核酸检测和基因编辑的新工具：CRISPR/Cas13系统

成簇的规律间隔的短回文重复序列(clustered regularly interspaced short palindromic repeats,CRISPR)/CRISPR相关蛋白(CRISPR-associated proteins, Cas)系统是目前基因编辑、基因表达研究的热点,其中,研究较成熟的为CRISPR/Cas9系统,其在单链RNA的引导下可特异地切割靶DNA的特定位点,实现DNA水平的操作。而靶向RNA的CRISPR/Cas13系统开启了在RNA水平研究、诊疗的新时代。活化的Cas13具有独特的核酸酶活性,可特异性地切割靶向RNA,同时非特异性地切割周围环境中的RNA,利用以上特性可实现体外核酸检测。通过活性位点突变可产生无核酸酶活性但可与RNA结合的dCas13(dead Cas13),将dCas13与其他功能性蛋白质进行融合可进一步扩大dCas13的应用范围。该文主要概括了CRISPR/Cas13系统在核酸检测以及在RNA水平作为基因编辑工具的新进展。     

CRISPR/Cas12系统在肿瘤检测中的应用

快速、灵敏、特异的检测方法对于提高肿瘤患者的生存率并改善预后至关重要。除了在基因编辑领域的贡献，近年来成簇规律间隔短回文重复序列(CRISPR)/CRISPR相关蛋白(Cas)系统以其特有的靶标核酸识别切割能力和反式切割活性为特点，已成为新一代核酸检测工具被广泛应用于病原体、肿瘤和转基因等检测领域。基于此，该文对CRISPR/Cas12系统原理及其在不同肿瘤标志物中的检测应用进展作一综述，并对其应用前景进行展望，以期为CRISPR/Cas系统更好地应用于肿瘤筛查和诊断提供参考及借鉴。     

CRISPR/Cas系统在分子检测中的应用

近年来,成簇规律间隔短回文重复序列/成簇规律短回文重复序列相关蛋白(CRISPR/Cas)系统凭借其简单、高效的基因编辑能力,已被广泛应用于生物、医学等多个研究领域。随着CRISPR技术的快速发展,CRISPR/Cas系统已被开发为一种快速、便携、低成本、高灵敏度的分子检测工具,在病原体检测、耐药性分析、单核苷酸多态性(SNP)分型、肿瘤基因突变检测等方面取得重大突破。文章就不同Cas蛋白在分子检测中的最新研究进展进行综述,并对其应用前景进行展望,以期为从事相关领域的科研工作者提供参考与帮助。     

规律成簇的间隔短回文重复序列中间隔序列的噬菌体来源研究

目的 发现在现有的全基因组测序完成的原核生物中规律成簇的间隔短回文重复序列(CRISPR)系统中间隔序列分布规律以及间隔序列中噬菌体来源情况. 方法 整理现有CRISPR数据库中2762株细菌基因组中的CRISPR系统和其中的间隔序列数据,整理GenBank数据库中发表的1444个噬菌体基因组数据.利用BLASTN软件对间隔序列数据与噬菌体基因组进行相似性比较,计数资料比较使用2检验. 结果 在2762个细菌基因组中整理出1940个基因组存在确定或可能的CRISPR结构和90 096条间隔序列,多数基因组具有1~50条间隔序列(1414/1940,72.9%),间隔序列数量250条的仅有58个基因组(58/1940,3.0%).其中古细菌13株(13/150,8.6%),真细菌45株(45/2612,1.7%),差异有统计学意义(2=29.98,P 0.01).相似性比较结果共发现245个细菌基因组的1055条间隔序列,成功比对上363个噬菌体,比对成功率仅为0.12%. 结论 细菌基因组中的CRISPR系统中间隔序列数量存在较大差异,古细菌基因组中CRISPR系统存在更多的间隔序列.相似性比较中噬菌体来源的间隔序列所占比例低,提示与细菌和噬菌体基因组发现较少相关,进一步深入研究可以大幅度提高成功率.     

