CRISPR/Cas技术在核酸检测中的应用进展

CRISPR/Cas是大多数细菌及古细菌基于RNA的后天免疫系统。由CRISPR/Cas系统改造而成的CRISPR/Cas技术已成为一种强大的基因编辑工具,广泛应用于基因功能研究和基因修饰与治疗。除了作为基因编辑工具,Ⅱ类Cas蛋白具有的"附属切割"特性,已被开发成一种快速、低成本且高灵敏的核酸检测工具,在核酸分子诊断领域具有重要的应用潜力。该文总结了3种Ⅱ类Cas蛋白在核酸检测领域的代表性研究进展,并对CRISPR/Cas系统在该领域的应用前景进行了展望。     

基于CRISPR/Cas技术的传染性病原精准便携化检测系统

以核酸为检测靶向的分子诊断方法是传染性病原体检测的金标准,但将其应用于便携化或现场快速诊断时仍存在多种限制和挑战,如特异性差、操作繁琐和便携化难等。规律间隔成簇短回文重复序列（CRISPR）/CRISPR相关蛋白（Cas）-荧光检测方法有望大幅提升核酸识别的特异性和信噪比。本研究开发了环介导等温扩增（LAMP）-CRISPR/Cas-便携化灰度读取仪检测系统。在CRISPR RNA (crRNA)存在下,利用CRISPR/Cas12a体系实现了对自主设计的甲型和乙型流感病毒核酸LAMP扩增反应体系的精准荧光检测,验证了CRISPR/Cas体系的高选择性和通用性。配套自主开发的便携化灰度读取仪,可同时实现荧光信号的低成本采集和高可靠性可视化判读,检测结束后直接输出样品的阳/阴性结果。利用本检测系统对实际样本进行了分析,验证了其可靠性和实用性,证明了本系统能够实现流感病毒的高灵敏、高特异性便携化分析。     

基于CRISPR/Cas的生物传感器在食源性病原菌检测中的应用进展

食源性病原菌严重威胁食品安全及消费者的健康,因此,对食源性病原菌快速、准确的检测方法对保障食品安全尤为重要。规则成簇的短回文重复序列（Clustered regularly interspaced short palindromic repeats,CRISPR）/CRISPR相关系统（CRISPR-associated systems, Cas）生物传感器是在引导核糖核酸（RNA）的作用下,使Cas蛋白与RNA形成RNA复合体,并对靶标基因进行特异性识别,从而将靶标信号转化为可检测的物理和化学信号。CRISPR/Cas生物传感器具有特异性好、可编程以及使用简便等优势,在病原菌检测领域具有广阔的应用前景。本文根据Cas蛋白的种类和作用机制不同,分别介绍了不同CRISPR/Cas系统的作用原理和特点,概述了基于不同CRISPR/Cas的生物传感的信号识别、信号放大、信号输出策略及其在食源性病原菌检测中的应用进展,讨论了基于CRISPR/Cas的多重生物传感器的构建原理及其在多病原菌同时检测中的应用,分析了基于CRISPR/Cas的生物传感器在病原菌快速检测中面临的挑战及未来的发展趋势。     

CRISPR/Cas9基因编辑非病毒递送系统

规律间隔成簇短回文重复序列及相关蛋白9(CRISPR/Cas9)系统的基因编辑技术为哺乳细胞基因组的精准修饰与编辑研究提供了高效、快捷的工具,但其化学生物学应用依然面临着CRISPR基因编辑工具Cas9蛋白和g RNA的细胞及活体递送等问题.近年来,研究人员通过开发多种非病毒递送载体,实现了编码CRISPR/Cas9基因编辑工具的DNA和信使RNA(mRNA)以及Cas9/gRNA核糖核蛋白(RNP)复合物的递送,并应用于靶基因的化学修饰与编辑调控.本文主要概述了近期CRISPR/Cas9基因编辑递送的研究进展,并对其化学生物学应用前景进行了展望.     

