Inhibition of HIV-1 gene expression by retroviral vector-mediated small-guide RNAs that direct specific RNA cleavage by tRNase ZL

The tRNA 3′-processing endoribonuclease (tRNase Z or 3′ tRNase; EC 3.1.26.11) is an essential enzyme that removes the 3′ trailer from pre-tRNA. The long form (tRNase ZL) can cleave a target RNA in vitro at the site directed by an appropriate small-guide RNA (sgRNA). Here, we investigated whether this sgRNA/tRNase ZL strategy could be applied to gene therapy for AIDS. We tested the ability of four sgRNA-expression plasmids to inhibit HIV-1 gene expression in COS cells, using a transient-expression assay. The three sgRNAs guide inhibition of HIV-1 gene expression in cultured COS cells. Analysis of the HIV-1 mRNA levels suggested that sgRNA directed the tRNase ZL to mediate the degradation of target RNA. The observation that sgRNA was localized primarily in nuclei suggests that tRNase ZL cleaves the HIV-1 mRNA when complexed with sgRNA in this location. We also examined the ability of two retroviral vectors expressing sgRNA to suppress HIV-1 expression in HIV-1-infected Jurkat T cells. sgRNA-SL4 suppressed HIV-1 expression almost completely in infected cells for up to 18 days. These results suggest that the sgRNA/tRNase ZL approach is effective in downregulating HIV-1 gene expression.     

Double-strand break toxicity is chromatin context independent

Cells respond to double-strand breaks (DSBs) by activating DNA damage response pathways, including cell cycle arrest. We have previously shown that a single double-strand break generated via CRISPR/Cas9 is sufficient to delay cell cycle progression and compromise cell viability. However, we also found that the cellular response to DSBs can vary, independent of the number of lesions. This implies that not all DSBs are equally toxic, and raises the question if the location of a single double-strand break could influence its toxicity. To systematically investigate if DSB-location is a determinant of toxicity we performed a CRISPR/Cas9 screen targeting 6237 single sites in the human genome. Next, we developed a data-driven framework to design CRISPR/Cas9 sgRNA (crRNA) pools targeting specific chromatin features. The chromatin context was defined using ChromHMM states, Lamin-B1 DAM-iD, DNAseI hypersensitivity, and RNA-sequencing data. We computationally designed 6 distinct crRNA pools, each containing 10 crRNAs targeting the same chromatin state. We show that the toxicity of a DSB is highly similar across the different ChromHMM states. Rather, we find that the major determinants of toxicity of a sgRNA are cutting efficiency and off-target effects. Thus, chromatin features have little to no effect on the toxicity of a single CRISPR/Cas9-induced DSB.     

基于CRISPR/Cas9技术的高通量筛选平台:发掘病毒复制相关宿主分子的新途径

病毒作为严格的细胞内寄生生物,需要多种宿主蛋白辅助其完成生命周期。寻找与病毒复制相关的宿主因子并揭示其作用机制,将有助于阐明病毒的感染机制,为疫病的防治提供新靶标。与RNA干扰技术相比,近年来兴起的CRISPR/Cas9技术能更特异、高效、准确地实现基因组编辑,因而在功能基因研究中得到更广泛应用。而基于CRISPR/Cas9系统的宿主全基因组sgRNA文库高通量筛选技术平台,可快速发现参与病毒侵入、复制等生物学过程的关键宿主因子,通过明确病毒-宿主分子相互作用进而揭示病毒的生命周期,为分子病毒学和免疫学提供了强大的研究工具。本文主要总结了基于CRISPR/Cas9技术的高通量筛选平台的具体筛选流程,归纳和讨论了该平台在筛选调控病毒复制相关宿主因子中的应用现状和发展前景。     

Updates on CRISPR-based gene editing in HIV-1/AIDS therapy

Although tremendous efforts have been made to prevent and treat HIV-1 infection, HIV-1/AIDS remains a major threat to global human health. The combination antiretroviral therapy (cART), although able to suppress HIV-1 replication, cannot eliminate the proviral DNA integrated into the human genome and thus requires lifelong treatment that may lead to various side effects. In recent years, clustered regularly interspaced short palindromic repeat (CRISPR)-associated nuclease 9 (Cas9) related gene-editing systems have been developed and designed as effective ways to treat HIV-1 infection. However, new gene-targeting tools derived from or functioning like CRISPR/Cas9, including base editor, prime editing, SHERLOCK, DETECTR, PAC-MAN, ABACAS, pfAGO, have been developed and optimized for pathogens detection and diseases correction. Here, we summarize recent studies on HIV-1/AIDS gene therapy and provide more gene-editing targets based on studies relating to the molecular mechanism of HIV-1 infection. We also identify the strategies and potential applications of these new gene-editing technologies for HIV-1/AIDS treatment in the future. Moreover, we discuss the caveats and problems that should be addressed before the clinical use of these versatile CRISPR-based gene targeting tools. Finally, we offer alternative solutions to improve the practice of gene targeting in HIV-1/AIDS gene therapy.     

