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/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。     

家蚕CRISPR/Cpf1基因编辑系统建立

自CRISPR/Cas9基因编辑系统成功应用于模式生物以来,因其快速、高效、便捷等特点,广泛应用于基因功能研究、基因治疗和基因工程等研究领域.与此同时,CRISPR/Cas系统不断在微生物界的发现也加速了新的基因编辑工具的不断涌现.CRISPR/Cpf1是第二类(Ⅴ型)能够编辑哺乳动物基因组的CRISPR系统,相比于C...     

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。     

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...     

Leydig-like cells derived from reprogrammed human foreskin fibroblasts by CRISPR/dCas9 increase the level of serum testosterone in castrated male rats

In the past few years, Leydig cell (LC) transplantation has been regarded as an effective strategy for providing physiological patterns of testosterone in vivo. Recently, we have successfully converted human foreskin fibroblasts (HFFs) into functional Leydig-like cells (iLCs) in vitro by using the CRISPR/dCas9 system, which shows promising potential for seed cells. However, it is not known whether the reprogrammed iLCs can survive or restore serum testosterone levels in vivo. Therefore, in this study, we evaluate whether reprogrammed iLCs can restore the serum testosterone levels of castrated rats when they are transplanted into the fibrous capsule. We first developed the castrated Sprague Dawley rat model through bilateral orchiectomy and subsequently injected extracellular matrix gel containing transplanted cells into the fibrous capsule of castrated rats. Finally, we evaluated dynamic serum levels of testosterone and luteinizing hormone (LH) in castrated rats, the survival of implanted iLCs, and the expression levels of Leydig steroidogenic enzymes by immunofluorescence staining and Western blotting. Our results demonstrated that implanted iLCs could partially restore the serum testosterone level of castrated rats, weakly mimic the role of adult Leydig cells in the hypothalamic-pituitary-gonadal axis for a short period, and survive and secrete testosterone, through 6 weeks after transplantation. Therefore, this study may be valuable for treating male hypogonadism in the future.     

CRISPR/Cas9-based knockout reveals that the clock gene timeless is indispensable for regulating circadian behavioral rhythms in Bombyx mori

Circadian rhythms, which are ubiquitous and adaptive, occur across all species, from microbes to humans, in which they organize and modify behavior and physiology. timeless (tim) is a canonical clock gene. The core composition of the Drosophila melanogaster endogenous circadian clock has been extensively investigated; however, in lepidopteran insects, including Bombyx mori, the mechanism is complicated and little is known regarding the participation of tim in the negative feedback loop responsible for behavioral activities. To arrive at a comprehensive understanding of the role of tim in the B. mori endogenous circadian clock, we exploited the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 gene editing system. We attempted to elucidate the functions of tim in the circadian clock of B. mori using Bmtim mutants. The knockouts affected two circadian behavioral activities: adult emergence and embryo hatching rhythms. Quantitative real-time polymerase chain reaction results confirmed that tim-knockouts induced relative reductions in the expression levels, and thereby the oscillation amplitudes, of Bmper and Bmclk messenger RNAs during both the photophase and scotophase. Additionally, the daily rhythmic expression of Bmdbt was upregulated in the photophase and downregulated in the scotophase in a tim-knockout. Our study reveals that tim is integral to the B. mori circadian clock and may be involved in regulating eclosion and hatching rhythms.     

Construction of a One-Vector Multiplex CRISPR/Cas9 Editing System to Inhibit Nucleopolyhedrovirus Replication in Silkworms

Recently the developed single guide(sg)RNA-guided clustered regularly interspaced short palindromic repeats/associated protein 9 nuclease(CRISPR/Cas9) technology has opened a new avenue for antiviral therapy. The CRISPR/Cas9 system uniquely allows targeting of multiple genome sites simultaneously. However, there are relatively few applications of CRISPR/Cas9 multigene editing to target insect viruses. To address the need for sustained delivery of a multiplex CRISPR/Cas9-based genome-editing vehicle against insect viruses, we developed a one-vector(pSL1180-Cas9-U6-sgRNA) system that expresses multiple sgRNA and Cas9 protein to excise Bombyx mori nucleopolyhedrovirus(BmNPV) in insect cells.We screened the immediate-early-1 gene(ie-1), the major envelope glycoprotein gene(gp64), and the late expression factor gene(lef-11), and identified multiple sgRNA editing sites through flow cytometry and viral DNA replication analysis. In addition, we constructed a multiplex editing vector(PSL1180-Cas9-sgIE1-sgLEF11-sgGP64, sgMultiple) to efficiently regulate multiplex gene-editing and inhibit BmNPV replication after viral infection. This is the first report of the application of a multiplex CRISPR/Cas9 system to inhibit insect virus replication. This multiplex system can significantly enhance the potential of CRISPR/Cas9-based multiplex genome engineering in insect virus.     

