Novel structural and regulatory features of rhoptry secretory kinases in Toxoplasma gondii

Serine/threonine kinases secreted from rhoptry organelles constitute important virulence factors of Toxoplasma gondii. Rhoptry kinases are highly divergent and their structures and regulatory mechanism are hitherto unknown. Here, we report the X‐ray crystal structures of two related pseudokinases named ROP2 and ROP8, which differ primarily in their substrate‐binding site. ROP kinases contain a typical bilobate kinase fold and a novel N‐terminal extension that both stabilizes the N‐lobe and provides a unique means of regulation. Although ROP2 and ROP8 were catalytically inactive, they provided a template for homology modelling of the active kinase ROP18, a major virulence determinant of T. gondii. Autophosphorylation of key residues in the N‐terminal extension resulted in ROP18 activation, which in turn phosphorylated ROP2 and ROP8. Mutagenesis and mass spectrometry experiments revealed that ROP18 was maximally activated when this phosphorylated N‐terminus relieved autoinhibition resulting from extension of aliphatic side chains into the ATP‐binding pocket. This novel means of regulation governs ROP kinases implicated in parasite virulence.     

Identification of three novel Toxoplasma gondii rhoptry proteins

The rhoptries are key secretory organelles from apicomplexan parasites that contain proteins involved in invasion and modulation of the host cell. Some rhoptry proteins are restricted to the posterior bulb (ROPs) and others to the anterior neck (RONs). As many rhoptry proteins have been shown to be key players in Toxoplasma invasion and virulence, it is important to identify, understand and characterise the biological function of the components of the rhoptries. In this report, we identified putative novel rhoptry genes by identifying Toxoplasma genes with similar cyclical expression profiles as known rhoptry protein encoding genes. Using this approach we identified two new rhoptry bulb (ROP47 and ROP48) and one new rhoptry neck protein (RON12). ROP47 is secreted and traffics to the host cell nucleus, RON12 was not detected at the moving junction during invasion. Deletion of ROP47 or ROP48 in a type II strain did not show major influence in in vitro growth or virulence in mice.     

Eimeria tenella ROP kinase EtROP1 induces G0/G1 cell cycle arrest and inhibits host cell apoptosis

Coccidia are obligate intracellular protozoan parasites responsible for human and veterinary diseases. Eimeria tenella, the aetiologic agent of caecal coccidiosis, is a major pathogen of chickens. In Toxoplasma gondii, some kinases from the rhoptry compartment (ROP) are key virulence factors. ROP kinases hijack and modulate many cellular functions and pathways, allowing T. gondii survival and development. E. tenella's kinome comprises 28 putative members of the ROP kinase family; most of them are predicted, as pseudokinases and their functions have never been characterised. One of the predicted kinase, EtROP1, was identified in the rhoptry proteome of E. tenella sporozoites. Here, we demonstrated that EtROP1 is active, and the N‐terminal extension is necessary for its catalytic kinase activity. Ectopic expression of EtROP1 followed by co‐immunoprecipitation identified cellular p53 as EtROP1 partner. Further characterisation confirmed the interaction and the phosphorylation of p53 by EtROP1. E. tenella infection or overexpression of EtROP1 resulted both in inhibition of host cell apoptosis and G0/G1 cell cycle arrest. This work functionally described the first ROP kinase from E. tenella and its noncanonical structure. Our study provides the first mechanistic insight into host cell apoptosis inhibition by E. tenella. EtROP1 appears as a new candidate for coccidiosis control.     

Development of an Agrobacterium‐delivered CRISPR/Cas9 system for wheat genome editing

CRISPR/Cas9 has been widely used for genome editing in many organisms, including important crops like wheat. Despite the tractability in designing CRISPR/Cas9, efficacy in the application of this powerful genome editing tool also depends on DNA delivery methods. In wheat, the biolistics based transformation is the most used method for delivery of the CRISPR/Cas9 complex. Due to the high frequency of gene silencing associated with co‐transferred plasmid backbone and low edit rate in wheat, a large T₀ transgenic plant population are required for recovery of desired mutations, which poses a bottleneck for many genome editing projects. Here, we report an Agrobacterium‐delivered CRISPR/Cas9 system in wheat, which includes a wheat codon optimized Cas9 driven by a maize ubiquitin gene promoter and a guide RNA cassette driven by wheat U6 promoters in a single binary vector. Using this CRISPR/Cas9 system, we have developed 68 edit mutants for four grain‐regulatory genes, TaCKX2‐1, TaGLW7, TaGW2, and TaGW8, in T₀, T₁, and T₂ generation plants at an average edit rate of 10% without detecting off‐target mutations in the most Cas9‐active plants. Homozygous mutations can be recovered from a large population in a single generation. Different from most plant species, deletions over 10 bp are the dominant mutation types in wheat. Plants homozygous of 1160‐bp deletion in TaCKX2‐D1 significantly increased grain number per spikelet. In conclusion, our Agrobacterium‐delivered CRISPR/Cas9 system provides an alternative option for wheat genome editing, which requires a small number of transformation events because CRISPR/Cas9 remains active for novel mutations through generations.     