细胞内表达CCR5Delta32蛋白对HIV-1感染抑制作用的研究

人CCR5Delta32突变个体能有效抵制HIV-1感染,主要是由于该个体淋巴细胞内表达的CCR5Delta32突变蛋白能通过反式显性失活效应(TDN)抑制细胞表面HIV-1辅受体CCR5和CXCR4的产生．通过构建CCR5Delta32慢病毒载体,体外转染人外周血单个核细胞(PBMCs),研究细胞内表达CCR5Delta32蛋白对HIV-1感染的抑制作用．结果表明,表达CCR5Delta32蛋白的人PBMCs对HIV-1 R5、X4及R5X4毒株感染均具有显著的抑制作用．这些工作为后续的AIDS基因治疗研究奠定了基础．     

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.     

CRISPR/Cas9基因组编辑技术在HIV-1感染治疗中的应用进展

基因治疗是将外源正常基因通过一定方式导入人体靶细胞以纠正或补偿因基因缺陷和异常引起的疾病,从而达到治疗目的。因此,基因治疗的技术方法在研究持续感染HIV-1或潜伏感染HIV-1原病毒患者的治疗中具有重大的现实意义。目前,现有的基因治疗方法存在识别靶向位点有限及脱靶几率大等主要问题。最新研究表明来源于细菌和古菌的规律间隔成簇短回文重复序列及其相关核酸酶9系统[Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9), CRISPR/Cas9]已被成功改造成基因组定点编辑工具。因此,如何利用CRISPR/Cas9系统实现对HIV-1病毒基因组进行高效靶向修饰,从而达到治疗HIV-1感染病患的目的已经成为当前研究的热点。本文参考最新国内外研究成果,重点介绍了 CRISPR/Cas9基因组编辑技术在HIV-1感染疾病治疗中的应用,主要包括CCR5基因编辑、清除HIV-1原病毒以及活化HIV-1原病毒,以期为HIV-1感染疾病的预防与治疗提供重要研究参考。     

Unexpected gene activation following CRISPR‐Cas9‐mediated genome editing

The discovery of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and its development as a genome editing tool has revolutionized the field of molecular biology. In the DNA damage field, CRISPR has brought an alternative to induce endogenous double‐strand breaks (DSBs) at desired genomic locations and study the DNA damage response and its consequences. Many systems for sgRNA delivery have been reported in order to efficiently generate this DSB, including lentiviral vectors. However, some of the consequences of these systems are not yet well understood. Here, we report that lentiviral‐based sgRNA vectors can integrate into the endogenous genomic target location, leading to undesired activation of the target gene. By generating a DSB in the regulatory region of the ABCB1 gene using a lentiviral sgRNA vector, we can induce the formation of Taxol‐resistant colonies. We show that these colonies upregulate ABCB1 via integration of the EEF1A1 and the U6 promoters from the sgRNA vector. We believe that this is an unreported CRISPR/Cas9 on‐target effect that researchers need to be aware of when using lentiviral vectors for genome editing.     

Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis,tobacco, sorghum and rice

The type II CRISPR/Cas system from Streptococcus pyogenes and its simplified derivative, the Cas9/single guide RNA (sgRNA) system, have emerged as potent new tools for targeted gene knockout in bacteria, yeast, fruit fly, zebrafish and human cells. Here, we describe adaptations of these systems leading to successful expression of the Cas9/sgRNA system in two dicot plant species, Arabidopsis and tobacco, and two monocot crop species, rice and sorghum. Agrobacterium tumefaciens was used for delivery of genes encoding Cas9, sgRNA and a non-fuctional, mutant green fluorescence protein (GFP) to Arabidopsis and tobacco. The mutant GFP gene contained target sites in its 5′ coding regions that were successfully cleaved by a CAS9/sgRNA complex that, along with error-prone DNA repair, resulted in creation of functional GFP genes. DNA sequencing confirmed Cas9/sgRNA-mediated mutagenesis at the target site. Rice protoplast cells transformed with Cas9/sgRNA constructs targeting the promoter region of the bacterial blight susceptibility genes, OsSWEET14 and OsSWEET11, were confirmed by DNA sequencing to contain mutated DNA sequences at the target sites. Successful demonstration of the Cas9/sgRNA system in model plant and crop species bodes well for its near-term use as a facile and powerful means of plant genetic engineering for scientific and agricultural applications.     

