CRISPR/dCas9系统在活细胞成像领域的研究进展

研究不同基因、染色体以及基因与染色体之间的时空关系在遗传学、发育生物学和生物医学等领域具有重要意义。CRISPR/Cas9基因编辑技术具有优异的靶向性,已经成为应用最广泛的基因编辑工具。近年来,研究人员基于Cas9的核酸酶失活突变体dCas9发展了一系列先进的活细胞成像技术,为染色质、基因组特定位点的高分辨成像提供了快速、方便的研究工具。文中从细胞递送方式、荧光信号优化以及正交多色成像3个方面对CRISPR/dCas9系统在活细胞成像中的研究进展进行了综述,并对该领域的发展趋势进行了展望。     

A modified pCas/pTargetF system for CRISPR-Cas9-assisted genome editing in Escherichia coli

The clustered regularly interspaced short palindromic repeats(CRISPR)-associated nuclease 9(Cas9)-based genome editing tool pCas/pTargetF system that we establi...     

基于CRISPR/dCas9技术活细胞成像基因组位点的研究进展

基因组的三维空间组织在调节其功能、维持表观遗传和稳定性方面起着重要作用.虽然基于荧光原位杂交的标记技术或单细胞显微观察等方法对此进行了广泛研究,但是细胞内部的时空动态在很大程度上仍难以捉摸.目前标记和观察活细胞中特定内源基因组位点的方法操作难度大且对生物的侵入性伤害较大.CRISPR的发现彻底改变了基因组工程领域.除了...     

The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli

The CRISPR/Cas adaptive immune system provides resistance against phages and plasmids in Archaea and Bacteria. CRISPR loci integrate short DNA sequences from invading genetic elements that provide small RNA-mediated interference in subsequent exposure to matching nucleic acids. In Streptococcus thermophilus, it was previously shown that the CRISPR1/Cas system can provide adaptive immunity against phages and plasmids by integrating novel spacers following exposure to these foreign genetic elements that subsequently direct the specific cleavage of invasive homologous DNA sequences. Here, we show that the S. thermophilus CRISPR3/Cas system can be transferred into Escherichia coli and provide heterologous protection against plasmid transformation and phage infection. We show that interference is sequence-specific, and that mutations in the vicinity or within the proto-spacer adjacent motif (PAM) allow plasmids to escape CRISPR-encoded immunity. We also establish that cas9 is the sole cas gene necessary for CRISPR-encoded interference. Furthermore, mutation analysis revealed that interference relies on the Cas9 McrA/HNH- and RuvC/RNaseH-motifs. Altogether, our results show that active CRISPR/Cas systems can be transferred across distant genera and provide heterologous interference against invasive nucleic acids. This can be leveraged to develop strains more robust against phage attack, and safer organisms less likely to uptake and disseminate plasmid-encoded undesirable genetic elements.     

酿酒酵母中基于CRISPR/dCas9的基因转录调控工具的开发与应用

酿酒酵母(Saccharomyces cerevisiae)是重要的模式真核微生物,广泛用于基础研究和工业发酵。基于CRISPR/dCas9系统开发的转录调控方法具有可编程、多重性和正交性等优点,在酿酒酵母的基因调控、功能基因组学、代谢工程等研究领域具有巨大潜力。本文关注酿酒酵母中CRISPR/dCas9基因转录调控工具的研究进展,阐述了不同转录调节结构域对dCas9或gRNA活性的调节,设计与优化dCas9和gRNA表达的方法,影响CRISPR/dCas9系统转录调控效率、特异性和通量的靶向性因素,最后总结了该工具在酿酒酵母代谢工程中的应用,并对该技术的未来发展提出了展望。     

CRISPR/Cas9 editing of carotenoid genes in tomato

CRISPR/Cas9 technology is rapidly spreading as genome editing system in crop breeding. The efficacy of CRISPR/Cas9 in tomato was tested on Psy1 and CrtR-b2, two key genes of carotenoid biosynthesis. Carotenoids are plant secondary metabolites that must be present in the diet of higher animals because they exert irreplaceable functions in important physiological processes. Psy1 and CrtR-b2 were chosen because their impairment is easily detectable as a change of fruit or flower color. Two CRISPR/Cas9 constructs were designed to target neighboring sequences on the first exon of each gene. Thirty-four out of forty-nine (69%) transformed plants showed the expected loss-of-function phenotypes due to the editing of both alleles of a locus. However, by including the seven plants edited only at one of the two homologs and showing a normal phenotype, the editing rate reaches the 84%. Although none chimeric phenotype was observed, the cloning of target region amplified fragments revealed that in the 40% of analyzed DNA samples were present more than two alleles. As concerning the type of mutation, it was possible to identify 34 new different alleles across the four transformation experiments. The sequence characterization of the CRISPR/Cas9-induced mutations showed that the most frequent repair errors were the insertion and the deletion of one base. The results of this study prove that the CRISPRCas9 system can be an efficient and quick method for the generation of useful mutations in tomato to be implemented in breeding programs.     

CRISPR/Cas9-mediated engineering of Escherichia coli for n-butanol production from xylose in defined medium

Journal of Industrial Microbiology & Biotechnology - Butanol production from agricultural residues is the most promising alternative for fossil fuels. To reach the economic viability of...     