应用CRISPR/Cas9基因编辑系统对HEK293细胞系TSC1基因稳定敲除效果的实验鉴定

目的　利用成簇规律间隔短回文重复序列/ 成簇规律间隔短回文重复序列相关蛋白（clustered regularly interspacedshort palindromic repeat/CRISPR-associated protein,CRISPR/Cas9）基因编辑系统在HEK293 细胞系中对TSC1 基因稳定敲除,并对其敲除效果进行鉴定。方法　根据TSC1 基因的序列设计sgRNA,将sgRNA 克隆到载体lentilCRISPRv2 上,将连接产物转化至Stbl3 感受态细胞,对菌液进行测序鉴定。将鉴定成功的lentiCRISPRV2-sgTSC1 质粒转染至HEK293细胞,加入嘌呤霉素进行筛选,挑选单细胞克隆进行PCR 鉴定,选取条带单一的细胞株用Western blot 鉴定TSC1 基因的表达。结果　成功构建lentiCRISPRV2-sgTSC1-1/2 重组质粒,并利用该质粒完成对TCS1 基因的定点切割,Westernblot 结果显示细胞中无TSC1 表达。结论　用CRISPR/Cas9 系统成功构建TSC1 基因敲除的稳定细胞株,为后续实验奠定了基础。     

基于CRISPR-Cas12a蛋白的副溶血弧菌及tdh基因检测分析

目的 将重组酶介导等温核酸扩增技术(RAA)与成簇的规律间隔短回文重复序列(CRISPR)系统相结合,建立一种快速检测并分型副溶血弧菌的检测方法.方法 通过纯化CRISPR相关蛋白Cas12a,设计和合成RAA引物、crRNA和单链DNA报告分子,建立副溶血弧菌的荧光和试纸条检测方法.采用煮沸法提取细菌核酸,经RAA扩...     

基于CRISPR/Cas蛋白的副溶血弧菌检测方法的研究

目的利用规律间隔性成簇短回文重复序列（CRISPR）免疫原理及Cas12a酶的特点构建一种快速检测副溶血弧菌（VP）的方法,实现对病原菌准确快速的检测和识别。方法本研究通过制备纯化Cas12a蛋白,筛选构建VP的gRNA,建立CRISPR-VP荧光检测系统,根据最终荧光扩增曲线判定CRISPR-VP检测方法的有效性。结果在CRISPR-VP检测方法中只在VP的序列存在时才会产生明显的荧光信号。结论本实验初步建立了基于CRISPR/Cas蛋白的VP的检测方法,为后续简易检测试剂的研制提供理论依据。     

CRISPR/Cas系统在新型冠状病毒肺炎快速诊断中的应用

近年来,基于规律成簇间隔的短回文重复序列（ clustered regularly interspaced short palindromic repeats, CRISPR）及其相关蛋白（ CRISPR-associated protein, Cas）系统的新型分子诊断工具,为病原体的诊断开辟了新的机遇。该文将关注现有和正在研究的 CRISPR/Cas系统用于新型冠状病毒肺炎（coronavirus disease 2019, COVID-19）快速诊断的潜在能力,并重点探讨其在临床中的应用和面临的挑战。     

RNA靶向系统Crispr/Cas13在分子诊断及临床治疗中的研究进展

成簇的规律间隔的短回文重复序列(clustered regularly interspaced short palindromic repeats, Crispr)/Crispr相关蛋白(Crispr-associated proteins, Cas)系统彻底变革了基因编辑和基因表达的方式,其中靶向切割DNA特定位点的Crispr/Cas9系统的研究日趋成熟,而目前靶向切割RNA的Crispr/Cas13系统研究较少。Cas13由crRNA(Crispr RNA, crRNA)引导,特异性识别靶向RNA片段,激活后特异性剪切靶向片段,随后对附近的RNA片段进行无差别的"附带剪切",在分子诊断及个体化治疗上具有广阔的应用前景。该文主要对靶向RNA的Crispr/Cas13系统的作用特点及机制进行阐述,并对在临床分子诊断及疾病治疗中的研究进展进行综述。     

