Establishing RNA virus resistance in plants by harnessing CRISPR immune system

Recently, CRISPR‐Cas (clustered, regularly interspaced short palindromic repeats–CRISPR‐associated proteins) system has been used to produce plants resistant to DNA virus infections. However, there is no RNA virus control method in plants that uses CRISPR‐Cas system to target the viral genome directly. Here, we reprogrammed the CRISPR‐Cas9 system from Francisella novicida to confer molecular immunity against RNA viruses in Nicotiana benthamiana and Arabidopsis plants. Plants expressing FnCas9 and sgRNA specific for the cucumber mosaic virus (CMV) or tobacco mosaic virus (TMV) exhibited significantly attenuated virus infection symptoms and reduced viral RNA accumulation. Furthermore, in the transgenic virus‐targeting plants, the resistance was inheritable and the progenies showed significantly less virus accumulation. These data reveal that the CRISPR/Cas9 system can be used to produce plant that stable resistant to RNA viruses, thereby broadening the use of such technology for virus control in agricultural field.     

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基因编辑技术在病毒感染疾病治疗中的应用

CRISPR-Cas9基因编辑技术是基于细菌或古细菌CRISPR介导的获得性免疫系统衍生而来,由一段RNA通过碱基互补配对识别DNA,指导Cas9核酸酶切割识别的双链DNA,诱发同源重组或非同源末端链接,进而实现在目的DNA上进行编辑。病毒通过特异的受体侵染细胞,其基因组在细胞内发生复制、转录、翻译等过程完成其生活周期,某些DNA病毒或逆转录病毒基因组会整合到宿主基因组中。基因治疗是病毒感染疾病治疗的新趋势。因此,基因编辑技术在持续感染的病毒或潜伏感染病毒疾病治疗中具有重大的潜在意义。文章主要从CRISPR-Cas9作用机制以及在病毒感染疾病治疗中的应用等方面进行了综述。     

NmeCas9 is an intrinsically high-fidelity genome-editing platform

Background

The development of CRISPR genome editing has transformed biomedical research. Most applications reported thus far rely upon the Cas9 protein from Streptococcus pyogenes SF370 (SpyCas9). With many RNA guides, wildtype SpyCas9 can induce significant levels of unintended mutations at near-cognate sites, necessitating substantial efforts toward the development of strategies to minimize off-target activity. Although the genome-editing potential of thousands of other Cas9 orthologs remains largely untapped, it is not known how many will require similarly extensive engineering to achieve single-site accuracy within large genomes. In addition to its off-targeting propensity, SpyCas9 is encoded by a relatively large open reading frame, limiting its utility in applications that require size-restricted delivery strategies such as adeno-associated virus vectors. In contrast, some genome-editing-validated Cas9 orthologs are considerably smaller and therefore better suited for viral delivery.

Results

Here we show that wildtype NmeCas9, when programmed with guide sequences of the natural length of 24 nucleotides, exhibits a nearly complete absence of unintended editing in human cells, even when targeting sites that are prone to off-target activity with wildtype SpyCas9. We also validate at least six variant protospacer adjacent motifs (PAMs), in addition to the preferred consensus PAM (5′-N₄GATT-3′), for NmeCas9 genome editing in human cells.

Conclusions

Our results show that NmeCas9 is a naturally high-fidelity genome-editing enzyme and suggest that additional Cas9 orthologs may prove to exhibit similarly high accuracy, even without extensive engineering.

     

Improvement of baculovirus as protein expression vector and as biopesticide by CRISPR/Cas9 editing

The clustered regularly interspaced short palindromic repeats (CRISPR) system–associated Cas9 endonuclease is a molecular tool that enables specific sequence editing with high efficiency. In this study, we have explored the use of CRISPR/Cas9 system for the engineering of baculovirus. We have shown that the delivering of Cas9-single guide RNA ribonucleoprotein (RNP) complex with or without DNA repair template into Sf21 insect cells through lipofection might be efficient to produce knockouts as well as knock-ins into the baculovirus. To evaluate potential application of our CRISPR/Cas9 method to improve baculovirus as protein expression vector and as biopesticide, we attempted to knockout several genes from a recombinant AcMNPV form used in the baculovirus expression system as well as in a natural occurring viral isolate from the same virus. We have additionally confirmed the adaptation of this methodology for the generation of viral knock-ins in specific regions of the viral genome. Analysis of the generated mutants revealed that the editing efficiency and the type of changes was variable but relatively high. Depending on the targeted gene, the editing rate ranged from 10% to 40%. This study established the first report revealing the potential of CRISPR/Cas9 for genome editing in baculovirus, contributing to the engineering of baculovirus as a protein expression vector as well as a biological control agent.     

