Background::The ability to generate functional hepatocytes without relying on donor liver organs holds significant therapeutic promise in the fields of regenerative medicine and potential liver disease treatments. Clustered regularly interspaced short palindromic repeats (CRISPR) activator (CRISPRa) is a powerful tool that can conveniently and efficiently activate the expression of multiple endogenous genes simultaneously, providing a new strategy for cell fate determination. The main purpose of this study is to explore the feasibility of applying CRISPRa for hepatocyte reprogramming and its application in the treatment of mouse liver fibrosis.Method::The differentiation of mouse embryonic fibroblasts (MEFs) into functional induced hepatocyte-like cells (iHeps) was achieved by utilizing the CRISPRa synergistic activation mediator (SAM) system, which drove the combined expression of three endogenous transcription factors— Gata4, Foxa3, and Hnf1a—or alternatively, the expression of two transcription factors, Gata4 and Foxa3. In vivo, we injected adeno-associated virus serotype 6 (AAV6) carrying the CRISPRa SAM system into liver fibrotic Col1a1-Cre ER; Cas9 fl/fl mice, effectively activating the expression of endogenous Gata4 and Foxa3 in fibroblasts. The endogenous transcriptional activation of genes was confirmed using real-time quantitative polymerase chain reaction (RT-qPCR) and RNA-seq, and the morphology and characteristics of the induced hepatocytes were observed through microscopy. The level of hepatocyte reprogramming in vivo is detected by immunofluorescence staining, while the improvement of liver fibrosis is evaluated through Sirius red staining, alpha-smooth muscle actin (α-SMA) immunofluorescence staining, and blood alanine aminotransferase (ALT) examination. Results::Activation of only two factors, Gata4 and Foxa3, via CRISPRa was sufficient to successfully induce the transformation of MEFs into iHeps. These iHeps could be expanded in vitro and displayed functional characteristics similar to those of mature hepatocytes, such as drug metabolism and glycogen storage. Additionally, AAV6-based delivery of the CRISPRa SAM system effectively induced the hepatic reprogramming from fibroblasts in mice with live fibrosis. After 8 weeks of induction, the reprogrammed hepatocytes comprised 0.87% of the total hepatocyte population in the mice, significantly reducing liver fibrosis. Conclusion::CRISPRa-induced hepatocyte reprogramming may be a promising strategy for generating functional hepatocytes and treating liver fibrosis caused by hepatic diseases.

The clustered regularly interspaced short palindromic repeats(CRISPR)-Cas9 system has been widely used for genome engineer-ing and transcriptional regulation in many different organisms.Current CRISPR-activation(CRISPRa)platforms often require multi-ple components because of inefficient transcriptional activation.Here,we fused different phase-separation proteins to dCas9-VPR(dCas9-VP64-P65-RTA)and observed robust increases in transcriptional activation efficiency.Notably,human NUP98(nucleoporin 98)and FUS(fused in sarcoma)IDR domains were best at enhancing dCas9-VPR activity,with dCas9-VPR-FUS IDR(VPRF)outperforming the other CRISPRa systems tested in this study in both activation efficiency and system simplicity.dCas9-VPRF overcomes the target strand bias and widens gRNA designing windows without affecting the off-target effect of dCas9-VPR.These findings demonstrate the feasibility of using phase-separation proteins to assist in the regulation of gene expression and support the broad appeal of the dCas9-VPRF system in basic and clinical applications.

CRISPR-Cas系统作为细菌、真菌体内的适应性免疫系统用以抵御外来噬菌体的感染.经过改造的CRISPR-Cas9已成为继ZFN、TALEN之后的第三代基因编辑技术.在CRISPR-Cas9基础上衍生的CRISPRi、CRISPRa等系统可以实现暂时抑制、激活或改变基因表观遗传学修饰的功能.相比传统基因编辑技术,CRISPR-Cas9具有效率高、操作简便、价格低等优势,因而在基因制备动物模型,治疗感染性疾病、遗传性疾病以及癌症等方面均有广阔的应用前景.本文主要对CRISPR-Cas9技术的发展与临床应用进展进行综述.

Gene expression regulation,including loss-of-function and gain-of-function assays,is a powerful method to study developmental and disease mechanisms.Drosophila melanogaster is an ideal model system particularly well-equipped with many genetic tools.In this review,we describe and discuss the gene expression regulation techniques recently developed and their applications,including the CRISPR/Cas9-triggered heritable mutation system,CRISPR/dCas9-based transcriptional activation (CRISPRa) system,and CRISPR/dCas9-based transcriptional repression (CRISPRi) system,as well as the next-generation transgenic RNAi system.The main purpose of this review is to provide the fly research community with an updated summary of newly developed gene expression regulation techniques and help the community to select appropriate methods and optimize the research strategy.