基于CRISPR/Cas系统诊断平台的研究进展

目前核酸检测技术已被广泛应用于临床实验室诊断,常规检测技术如实时荧光定量PCR技术耗时长且依赖特定的仪器设备。成簇规律间隔短回文重复序列(CRISPR)/CRISPR相关蛋白(Cas)系统是细菌和古细菌在与病毒斗争过程中获得的适应性免疫防御机制,已被发展成强大的基因组编辑技术。最近CRISPR领域的先驱团队基于Cas13a、Cas12a和新发现的Cas14蛋白开发出SHERLOCK、DETECTR等新型核酸检测工具,在传染性疾病的快速诊断、癌症中基因突变的检测和基因分型等方面意义重大,其灵敏度高、特异性强且快速经济,在临床分子诊断领域具有巨大潜力。本文综述了CRISPR/Cas系统的作用机制及新型诊断平台的原理和应用进展,总结了新型检测技术的优缺点并对其发展前景进行展望。     

Personalized therapeutic strategies for patients with retinitis pigmentosa

Introduction: Retinitis pigmentosa (RP) encompasses many different hereditary retinal degenerations that are caused by a vast array of different gene mutations and have highly variable disease presentations and severities. This heterogeneity poses a significant therapeutic challenge, although an answer may eventually be found through two recent innovations: induced pluripotent stem cells (iPSCs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas genome editing.

Areas covered: This review discusses the wide-ranging applications of iPSCs and CRISPR–including disease modelling, diagnostics and therapeutics – with an ultimate view towards understanding how these two technologies can come together to address disease heterogeneity and orphan genes in a novel personalized medicine platform. An extensive literature search was conducted in PubMed and Google Scholar, with a particular focus on high-impact research published within the last 1 – 2 years and centered broadly on the subjects of retinal gene therapy, iPSC-derived outer retina cells, stem cell transplantation and CRISPR/Cas gene editing.

Expert opinion: For the retinal pigment epithelium, autologous transplantation of gene-corrected grafts derived from iPSCs may well be technically feasible in the near future. Photoreceptor transplantation faces more significant unresolved technical challenges but remains an achievable, if more distant, goal given the rapid pace of advancements in the field.     

经皮肝穿刺多弹头射频治疗肝癌 (附31 例报告)

目的　探讨并总结多弹头射频治疗肝癌的临床近期疗效。方法　对 31例肝癌患者在B超引导下进行多弹头射频治疗 ,通过对手术前后CT、B超检查的比较及AFP、肝功能、机体免疫力、体重和自觉症状的改变进行对比观察。结果　 31例于治疗后 7天、1 0例于治疗后 30天经B超及CT检查 :7天后 80 %的病人肿瘤体积缩小 30 %以上 ,30天后 1 0例病人肿瘤体积缩小 40 %以上 ,与术前相比瘤体内血供消失。 84%病人自觉症状改善 ,AFP值下降者占 77 8% ,肝功能酶谱及胆红素均恢复正常 ,白蛋白平均增加 1 1 0 g/L。体重增加 5 6kg ,T淋巴细胞转化率平均增加 2 1 %。IgG平均增加 2 0 g/L ,无手术死亡。结论　多弹头射频治疗肝癌的适应证广、安全可靠、疗效满意     

集束电极射频治疗肺癌CT 评价

目的 用ＣＴ评价集束电极射频治疗肺癌的近期效果,方法 对31例肺癌34个病灶手术前、后肿瘤大小、密度及并发症进行观察。结果 射频治疗后5min79%(27/34)的肺癌病灶半大,21％（7／34）大小无变化,＜5cm的肿瘤密度增高,＞5cm的肿瘤密度均减低;2个月后17例复查,64％（11／17）病灶较原病灶缩小,18％（3／17）病灶较术前增大,18％（3／17）病灶大小无变化,肿瘤的CT值均减低约20Hu左右。结论 CT可准确评价集束电极射频治疗后肺癌的病理变化。     