非病毒CRISPR/Cas9系统载体的研究进展

近年来,规律成簇的间隔短回文重复序列(clustered regularly interspaced short palindromic repeats sequences, CRISPR)/CRISPR相关蛋白9(CRISPR associated protein 9, Cas9)基因编辑系统已经成为生物医学基础研究中最强大的技术之一.但是如何将CRISPR/Cas9系统高效地递送至靶器官或细胞仍是一个巨大的挑战.随着非病毒载体的快速发展,基于脂质体、聚合物、无机纳米粒子等化学方法的纳米载体已经显现出巨大的应用潜力.本文首先对CRISPR/Cas9系统在疾病建模和疾病治疗中的应用潜力以及其局限性进行了阐述,然后分析了以质粒DNA、mRNA或蛋白质形式递送CRISPR/Cas9系统的优缺点并详细概述了已经出现的非病毒载体,最后总结了当前非病毒载体在递送CRISPR/Cas9系统过程中面临的关键障碍,并提出了有望克服这些障碍的策略.     

化学调控CRISPR/Cas9基因编辑技术的研究进展

规律间隔成簇短回文重复序列及其相关蛋白9(Clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9, CRISPR/Cas9)基因编辑技术作为一项基因工程领域革新式的技术,为癌症、遗传性疾病及感染性疾病等多种重大疾病的治疗提供了极大的帮助.但如何在特定细胞和组织中实现时空调控的精准基因编辑,进而避免脱靶效应,依然是该技术在临床转化领域面临的重要挑战.近年来,通过化学分子和反应实现对CRISPR/Cas9活性的调控已经成为提升这项基因编辑技术效率的重要手段之一.本文综合评述了一些最近报道的化学调控CRISPR/Cas9基因编辑的方法,并对其在临床医学领域的应用前景进行了展望.     

非病毒基因编辑CRISPR/Cas9递送载体的研究进展

基因编辑技术是指能对目标基因片段进行编辑,包括敲除、敲入等。近年来,基因编辑技术得到了很大的发展,包括规律成簇的间隔短回文重复序列(CRISPR)/CRISPR相关蛋白9(Cas9)在内的一系列基因编辑技术受到了广泛的关注。CRISPR/Cas9主要利用限制性内切酶,在蛋白或RNA的引导下,使目标位置的DNA双链断裂,在治疗基因表达异常引起的疾病方面表现出了巨大的潜力。应用这项技术进行基因编辑的关键点之一是,必须要将相关的蛋白或核酸高效地递送到细胞核中。相对于病毒载体,非病毒载体在安全性及规模化生产方面表现出了更高的优越性,是目前基因编辑递送载体发展的重点。本文对近期基因编辑CRISPR/Cas9非病毒递送载体的重要进展进行综述,介绍了非病毒载体的优势,在非病毒载体体系中,着重介绍脂质体和高分子载体在CRISPR/Cas9技术应用中的近期进展,并展望了非病毒CRISPR/Cas9递送载体在未来的发展方向。     

病原微生物的核酸检测分析研究进展

2019年12月,全球爆发新型冠状病毒肺炎疫情(COVID-19),使生物安全面临着巨大危机.病原微生物的快速、准确、低成本检测是保障生物安全的重要组成元素之一,是疫情预防、控制和诊断的关键.本综述介绍了核酸测序技术、等温扩增技术和基因编辑技术(CRISPR/Cas)于便携式一体化设备中的应用,为研发"低成本,高效率,...     

基于CRISPR的生物分析化学技术

CRISPR(Clustered regularly interspaced short palindromic repeats)技术是一种革命性的基因编辑和调控工具,问世之后迅速成为了生物医学领域的前沿热点,广泛用于基因功能研究和治疗。CRISPR具有优异的序列识别性质,核酸切割能力,并且易于编程设计改造,在生物分析化学领域展示出独特的魅力,在病毒检测、临床诊断和单细胞成像分析等方面都取得了突破性进展。目前基于CRISPR技术的检测方法种类繁多,本文综述了CRISPR-Cas分析检测方法的研究进展,并且展望了该技术的发展趋势。     