Delivery of CRISPR/Cas9 for therapeutic genome editing

The clustered, regularly‐interspaced, short palindromic repeat (CRISPR)‐associated nuclease 9 (CRISPR/Cas9) is emerging as a promising genome‐editing tool for treating diseases in a precise way, and has been applied to a wide range of research in the areas of biology, genetics, and medicine. Delivery of therapeutic genome‐editing agents provides a promising platform for the treatment of genetic disorders. Although viral vectors are widely used to deliver CRISPR/Cas9 elements with high efficiency, they suffer from several drawbacks, such as mutagenesis, immunogenicity, and off‐target effects. Recently, non‐viral vectors have emerged as another class of delivery carriers in terms of their safety, simplicity, and flexibility. In this review, we discuss the modes of CRISPR/Cas9 delivery, the barriers to the delivery process and the application of CRISPR/Cas9 system for the treatment of genetic disorders. We also highlight several representative types of non‐viral vectors, including polymers, liposomes, cell‐penetrating peptides, and other synthetic vectors, for the therapeutic delivery of CRISPR/Cas9 system. The applications of CRISPR/Cas9 in treating genetic disorders mediated by the non‐viral vectors are also discussed.     

A simplified and efficient germline-specific CRISPR/Cas9 system for Drosophila genomic engineering

The type II CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeats/CRISPR-associated) has recently emerged as an efficient and simple tool for site-specific engineering of eukaryotic genomes. To improve its applications in Drosophila genome engineering, we simplified the standard two-component CRISPR/Cas9 system by generating a stable transgenic fly line expressing the Cas9 endonuclease in the germline (Vasa-Cas9 line). By injecting vectors expressing engineered target-specific guide RNAs into Vasa-Cas9 fly embryos, mutations were generated from site-specific DNA cleavages and efficiently transmitted into progenies. Because Cas9 endonuclease is the universal component of the type II CRISPR/Cas9 system, site-specific genomic engineering based on this improved platform can be achieved with lower complexity and toxicity, greater consistency, and excellent versatility.     

A simplified and efficient germline-specific CRISPR/Cas9 system for Drosophila genomic engineering

The type II CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeats/CRISPR-associated) has recently emerged as an efficient and simple tool for site-specific engineering of eukaryotic genomes. To improve its applications in Drosophila genome engineering, we simplified the standard two-component CRISPR/Cas9 system by generating a stable transgenic fly line expressing the Cas9 endonuclease in the germline (Vasa-Cas9 line). By injecting vectors expressing engineered target-specific guide RNAs into Vasa-Cas9 fly embryos, mutations were generated from site-specific DNA cleavages and efficiently transmitted into progenies. Because Cas9 endonuclease is the universal component of the type II CRISPR/Cas9 system, site-specific genomic engineering based on this improved platform can be achieved with lower complexity and toxicity, greater consistency, and excellent versatility.     

Anti‐HIV‐1 potency of the CRISPR/Cas9 system insufficient to fully inhibit viral replication

The range of genome‐editing tools has recently been expanded. In particular, an RNA‐guided genome‐editing tool, the clustered regularly interspaced short palindromic repeat (CRISPR)‐associated 9 (Cas9) system, has many applications for human diseases. In this study, guide RNA (gRNA) to target gag, pol and a long terminal repeat of HIV‐1 was designed and used to generate gRNA‐expressing lentiviral vectors. An HIV‐1‐specific gRNA and Cas9 were stably dually transduced into a highly HIV‐1‐susceptible human T‐cell line and the inhibitory ability of the anti‐HIV‐1 CRISPR/Cas9 lentiviral vector assessed. Although clear inhibition of the early phase of HIV‐1 infection was observed, as evaluated by a VSV‐G‐pseudotyped HIV‐1 reporter system, the anti‐HIV‐1 potency in multiple rounds of wild type (WT) viral replication was insufficient, either because of generation of resistant viruses or overcoming of the activity of the WT virus. Thus, there are potential difficulties that must be addressed when considering anti‐HIV‐1 treatment with the CRISPR/Cas9 system alone.     