The recently developed clustered regularly interspaced short palindromic repeats (CRISPR)-based techniques have made it possible to reprogram target gene expression without cloning complementary DNA or disturbing genomic sequence in mammalian cells and several multicellular organisms.We previously showed that CRISPR-associated protein 9 (Cas9) and CRISPR fromPrevotella andFrancisella 1 (Cpf1) could induce target mutations,deletions,inversions,and duplications both singly and multiplex in silkworm,Bombyx mori.However,it remains unknown whether the CRISPR activation (CRISPRa) system can be used in B.mori.In this study,we investigated the CRISPRa system,in which a nuclease dead Streptococcuspyogenes Cas9 (SpCas9) is fused to two transcription activation domains,including VP64 (a tetramer of the herpes simplex VP 16 transcriptional activator domain),and VPR (a tripartite activator,composed of VP64,p65,and Rta).The results showed that both dCas9-VP64 and dCas9-VPR systems could be used in B.mori cells,of which the latter showed significantly higher activity.The dCas9-VPR system showed considerable activity on all five tested target genes,and further analysis revealed that the up-regulation of genes was negatively correlated to their basal expression level.We also observed that this system could be used to upregulate a range of target genes.Taken together,our findings demonstrate that CRISPRa can be a powerful tool to study gene functions in B.mori and perhaps other non-drosophila insects.

CRISPR-Cas9是一种强大的基因组编辑系统,随着研究的深入,科学家建立了调控基因组转录的CRISPR-dCas9系统.该系统的建立基于dCas9的发现,dCas9丧失核酸酶活性,虽不具有DNA切割活性,但仍然具有DNA结合活性,其可在sgRNA的引导下靶向目的基因,将特定的转录激活因子(或抑制因子)携带至目的基因上游,实现对目的基因的转录激活(CRISPRa)或转录抑制(CRISPRi).目前该系统已用于遗传病治疗的实验研究,取得了可喜的进展,具有潜在的临床应用价值.该文从CRISPR-dCas9系统建立、发展以及在几种遗传病治疗领域的研究进行了综述.

CRISPR and adeno-associated virus are becoming powerful tools to remedy genetic disorders in somatic cells of adulthood.A recent study published in Science (Matharu et al.,2019) safely targeted the non-coding genomic region of Sim1 and MC4R with rAAV packed with CRISPRa,and successfully rescued the obesity syndrome caused by haploinsufficiency in a murine model,which shed light on their potential therapeutic applications in the future (Matharu et al.,2019).

由Cas9核酸酶和单向导RNA(sgRNA)组成的CRISPR/Cas9系统,可对sgRNA靶向的DNA序列进行缺失、插入和点突变等基因重编程操作,是新兴的基因编辑技术.此外,CRISPR/dCas9(Cas9核酸酶活性丧失的突变体),仍保留sgRNA靶向结合DNA的能力,dCas9蛋白融合转录激活物(CRISPRa)后可激活目标基因表达,也可通过融合转录阻遏物(CRISPRi)抑制目标基因表达.CRISPR/Cas9系统的高效输送是限制其临床广泛应用的主要问题之一.病毒载体被广泛用于递送CRISPR/Cas9元件;但就安全性、简便性和灵活性而言,非病毒载体研究更具吸引力.本文主要总结了CRISPR技术的原理和研究进展,包括CRISPR/Cas9的递送载体,递送模式以及递送过程的障碍,并回顾基于CRISPR技术在骨和软骨组织工程中的研究进展,讨论CRISPR技术在骨和软骨组织工程应用中面临的挑战和未来.

随着结构基因组学的不断成熟,基因组学已经进入功能基因组学时代.功能基因组学的主要目标是明确各基因及蛋白质的生物学功能及相互作用网络.该领域常用的研究策略是在基因组范围内通过人为改变生物体或细胞内基因的序列或表达状态,观察干预后的表型,从而建立基因型与表型之间的关联,阐述基因的功能.根据人为干预方式的不同,功能基因组筛选策略可分为功能缺失型筛选和功能获得型筛选两大类.本文将分别阐述这两类筛选策略的最新技术进展.其中,功能缺失型筛选包括小干扰RNA(small interfering RNA,siRNA)筛选、短发夹RNA(small hairpin RNA,shRNA)筛选、CRISPR(clustered regularly interspaced shortpalindromic repeats)-Cas(CRISPR-association proteins)基因敲除及CRISPR转录抑制(CRISPR interference,CRISPRi)筛选.功能获得型筛选包括cDNA/开放阅读框(open reading frame,ORF)文库过表达筛选及CRISPR转录激活(CRISPR activation,CRISPRa)筛选.本文还在阐述各种筛选体系作用机制的基础上讨论了不同体系的优缺点.另外,本文对点阵式筛选及混合式筛选两种筛选模式进行比较,并重点阐述了高内涵成像分析系统及二代测序技术在功能基因组学领域的应用及对该领域的推动作用.

基因编辑技术是通过核酸内切酶对基因组DNA进行定向改造的技术,可以实现对特定DNA碱基的缺失、替换等,常用的四种基因编辑工具分别是:巨型核酸酶、锌指核酸酶、转录激活因子样效应物核酸酶以及CRISPR/Cas9系统.其中CRISPR/Cas9系统作为一种新型的基因组编辑技术具有组成简单、特异性好、切割效率高的优点.该文对CRISPR/Cas9系统的结构组成和功能机制,动植物基因靶向编辑和人类在遗传性疾病、病毒感染性疾病以及肿瘤方面进行综述,旨在对CRISPR/Cas9系统的现状和发展进行总结和展望.