基于小分子化学反应工具构建CRISPR-Cas9功能调控体系的研究进展

CRISPR技术是目前基因编辑领域的一大研究热点,已在疾病治疗、作物改良等领域得到广泛的应用,CRISPR-Cas9系统是其中研究最为深入的一种类型.如何降低Cas9/sgRNA复合物在细胞内作用的脱靶性是CRISPR-Cas9技术发展面临的主要挑战之一.利用光或活性小分子诱发的小分子化学反应工具构建CRISPRCas9功能调控体系,通过对sgRNA, Cas9或Cas9/sgRNA复合物的功能进行调控,可以在细胞乃至活体水平上一定程度实现对CRISPR-Cas9作用的时间或空间特异性的操纵,大大降低非特异性基因编辑作用发生的概率,同时小分子对原体系的干扰较小,因此小分子化学反应逐渐成为操纵CRISPR-Cas9体系的一种重要的研究工具.本文总结和介绍了小分子反应工具用于CRISPR-Cas9功能调控系统构建的主要研究进展,并对其未来的发展进行了展望.     

二氧化碳和丙烯直接合成甲基丙烯酸的NiPMo/TiO2催化剂的研究

用溶胶-凝胶法以磷钼酸(MPA)的镍盐溶液水解钛酸四丁酯制备了NiPMo/TiO2催化剂.使用ICP、 XRD、 TG-DTA、 IR、 TPD-MS和微反应技术研究了催化剂的化学组成、热稳定性、化学吸附性质和催化反应性能.杂多钼酸盐与TiO2通过O2-在TiO2表面发生了键合.在623 K下,杂多阴离子仍保持原有的Keggin结构.CO2在Lewis酸位Ni(Ⅱ)和Lewis碱位Ni-O-Mo的桥氧协同作用下生成CO2卧式吸附态Ni(Ⅱ)←O-(CO)←(O--Ni).丙烯有多种吸附态在催化剂上吸附.在563 K、 1 MPa和空速1500 h-1的反应条件下,丙烯的摩尔转化率为3.2%,产物MAA选择性为95%.     

CoFe2O4自形成磁性液体场致结构化对磁化的影响

The Langevin paramagnetic theory can’t describe the relation between magnetization of ferrofluids and applied magnetic field. The structuralization of ferrofluids, which is considered the main influence factor of the magnetization, is regarded. The part of magnetization works is deposited when the structure is forming. This action influences the magnetization of ferrofluids directly or indirectly. On the base of the “compressing” model, the Langevin function that usually describes the magnetization of ferrofluid is modified, and a well-fitted curve is obtained. An equation of the relation between the equivalent volume fraction after being “compressed” and the intensity of magnetic field is discovered, which approximately describes the process of magnetization. The relation between the approximate initial susceptibility and the volume fraction can be obtained from modified formula.     

Highly regioselective Buchwald–Hartwig amination at C-2 of 2,4-dichloropyridine enabling a novel approach to 2,4-bisanilinopyridine (BAPyd) libraries

The highly regioselective Buchwald–Hartwig amination at C-2 of the cheap and readily accessible reagent, 2,4-dichloropyridine with a range of anilines and heterocyclic amines is described. This new methodology is robust and provides a facile access to 4-chloro-N-phenylpyridin-2-amines on 0.25 mol scale. These intermediates undergo a further Buchwald–Hartwig amination at higher temperature to enable rapid exploration of the chemical space at C-4 and to provide a library of 2,4-bisaminopyridines.     

Sulfur-directed metal-free and regiospecific methyl C(sp3)–H imidation of thioanisoles

Regiospecific and direct imidation of the methyl C(sp³)–H bond of thioanisoles is realized under mild and metal-free conditions with N-fluorobis(benzenesulfonyl)imide as an oxidant and nitrogen source. Proposed mechanism suggests that thionium ion intermediates and a Pummerer-type reaction are involved. The imidation has advantages such as high step-economy, excellent functionality tolerance, and regiospecificity, giving structurally diverse imidation products.     