Exercise-related QT interval shortening with a peaked T wave in a healthy boy with a family history of sudden cardiac death

An asymptomatic 15-year-old boy, who had a family history of sudden cardiac death, was referred for screening for cardiac disease. The 12-lead electrocardiogram at rest showed a short QT/QTc(Bazett)/QTc(Fredericia) interval of 320/388/364 ms, but the intervals were further shortened to 200/339/284 ms after the treadmill test concomitant with appearance of a peaked T wave. Other conventional cardiac examinations were normal, but effective refractory period was less than 180 ms in both ventricles, and double ventricular extrastimulation reproducibly induced nonsustained polymorphic ventricular tachycardia. Intravenous administration of epinephrine also induced a short QT interval and a peaked T wave, and a hump was manifested on the T wave of the first postpacing beat with a longer preceding R-R interval. Furthermore, a couple of premature ventricular complexes originated from a similar timing as the hump. Genetic analysis did not show the mutation in KCNQ1, KCNH2, KCNE1, KCNE2, KCNJ2, SCN5A genes but revealed single nucleotide polymorphism (C5457T) in SCN5A gene.     

ClusterSeg: A crowd cluster pinpointed nucleus segmentation framework with cross-modality datasets

The detection and segmentation of individual cells or nuclei is often involved in image analysis across a variety of biology and biomedical applications as an indispensable prerequisite. However, the ubiquitous presence of crowd clusters with morphological variations often hinders successful instance segmentation. In this paper, nuclei cluster focused annotation strategies and frameworks are proposed to overcome this challenging practical problem. Specifically, we design a nucleus segmentation framework, namely ClusterSeg, to tackle nuclei clusters, which consists of a convolutional-transformer hybrid encoder and a 2.5-path decoder for precise predictions of nuclei instance mask, contours, and clustered-edges. Additionally, an annotation-efficient clustered-edge pointed strategy pinpoints the salient and error-prone boundaries, where a partially-supervised PS-ClusterSeg is presented using ClusterSeg as the segmentation backbone. The framework is evaluated with four privately curated image sets and two public sets with characteristic severely clustered nuclei across a variety range of image modalities, e.g., microscope, cytopathology, and histopathology images. The proposed ClusterSeg and PS-ClusterSeg are modality-independent and generalizable, and superior to current state-of-the-art approaches in multiple metrics empirically. Our collected data, the elaborate annotations to both public and private set, as well the source code, are released publicly at https://github.com/lu-yizhou/ClusterSeg.     

Association of Ultrasonographic Findings of Synovitis With Anti–Cyclic Citrullinated Peptide Antibodies and Rheumatoid Factor in Patients With Palindromic Rheumatism During Active Episodes

Objective. The purpose of this study was to investigate whether the presence of ultrasonographic findings of synovitis is associated with the presence of anti–cyclic citrullinated peptide (CCP) antibodies and rheumatoid factor (RF) in patients with palindromic rheumatism (PR) during active episodes. Methods. Clinically involved regions of 84 patients with PR during active episodes were examined with high‐resolution ultrasonography. Serum levels of anti‐CCP antibodies were determined by an enzyme‐linked immunosorbent assay, and RF levels were measured by nephelometry. Results. Thirty patients (36%) had ultrasonographic findings of synovitis during active episodes. Significantly higher positive rates of anti‐CCP antibodies and RF were observed in patients with PR who had ultrasonographic findings of synovitis compared with those who had no ultrasonographic findings of synovitis (26.7% versus 5.6%; odds ratio, 6.18; P < .05; and 30.0% versus 5.6%; odds ratio, 7.29; P < .01, respectively). The intraobserver and interobserver agreement for the detection of synovitis and Doppler signals by ultrasonographic assessment was excellent. Conclusions. Ultrasonography is a reliable method for assessing the presence of synovitis in patients with PR during active episodes. The ultrasonographic findings of synovitis are associated with the presence of anti‐CCP antibodies and RF in patients with PR.