Bombyx mori kynurenine 3-monooxygenase gene editing and insect molecular breeding using the clustered regularly interspaced short palindromic repeat/CRISPR associated protein 9 system

Genome editing by clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR associated protein (Cas)9, a third-generation gene scissors, and molecular breeding at the genome level are attracting considerable attention as future breeding techniques. In the present study, genetic and phenotypic analyses were conducted to examine the molecular breeding of Bombyx mori through CRISPR/Cas9-mediated editing of the kynurenine 3-monooxygenase (KMO) gene. The synthesized guide RNAs (gRNAs) were analyzed using T7 endonuclease I after introduction into the BM-N silkworm cell line. To edit the silkworm gene, K1P gRNA, and Cas9 complexes were microinjected into silkworm embryos. After microinjection, the hatching rate and the incidence of mutation were determined as 18.1% and 60%, respectively. Gene mutation was verified in the heterozygous G0 generation, but no phenotypic change was observed; however, certain embryos and moths produced through sib-mating had significant differences compared to the wild-type. In successive generations, a distinct phenotypic change was also observed by continuous mating. Thus, although there are limitations in the phenotypic expression in breeding through the induction of deletion mutations, as in the present study, the process is believed to yield successful results within a shorter period compared to traditional breeding and is safer than transgenic technology.     

基于CRISPR/Cas9系统构建绵羊VASA基因敲入载体及验证

本研究旨在探索VASA基因在绵羊睾丸发育中的表达变化,并通过构建VASA基因敲入载体,为下一步进行绵羊生殖细胞体外诱导分化研究提供基础。采集性成熟前后即3月龄（3-month-old,3M）和9月龄（9-month-old,9M）绵羊睾丸组织,利用实时荧光定量PCR （quantitative real-time PCR,qPCR）和Western blotting技术分析VASA基因的差异表达,并利用免疫组织化学技术对VASA基因的表达定位进行分析。设计靶向VASA基因的向导RNA （guide RNA,gRNA）,并构建同源重组载体,进行质粒转染绵羊耳成纤维细胞。结合CRISPR/dCas9技术对VASA基因进行激活,进一步验证载体效率。结果表明,VASA基因随着绵羊睾丸发育,表达水平极显著增加（P<0.01）,且主要定位在精母细胞和圆形精子细胞中。利用CRISPR/Cas9系统构建了VASA基因敲入载体,联合pEGFP-PGK puro-VASA载体转染耳成纤维细胞,CRISPR/dCas9系统激活后,耳成纤维细胞成功表达VASA基因。结果提示,VASA基因在绵羊睾丸发育和精子发生中发挥潜在功能,且通过CRISPR/Cas9系统可在体外构建VASA基因敲入载体,为下一步探究VASA基因对绵羊雄性生殖细胞的发育和分化提供有效的研究手段。     

Functional analysis of Bombyx Wnt1 during embryogenesis using the CRISPR/Cas9 system

Recently established, custom-designed nuclease technologies such as the clustered regularly interspaced short palindromic repeat (CRISPR)-associated system provide attractive genome editing tools. Targeted gene mutagenesis using the CRISPR/Cas9 system has been achieved in several orders of insects. However, outside of studies on Drosophila melanogaster and the lepidopteron model insect Bombyx mori, little success has been reported, which is largely due to a lack of effective genetic manipulation tools that can be used in other insect orders. To create a simple and effective method of gene knockout analysis, especially for dissecting gene functioning during insect embryogenesis, we performed a functional analysis of the Bombyx Wnt1 (BmWnt1) gene using Cas9/sgRNA-mediated gene mutagenesis. The Wnt1 gene is required for embryonic patterning in various organisms, and its crucial roles during embryogenesis have been demonstrated in several insect orders. Direct injection of Cas9 mRNA and BmWnt1-specific sgRNA into Bombyx embryos induced a typical Wnt-deficient phenotype: injected embryos could not hatch and exhibited severe defects in body segmentation and pigmentation in a dose-dependent manner. Quantitative real-time PCR (qRT-PCR) analysis revealed that Hox genes were down-regulated after BmWnt1 depletion. Furthermore, large deletion, up to 18 Kb, ware generated. The current study demonstrates that using the CRISPR/Cas9 system is a promising approach to achieve targeted gene mutagenesis during insect embryogenesis.     