利用CRISPR 系统介导人类mir-505基因位点的编辑

目的:通过针对人源mir-505基因,设计不同sgRNA,从而利用CRIPSR系统对mir-505基因位点进行编辑,并探讨CRISPR系统对靶点的剪切效率的影响因素。方法:针对人源mir-505基因设计两条具有不同GC%的sgRNA,随后分别将其构建入41824-sgRNA表达载体和42230-Cas9蛋白/sgRNA共表达载体,并将表达载体利用脂质体转染法转染入人类细胞,通过T7E1assay检测不同CRISPR系统对靶点剪切效率的影响。结果:对靶点的剪切与Cas9蛋白和sgRNA的剂量成正比;当sgRNA与Cas9蛋白共传递时,也能够明显提高靶点的剪切效率;而较低的sgRNA的GC%会降低其对靶点的剪切效率。结论:本研究利用CRISPR系统靶向人源细胞的mir-505基因,使得mir-505基因发生突变。CRISPR系统对靶点的剪切效率和sgRNA和Cas9蛋白的剂量、sgRNA是否和Cas9蛋白共传递以及sgRNA的GC%有关。     

CRISPR/Cas9基因编辑技术在致瘤病毒感染研究中的应用

成簇的规律间隔的短回文重复序列及其相关蛋白9〔clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9),CRISPR/Cas9〕基因编辑技术的发现源于真细菌和古细菌中CRISPR/Cas系统介导的适应性免疫机制研究。该技术利用特异性向导RNA识别靶点基因,引导核酸内切酶Cas9对其切割,并通过同源重组或非同源末端连接完成对目的DNA的编辑。某些病毒感染机体后,可将其基因组整合到宿主细胞基因组中或潜伏于组织中而无法被彻底清除,从而引起持续性感染。本文参考2013年以来CRISPR/Cas9基因组编辑技术的最新相关研究报道,重点综述其在人类免疫缺陷病毒1型(human immunodeficiency virus type 1,HIV-1)、人乳头瘤病毒(human papillomavirus,HPV )、乙型肝炎病毒(hepatitis B virus, HBV)、 Epstein-Barr病毒(Epstein-Barr virus,EBV)等致瘤病毒感染相关疾病研究中的应用,并概括其作用于这些病毒的有效靶点。     

Optimized paired‐sgRNA/Cas9 cloning and expression cassette triggers high‐efficiency multiplex genome editing in kiwifruit

Kiwifruit is an important fruit crop; however, technologies for its functional genomic and molecular improvement are limited. The clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR‐associated protein (Cas) system has been successfully applied to genetic improvement in many crops, but its editing capability is variable depending on the different combinations of the synthetic guide RNA (sgRNA) and Cas9 protein expression devices. Optimizing conditions for its use within a particular species is therefore needed to achieve highly efficient genome editing. In this study, we developed a new cloning strategy for generating paired‐sgRNA/Cas9 vectors containing four sgRNAs targeting the kiwifruit phytoene desaturase gene (AcPDS). Comparing to the previous method of paired‐sgRNA cloning, our strategy only requires the synthesis of two gRNA‐containing primers which largely reduces the cost. We further compared efficiencies of paired‐sgRNA/Cas9 vectors containing different sgRNA expression devices, including both the polycistronic tRNA‐sgRNA cassette (PTG) and the traditional CRISPR expression cassette. We found the mutagenesis frequency of the PTG/Cas9 system was 10‐fold higher than that of the CRISPR/Cas9 system, coinciding with the relative expressions of sgRNAs in two different expression cassettes. In particular, we identified large chromosomal fragment deletions induced by the paired‐sgRNAs of the PTG/Cas9 system. Finally, as expected, we found both systems can successfully induce the albino phenotype of kiwifruit plantlets regenerated from the G418‐resistance callus lines. We conclude that the PTG/Cas9 system is a more powerful system than the traditional CRISPR/Cas9 system for kiwifruit genome editing, which provides valuable clues for optimizing CRISPR/Cas9 editing system in other plants.     

CRISPR/Cas9系统中sgRNA设计与脱靶效应评估

基于CRISPR/Cas9系统介导的第三代基因组编辑技术,已成功应用于动物、植物和微生物等诸多物种的基因组改造。如何提高CRISPR/Cas9技术的基因组编辑效率和最大限度降低脱靶风险一直是本领域的研究热点,而使用高效且特异的sgRNA(Small guide RNA)是基因组改造成功的关键性因素之一。目前,已有多款针对CRISPR/Cas9技术的sgRNA设计和/或脱靶效应评估软件,但不同的软件各有优缺点。本文重点对16款sgRNA 设计和脱靶效应评估在线和单机版软件的特点进行了阐述,通过制定38项评估指标对不同软件进行了比较分析,最后对11种用于检测基因组编辑效率和脱靶的实验方法,以及如何筛选高效且特异的sgRNA进行了归纳总结。     

Implication of the lymphocyte-specific nuclear body protein Sp140 in an innate response to human immunodeficiency virus type 1

The viral infectivity factor (Vif) of human immunodeficiency virus type 1 (HIV-1) neutralizes an unidentified antiviral pathway that occurs only in nonpermissive (NP) cells. Using a yeast two-hybrid screen of a human lymphocyte cDNA library, we identified several potential Vif partners. One, the nuclear body protein Sp140, was found specifically in all NP cells (n = 12 cell lines tested; P < or = 0.001), and HIV-1 infection induced its partial dispersal from nuclear bodies into cytosolic colocalization with Vif. Our results implicate Sp140 in a response to HIV-1 that may be related to or coordinated with the pathway that inactivates HIV-1 lacking vif.     