CRISPR/dCas9干扰bcsD基因表达调控细菌纤维素结构

木葡糖酸醋杆菌(Gluconacetobacter xylinus)是细菌纤维素的主要生产菌株。在该菌中,BcsD是纤维素合酶的亚基之一,参与细菌纤维素的组装过程。利用CRISPR/dCas9系统调控bcsD基因的表达量,获得了一系列bcsD基因表达量不同的木葡糖酸醋杆菌。通过分析细菌纤维素的结构特征发现,细菌纤维素的结晶度和孔隙率随着木葡糖酸醋杆菌中bcsD表达量的变化而发生改变。其中孔隙率的变化范围在59.95%–84.05%之间,结晶度的变化范围在74.26%–93.75%之间,而细菌纤维素的产量并未因bcsD的表达量变化而发生显著下降。结果表明,bcsD的表达量低于55.34%后,细菌纤维素的孔隙率显著上升,并且细菌纤维素的结晶度与bcsD的表达量呈正相关。最终,通过干扰bcsD基因的表达,实现了一步发酵木葡糖酸醋杆菌获得了产量稳定且结构不同的细菌纤维素。     

CRISPR/dCas9‐mediated activation of multiple endogenous target genes directly converts human foreskin fibroblasts into Leydig‐like cells

Recently, Leydig cell (LC) transplantation has been revealed as a promising strategy for treating male hypogonadism; however, the key problem restricting the application of LC transplantation is a severe lack of seed cells. It seems that targeted activation of endogenous genes may provide a potential alternative. Therefore, the aim of this study was to determine whether targeted activation of Nr5a1, Gata4 and Dmrt1 (NGD) via the CRISPR/dCas9 synergistic activation mediator system could convert human foreskin fibroblasts (HFFs) into functional Leydig‐like cells. We first constructed the stable Hsd3b‐dCas9‐MPH‐HFF cell line using the Hsd3b‐EGFP, dCas9‐VP64 and MS2‐P65‐HSF1 lentiviral vectors and then infected it with single guide RNAs. Next, we evaluated the reprogrammed cells for their reprogramming efficiency, testosterone production characteristics and expression levels of Leydig steroidogenic markers by quantitative real‐time polymerase chain reaction or Western blotting. Our results showed that the reprogramming efficiency was close to 10% and that the reprogrammed Leydig‐like cells secreted testosterone rapidly and, more importantly, responded effectively to stimulation with human chorionic gonadotropin and expressed Leydig steroidogenic markers. Our findings demonstrate that simultaneous targeted activation of the endogenous NGD genes directly reprograms HFFs into functional Leydig‐like cells, providing an innovative technology that may have promising potential for the treatment of male androgen deficiency diseases.     

基于CRISPR/Cas9的激活系统研究进展

基因编辑技术自问世以来就一直作为生物技术领域的研究热点。基因编辑工具成簇的规律间隔短回文重复序列及其相关系统(CRISPR/Cas系统)具有特异性、简便性和灵活性等优点,为研究人员提供了丰富的遗传操作工具,也让CRISPR/Cas系统的应用在多种生物中得到了飞速发展。特别是将转录激活因子与失活的Cas蛋白结合,可在RNA转录水平实现基因表达特异性调控,为生物技术在医学研究及农业领域的发展做出了重要的贡献。外源基因的过表达是验证基因功能和基因调控的常用方法,然而由于载体容量的限制难以实现多基因过表达。基于CRISPR/Cas9激活系统可在不同向导RNA的引导下对多个基因进行调控,实现调控水平验证基因功能。本文通过对CRISPR/Cas9激活系统组成及不同激活策略进行总结,整理针对过度激活的解决方案,为CRISPR/Cas9激活系统应用于棉花遗传改良及除草剂抗性研究提供更多参考。     

Triosephosphates are toxic to superoxide dismutase-deficient Escherichia coli

Increase in the production of triosephosphates has been considered an important factor leading to diabetic complications. It might be expected that like the other short chain monosaccharides, triosephosphates autoxidize producing superoxide radical and alpha,beta-diketones. Since superoxide can also initiate the oxidation of short chain sugars, free radical chain reactions are possible. If such reactions occur in vivo, triosephosphates would be more deleterious to cells lacking superoxide dismutase (SOD) than to normal cells. Here we demonstrate that triosephosphates kill a SOD-deficient Escherichia coli mutant much more than the parental, SOD-proficient strain. The effect is oxygen-dependent and is partially suppressed by aminoguanidine. Increased production of superoxide and diketones appeared to be the cause of triosephosphates toxicity.     

Coupling the CRISPR/Cas9 System with Lambda Red Recombineering Enables Simplified Chromosomal Gene Replacement in Escherichia coli

To date, most genetic engineering approaches coupling the type II Streptococcus pyogenes clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system to lambda Red recombineering have involved minor single nucleotide mutations. Here we show that procedures for carrying out more complex chromosomal gene replacements in Escherichia coli can be substantially enhanced through implementation of CRISPR/Cas9 genome editing. We developed a three-plasmid approach that allows not only highly efficient recombination of short single-stranded oligonucleotides but also replacement of multigene chromosomal stretches of DNA with large PCR products. By systematically challenging the proposed system with respect to the magnitude of chromosomal deletion and size of DNA insertion, we demonstrated DNA deletions of up to 19.4 kb, encompassing 19 nonessential chromosomal genes, and insertion of up to 3 kb of heterologous DNA with recombination efficiencies permitting mutant detection by colony PCR screening. Since CRISPR/Cas9-coupled recombineering does not rely on the use of chromosome-encoded antibiotic resistance, or flippase recombination for antibiotic marker recycling, our approach is simpler, less labor-intensive, and allows efficient production of gene replacement mutants that are both markerless and “scar”-less.     

Multiple-step chromosomal integration of divided segments from a large DNA fragment via CRISPR/Cas9 in Escherichia coli

Journal of Industrial Microbiology & Biotechnology - Although CRISPR/Cas9-mediated gene editing technology has developed vastly in Escherichia coli, the chromosomal integration of large DNA...