CRISPR系统用于昆虫基因表达调控的研究进展与展望

昆虫基因功能研究因缺少相应的工具而受到明显限制,但CRISPR/Cas9系统的出现为昆虫基因编辑及转录调控研究提供巨大助力。将Cas9核酸酶的RuvC和HNH剪切结构域失活改造得到的dCas9系统近年来在基因转录调控方面得到了广泛应用,同时CRISPR/dCpf1和最新发现的CRISPR/Cas13(a/b)系统为基因功能研究提供更多选择。本文综述了dCas9, dCpf1及Cas13(a/b)系统作用机理及在果蝇中的转录调控研究进展,以期为相关昆虫研究提供参考。     

基于CRISPR转录激活系统构建内源性着丝粒蛋白F稳定过表达的肝细胞癌细胞模型

临床研究表明着丝粒蛋白F(centromere protein F,CENPF)与肝细胞癌(hepatocellular carcinoma,HCC)的发生相关。由于CENPF蛋白的分子量高达358 kDa,目前有关CENPF致癌机制研究使用的体内外模型主要基于CENPF表达敲低,尚缺乏将CENPF过表达以探究其与癌变因果关系的研究,CENPF过表达改变到底是“因”还是“果”仍不清楚。本研究旨在利用包括lentiMPHv2和lentiSAMv2两个载体的CRISPR/dCas9转录激活协同激活介体(synergistic activation mediator,SAM)系统构建内源性CENPF稳定过表达的细胞模型,为阐明CENPF过表达与HCC发生发展的因果关系提供有效工具。首先设计并合成能够特异性识别CENPF基因转录起始位点的单向导核糖核酸(single guide RNAs,sgRNA),并将其插入lentiSAMv2质粒中。利用慢病毒载体将lentiMPHv2质粒导入Huh-7和HCCLM3细胞株中,用潮霉素B筛选后再用慢病毒载体将连接好的携带sgRNA序列的lentiSAMv2质粒导入细胞中,并用杀稻瘟菌素S继续筛选。对2种抗生素筛选获得的Huh-7和HCCLM3细胞株样品进行CENPF mRNA和蛋白表达水平的检测,结果显示sgRNA1和sgRNA4序列均能够激活内源CENPF的表达,其中sgRNA4诱导转录效果更加明显。本研究利用CRISPR/dCas9系统成功构建内源性CENPF稳定过表达的HCC细胞模型,为研究CENPF过表达与肿瘤发生的因果关系奠定基础,并为构建大分子量蛋白过表达的细胞模型提供参考方案。     

CRISPR/Cas9 system in Plasmodium falciparum using the centromere plasmid

The CRISPR/Cas9 nuclease system is a powerful method to genetically modify the human malarial parasite, Plasmodium falciparum. Currently, this method is carried out by co-transfection with two plasmids, one containing the Cas9 nuclease gene, and another encoding the sgRNA and the donor template DNA. However, the efficiency of modification is currently low owing to the low frequency of these plasmids in the parasites. To improve the CRISPR/Cas9 nuclease system for P. falciparum, we developed a novel method using the transgenic parasite, PfCAS9, which stably expresses the Cas9 nuclease using the centromere plasmid. To examine the efficiency of genetic modification using the PfCAS9 parasite, we performed site-directed mutagenesis of kelch13 gene, which is considered to be involved in artemisinin resistance. Our results demonstrated that the targeted mutation could be introduced with almost 100% efficiency when the transfected PfCAS9 parasites were treated with two drugs to maintain both the centromere plasmid containing the Cas9 nuclease and the plasmid having the sgRNA. Therefore, the PfCAS9 parasite is a useful parasite line for the genetic modification of P. falciparum.     

A palmitoyltransferase Approximated gene Bm-app regulates wing development in Bombyx mori

The silkworm Bombyx mori is an important lepidopteran model insect in which many kinds of natural mutants have been identified.However,molecular mechanisms of most of these mutants remain to be explored.Here we report the identification of a gene Bm-app is responsible for the silkworm minute wing(mw)mutation which exhibits exceedingly small wings during pupal and adult stages.Compared with the wild type silkworm,relative messenger RNA expression of Bm-app is significantly decreased in the ul 1 mutant strain which shows mw phenotype.A 10 bp insertion in the putative promoter region of the Bm-app gene in mw mutant strain was identified and the dual luciferase assay revealed that this insertion decreased Bm-app promoter activity.Furthermore,clustered regularly interspaced short palindromic repeats/RNA-guided Cas9 nucleases-mediated depletion of the Bm-app induced similar wing defects which appeared in the mw mutant,demonstrating that Bm-app controls wing development in B.mori.Bm-app encodes a palmitoyltransferase and is responsible for the palmitoylation of selected cytoplasmic proteins,indicating that it is required for cell mitosis and growth during wing development.We also discuss the possibility that Bm-app regulates wing development through the Hippo signaling pathway in B.mori.