Generation of virus‐resistant potato plants by RNA genome targeting

CRISPR/Cas systems provide bacteria and archaea with molecular immunity against invading phages and foreign plasmids. The class 2 type VI CRISPR/Cas effector Cas13a is an RNA‐targeting CRISPR effector that provides protection against RNA phages. Here we report the repurposing of CRISPR/Cas13a to protect potato plants from a eukaryotic virus, Potato virus Y (PVY). Transgenic potato lines expressing Cas13a/sgRNA (small guide RNA) constructs showed suppressed PVY accumulation and disease symptoms. The levels of viral resistance correlated with the expression levels of the Cas13a/sgRNA construct in the plants. Our data further demonstrate that appropriately designed sgRNAs can specifically interfere with multiple PVY strains, while having no effect on unrelated viruses such as PVA or Potato virus S. Our findings provide a novel and highly efficient strategy for engineering crops with resistances to viral diseases.     

Recent advances in CRISPR/Cas9 mediated genome editing in <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Genome editing using engineered nucleases has rapidly transformed from a niche technology to a mainstream method used in various host cells. Its widespread adoption has been largely developed by the emergence of the clustered regularly interspaced short palindromic repeats (CRISPR) system, which uses an easily customizable specificity RNA-guided DNA endonuclease, such as Cas9. Recently, CRISPR/Cas9 mediated genome engineering has been widely applied to model organisms, including Bacillus subtilis, enabling facile, rapid high-fidelity modification of endogenous native genes. Here, we reviewed the recent progress in B. subtilis gene editing using CRISPR/Cas9 based tools, and highlighted state-of-the-art strategies for design of CRISPR/Cas9 system. Finally, future perspectives on the use of CRISPR/Cas9 genome engineering for sequence-specific genome editing in B. subtilis are provided.     

Attenuated and Replication-Competent Vaccinia Virus Strains M65 and M101 with Distinct Biology and Immunogenicity as Potential Vaccine Candidates against Pathogens

Replication-competent poxvirus vectors with an attenuation phenotype and with a high immunogenic capacity of the foreign expressed antigen are being pursued as novel vaccine vectors against different pathogens. In this investigation, we have examined the replication and immunogenic characteristics of two vaccinia virus (VACV) mutants, M65 and M101. These mutants were generated after 65 and 101 serial passages of persistently infected Friend erythroleukemia (FEL) cells. In cultured cells of different origins, the mutants are replication competent and have growth kinetics similar to or slightly reduced in comparison with those of the parental Western Reserve (WR) virus strain. In normal and immune-suppressed infected mice, the mutants showed different levels of attenuation and pathogenicity in comparison with WR and modified vaccinia Ankara (MVA) strains. Wide genome analysis after deep sequencing revealed selected genomic deletions and mutations in a number of viral open reading frames (ORFs). Mice immunized in a DNA prime/mutant boost regimen with viral vectors expressing the LACK (Leishmania homologue for receptors of activated C kinase) antigen of Leishmania infantum showed protection or a delay in the onset of cutaneous leishmaniasis. Protection was similar to that triggered by MVA-LACK. In immunized mice, both polyfunctional CD4⁺ and CD8⁺ T cells with an effector memory phenotype were activated by the two mutants, but the DNA-LACK/M65-LACK protocol preferentially induced CD4⁺ whereas DNA-LACK/M101-LACK preferentially induced CD8⁺ T cell responses. Altogether, our findings showed the adaptive changes of the WR genome during long-term virus-host cell interaction and how the replication competency of M65 and M101 mutants confers distinct biological properties and immunogenicity in mice compared to those of the MVA strain. These mutants could have applicability for understanding VACV biology and as potential vaccine vectors against pathogens and tumors.     