Selective syntheses of 2H-1,3-oxazines and 1H-pyrrol-3(2H)-ones via temperature-dependent Rh(II)-carbenoid-mediated 2H-azirine-ring expansion

The Rh(II)-catalyzed reaction of 2-carbonyl-substituted 2H-azirines with ethyl 2-cyano-2-diazoacetate or 2-diazo-3,3,3-trifluoropropionate provides an easy access to 2H-1,3-oxazines and 1H-pyrrol-3(2H)-ones. These compounds can be selectively prepared from the same starting material using temperature as the only varied parameter. The 2-azabuta-1,3-diene intermediate, a common precursor for both heterocyclic products, isomerizes into 2H-1,3-oxazine under kinetic control, while 1H-pyrrol-3(2H)-one is the sole product of the reaction at elevated temperatures. According to DFT-calculations a one-atom oxazine ring contraction involving ring-opening to a 2-azabuta-1,3-diene intermediate, followed by a 1,5- and 1,2-prototropic shift leads to the consecutive formation of imidoylketene and azomethine ylide, which then further undergo cyclization to the pyrrole derivative.     

Copper catalyzed/mediated synthetic methodology for ebselen and related isoselenazolones

Scope of the copper catalyzed/mediated selenium-nitrogen coupling reaction has been studied for the synthesis of isoselenazolones. It is noticed that the 2-chloro, 2-bromo-, and 2-iodo-aryl amides substrates can be exploited in the selenium-nitrogen coupling reaction by employing 25-100 mol % of CuI/1,10-phenanthroline (L) and potassium carbonate as a base in DMF. Furthermore, electron rich 2-chloro-arylamides also underwent selenium-nitrogen coupling reaction to give biologically important selenium-nitrogen heterocycles. Also, copper-catalyzed selenium-nitrogen coupling reaction has been meticulously applied for the synthesis of diaryl diselenides having methoxy, amine, and amide functionality from respective aryl iodides in the presence of stoichiometric amount of succinimide as an external Se-N coupling partner.     

Knoevenagel condensation catalyzed by novel Nmm-based ionic liquids in water

A series of novel N-methyl morpholine (Nmm) based ionic liquids with 1,2-propanediol group were synthesized and used as catalysts for Knoevenagel condensation at room temperature in water. Under the effect of the catalyst, various aldehydes or aliphatic ketones could react with a wide range of activated methylene compounds well, including malononitrile, alkyl cyanoacetate, cyanoacetamide, β-diketone, barbituric acid, 2-arylacetonitrile and thiazolidinedione. Furthermore, most of the products could be separated just by filtrating and washing with water. Additionally, the catalyst is recyclable and applicable for the large-scale synthesis.     

Construction of polyheterocyclic spirotetrahydrothiophene derivatives via sulfa-Michael/aldol cascade reaction

A series of polyheterocyclic spirotetrahydrothiophene derivatives were obtained in moderate to excellent yields via a catalyst-free sulfa-Michael/aldol cascade reaction of chalcones 1 and commercially available 1,4-dithiane-2,5-diol 2 under mild conditions. We also present the first asymmetric sulfa-Michael/aldol cascade reaction of chalcones 1 and commercially available 1,4-dithiane-2,5-diol 2 with moderate to good enantioselectivities catalyzed by readily available chiral phase-transfer catalysts (PTCs).     

Suzuki-Miyaura reactions of the soluble guanylate cyclase inhibitor NS2028: a non-product specific route to C-8 substituted analogues

Both soluble guanylate cyclase (sGC) inhibitors ODQ 1 and NS2028 2 are synthesized via improved protocols. In the former case treating 3,4-dihydroquinoxalin-2(1H)-one oxime 8, which can be prepared in two steps from 1,2-benzenediamine, with 1,1′-carbonyldiimidazole (CDI) gives the dihydro-ODQ 10 that in the presence of KMnO₄ oxidises to give ODQ 1 in an overall yield of 46% starting from 1,2-benzenediamine. In the latter case, the synthesis affords NS2028 2 from 2-amino-4-bromophenol 3 in three steps with an overall yield of 85% and avoids the need for chromatography. Furthermore, Suzuki-Miyaura reaction conditions are described that enable the preparation of 8-aryl and 8-heteroaryl derivatives of NS2028 directly from NS2028 2. Finally, demethylation of the 8-(methoxyphenyl) substituted analogues afforded the 8-(hydroxyphenyl) derivatives 40-42. All new products are fully characterised.