致病菌RNA,无需逆转录、PCR扩增和核酸标记,简化了实验流程,对于实现食品中单增李斯特菌的现场检测及生物安全控制具有重要意义。     

多操纵面飞机全局集合稳定非线性自适应动态控制分配

针对多操纵面飞机具有冗余操纵面的特点,考虑包含操纵面偏转角的位置约束和速率约束以及未知有界参数时的非线性控制分配问题,设计一种由上层虚拟控制律和自适应控制分配更新律组成的非线性角速度跟踪控制器.当系统满足充分激励条件时,基于集合稳定性理论,分别证明了上层虚拟控制子系统、控制分配子系统和整个闭环系统的全局一致渐近稳定性.对某多操纵面飞机的仿真结果验证了所提出方法的有效性,并且该方法能使参数估计收敛至真实值.     

Reversal of the Inflammatory Responses in Fabry Patient iPSC-Derived Cardiovascular Endothelial Cells by CRISPR/Cas9-Corrected Mutation

The late-onset type of Fabry disease (FD) with GLA IVS4 + 919G > A mutation has been shown to lead to cardiovascular dysfunctions. In order to eliminate variations in other aspects of the genetic background, we established the isogenic control of induced pluripotent stem cells (iPSCs) for the identification of the pathogenetic factors for FD phenotypes through CRISPR/Cas9 genomic editing. We adopted droplet digital PCR (ddPCR) to efficiently capture mutational events, thus enabling isolation of the corrected FD from FD-iPSCs. Both of these exhibited the characteristics of pluripotency and phenotypic plasticity, and they can be differentiated into endothelial cells (ECs). We demonstrated the phenotypic abnormalities in FD iPSC-derived ECs (FD-ECs), including intracellular Gb3 accumulation, autophagic flux impairment, and reactive oxygen species (ROS) production, and these abnormalities were rescued in isogenic control iPSC-derived ECs (corrected FD-ECs). Microarray profiling revealed that corrected FD-derived endothelial cells reversed the enrichment of genes in the pro-inflammatory pathway and validated the downregulation of NF-κB and the MAPK signaling pathway. Our findings highlighted the critical role of ECs in FD-associated vascular dysfunctions by establishing a reliable isogenic control and providing information on potential cellular targets to reduce the morbidity and mortality of FD patients with vascular complications.