CRISPR-Cas9应用于病毒性传染病防控的研究进展

病毒性传染病是威胁人类健康的重要因素,迫切需要新的治疗方法来降低由急性病毒感染如鼻病毒和登革热病毒以及慢性病毒感染如人类免疫缺陷病毒1和乙型肝炎病毒引起的发病率和死亡率.随着分子生物学技术的发展,靶向序列特异性的基因编辑技术成为传染病治疗的有力工具.其中规律成簇间隔短回文重复序列(clustered regularly interspaced short palindromic repeats,CRISPR)-CRISPR相关蛋白9(CRISPR associated protein 9,Cas9)凭借其高效、简便、高特异性等特点被广泛应用于细胞系和动物模型中的传染病治疗,从而成为有前景的新型传染病治疗模式.目前,利用病毒和非病毒载体将Cas9以DNA、m RNA或蛋白质的形式递送到细胞中的可行性研究和评估CRISPR-Cas9体内适用性的临床试验已经在进行中.本篇综述中,我们将对CRISPR-Cas9的原理,其应用于传染病治疗的最新研究进展以及该技术面临的挑战和可预测性的解决方法等加以概述,并进一步展望其未来的发展方向.     

Establishing a dual knock-out cell line by lentivirus based combined CRISPR/Cas9 and Loxp/Cre system

The clustered regulatory interspersed short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system has been widely used for gene knock-out. Lentiviral vectors have been commonly used as a delivery method for this system, however, prolonged Cas9/sgRNA expression due to lentiviral integration can lead to accumulating off-target mutations. To solve this issue in engineering a gene knock-out cell line, this study established a novel system, which was composed of two lentiviral vectors. One lentiviral vector carried simultaneously sgRNAs and CRISPR/Cas9 expression cassettes targeting single or multiple gene(s); the other lentiviral vector carried Cre that could remove excess sgRNAs and Cas9 expression cassettes in the genome after gene targeting was achieved. To prove the principle, two candidate genes, extracellular matrix protein 1 (ECM1) and progranulin (PGRN), both highly expressed in MDA-MB-231 cells, were selected for testing the novel system. A dual knock-out of ECM1 and PGRN was successfully achieved in MDA-MB-231 cell line, with the sgRNAs and Cas9 expression cassettes being removed by Cre. This system should have great potential in applications for multiple genes knock-out in vitro.     

酿酒酵母中基于CRISPR/Cas9工具的基因编辑与代谢调控研究进展

CRISPR/Cas9系统已广泛用于各种生物体的基因编辑和代谢工程。本文综述了CRISPR/Cas9在酿酒酵母中的基本原理和实际应用。首先总结了CRISPR/Cas9技术的发展历史、酿酒酵母基因组中基因缺失和多DNA片段插入的成功案例。这一先进的系统减少了劳动力,增强了对分子遗传学的理解,加速了微生物工程的发展。其次总结了基于CRISPR/Cas9的系统在生产高附加值化学品和提高酿酒酵母耐应激性方面的研究进展。该综述对酿酒酵母的遗传和合成生物学研究具有重要的参考价值。     

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。     

Activity and specificity of TRV-mediated gene editing in plants

Plant trait engineering requires efficient targeted genome-editing technologies. Clustered regularly interspaced palindromic repeats (CRISPRs)/ CRISPR associated (Cas) type II system is used for targeted genome-editing applications across eukaryotic species including plants. Delivery of genome engineering reagents and recovery of mutants remain challenging tasks for in planta applications. Recently, we reported the development of Tobacco rattle virus (TRV)-mediated genome editing in Nicotiana benthamiana. TRV infects the growing points and possesses small genome size; which facilitate cloning, multiplexing, and agroinfections. Here, we report on the persistent activity and specificity of the TRV-mediated CRISPR/Cas9 system for targeted modification of the Nicotiana benthamiana genome. Our data reveal the persistence of the TRV- mediated Cas9 activity for up to 30 d post-agroinefection. Further, our data indicate that TRV-mediated genome editing exhibited no off-target activities at potential off-targets indicating the precision of the system for plant genome engineering. Taken together, our data establish the feasibility and exciting possibilities of using virus-mediated CRISPR/Cas9 for targeted engineering of plant genomes.