CHO细胞基因组NW-003614092.1内稳定表达位点的发现*

目的:获得中国仓鼠卵巢细胞(Chinese hamster ovary cells,CHO)内稳定表达位点信息,为构建重组蛋白CHO稳定表达株、缩短研发时间线提供信息明确的稳定位点。方法:对慢病毒随机整合Zsgreen1基因的具有潜在稳定表达位点的CHO-K1-1d2细胞株进行连续传代培养,验证表达稳定性;通过染色体步移分析慢病毒载体整合位点,并利用CRISPR/Cas9技术验证位点的可编辑性。结果:CHO-K1-1d2细胞在连续贴壁培养20代、悬浮培养50代过程中,能够100 %发绿色荧光,且荧光强度稳定,能够稳定表达Zsgreen1蛋白;染色体步移分析测序结果表明,CHO-K1-1d2细胞中慢病毒载体整合于CHO细胞基因组NW-003614092.1上第1 159 463与1 159 467碱基间;共转染sgRNA与Cas9质粒后,测序结果表明,该位点可被CRISPR/Cas9技术编辑。结论:CHO细胞基因组NW-003614092.1内存在一个信息明确的、能够被CRISPR/Cas9技术编辑的稳定表达位点。     

Replication potentials of HIV-1/HSIV in PBMCs from northern pigtailed macaque (Macaca leonina)

The northern pig-tailed macaque(Macaca leonina) has been identified as an independent species of Old World monkey, and we previously found that PBMCs from M. leonina were susceptible to human immunodeficiency virus type 1(HIV-1), which may be due to the absence of a TRIM5 protein restricting HIV-1 replication. Here we investigated the infection potentials of six laboratory adapted HIV-1 strains and three primary HIV-1 isolates in PBMCs from M. leonina. The results indicate that these strains are characterized by various but low replication levels, and among which, HIV-1NL4-3 shows the highest replication ability. Based on the abundant evidence of species-specific interactions between restriction factors APOBEC3 and HIV/SIV-derived Vif protein, we subsequently examined the replication potentials of vif-substituted HIV-1(HSIV) in M. leonina PBMCs. Notably, HSIV-vifmac and stHIV-1SV chimeras, two HIV-1NL4-3-derived viruses encoding the viral infectivity factor(Vif) protein from SIVmac239, replicated robustly in cells from M. leonina, which suggests that HSIV could effectively antagonize the antiviral activity of APOBEC3 proteins expressed in cells of M. leonina. Therefore, our data demonstrate that M. leonina has the potential to be developed into a promising animal model for human AIDS.     

Combining orthogonal CRISPR and CRISPRi systems for genome engineering and metabolic pathway modulation in Escherichia coli

CRISPR utilizing Cas9 from Streptococcus pyogenes (SpCas9) and CRISPR interference (CRISPRi) employing catalytically inactive SpCas9 (SpdCas9) have gained popularity for Escherichia coli engineering. To integrate the SpdCas9-based CRISPRi module using CRISPR while avoiding mutual interference between SpCas9/SpdCas9 and their cognate single-guide RNA (sgRNA), this study aimed at exploring an alternative Cas nuclease orthogonal to SpCas9. We compared several Cas9 variants from different microorganisms such as Staphylococcus aureus (SaCas9) and Streptococcus thermophilius CRISPR1 (St1Cas9) as well as Cas12a derived from Francisella novicida (FnCas12a). At the commonly used E. coli model genes  LacZ, we found that SaCas9 and St1Cas9 induced DNA cleavage more effectively than FnCas12a. Both St1Cas9 and SaCas9 were orthogonal to SpCas9 and the induced DNA cleavage promoted the integration of heterologous DNA of up to 10 kb, at which size St1Cas9 was superior to SaCas9 in recombination frequency/accuracy. We harnessed the St1Cas9 system to integrate SpdCas9 and sgRNA arrays for constitutive knockdown of three genes, knock-in pyc and knockout adhE, without compromising the CRISPRi knockdown efficiency. The combination of orthogonal CRISPR/CRISPRi for metabolic engineering enhanced succinate production while inhibiting byproduct formation and may pave a new avenue to E. coli engineering.