Analysis of CRISPR–Cas system and antimicrobial resistance in Staphylococcus coagulans isolates

CRISPR–Cas system contributes adaptive immunity to protect the bacterial and archaeal genome against invading mobile genetic elements. In this study, an attempt was made to characterize the CRISPR–Cas system in Staphylococcus coagulans, the second most prevalent coagulase positive staphylococci causing skin infections in dogs. Out of 45 S. coagulans isolates, 42/45 (93·33%) strains contained CRISPR–Cas system and 45 confirmed CRISPR system was identified in 42 S. coagulans isolates. The length of CRISPR loci ranged from 167 to 2477 bp, and the number of spacers in each CRISPR was varied from two spacers to as high as 37 numbers. Direct repeat (DR) sequences were between 30 and 37, but most (35/45) of the DRs contained 36 sequences. The predominant S. coagulans strains 29/45 did not possess any antimicrobial resistant genes (ARG); 26/29 strains contained Type IIC CRISPR–Cas system. Three isolates from Antarctica seals neither contain CRISPR–Cas system nor ARG. Only 15/45 S. coagulans strains (33·33%) harboured at least one ARG and 13/15 of them were having mecA gene. All the methicillin susceptible S. coagulans isolates contained Type IIC CRISPR–Cas system. In contrast, many (10/13) S. coagulans isolates which were methicillin resistant had Type IIIA CRISPR–Cas system, and this Type IIIA CRISPR–Cas system was present within the SCCmec mobile genetic element. Hence, this study suggests that Type II CRISPR–Cas in S. coagulans isolates might have played a possible role in preventing acquisition of plasmid/phage invasion and Type IIIA CRISPR–Cas system may have an insignificant role in the prevention of horizontal gene transfer of antimicrobial resistance genes in S. coagulans species.     

Target-specific mutations efficiency at multiple loci of CRISPR/Cas9 system using one sgRNA in soybean

Soybean has a palaeopolyploid genome with nearly 75% of the genes present in multiple copies. Although the CRISPR/Cas9 system has been employed in soybean to generate site-directed mutagenesis, a systematical assessment of mutation efficiency of the CRISPR/Cas9 system for the multiple-copy genes is still urgently needed. Here, we successfully optimize one sgRNA CRISPR/Cas9 system in soybean by testing the efficiency, pattern, specificity of the mutations at multiple loci of GmFAD2 and GmALS. The results showed that simultaneous site-directed mutagenesis of two homoeologous loci by one sgRNA, the mutation frequency in the T0 generation were 64.71% for GmPDS, 60.0% for GmFAD2 and 42.86% for GmALS, respectively. The chimeric and heterozygous mutations were dominant types. Moreover, association of phenotypes with mutation pattern at target loci of GmPDS11 and GmPDS18 could help us further demonstrate that the CRISPR/Cas9 system can efficiently generate target specific mutations at multiple loci using one sgRNA in soybean, albeit with a relatively low transformation efficiency.

     

Rhoptries: an arsenal of secreted virulence factors

Apicomplexan parasites use actin-based motility coupled with regulated protein secretion from apical organelles to actively invade host cells. Crucial in this process are rhoptries, club-shaped secretory organelles that discharge their contents during parasite invasion into host cells. A proteomic analysis of the rhoptries in Toxoplasma gondii demonstrated that this organelle contains a number of novel rhoptry proteins (ROPs) including serine-threonine kinases and protein phosphatases. A subset of rhoptry proteins called RONs have been shown to target the moving junction, which plays a key role in invasion and parasitophorous vacuole formation. Other ROP proteins have various destinations in the host cell including the host cell nucleus and the parasitophorous vacuole, probably reflecting their distinct targets and roles. Forward genetic analysis recently revealed that secretory ROP kinases dramatically influence host gene expression and are the major parasite virulence factors. Thus, ROP proteins are functionally analogous (though not homologous) to effectors released by type III and IV secretion systems, which are factors that play an important role in bacterial virulence. Deciphering the role of ROP effectors may allow specific disruption of these factors, thus offering new options for preventing disease.     

CRISPR/Cas系统与结核病防控新措施相关性研究

结核分枝杆菌(Mycobacterium tuberculosis,MTB,以下简称结核杆菌)感染引起的结核病仍然是严重影响人类健康全球性重大传染病。全球约1/4人口是结核杆菌的潜伏感染者。2019年,世界卫生组织(Worldhealthorganization,WHO)报道全球约150万人死于结核病。深入研究结核杆菌生物学有望为结核病防控提供新工具。成簇规律性间隔短回文重复(Clusteredregularlyinterspacedshort palindromic repeats,CRISPR)/Cas是细菌免疫系统的重要成分,在结核杆菌等分枝杆菌中也广泛存在,同时,也是分枝杆菌基因编辑的重要工具。本文结合课题组研究工作,综述了结核杆菌III-A型CRISPR/Cas系统各组分的生物学功能以及与致病的相关性,CRISPR/Cas编辑工具在诊断治疗耐药结核杆菌和结核病防控新措施中的进展。     

The Polymorphic Pseudokinase ROP5 Controls Virulence in Toxoplasma gondii by Regulating the Active Kinase ROP18

Secretory polymorphic serine/threonine kinases control pathogenesis of Toxoplasma gondii in the mouse. Genetic studies show that the pseudokinase ROP5 is essential for acute virulence, but do not reveal its mechanism of action. Here we demonstrate that ROP5 controls virulence by blocking IFN-γ mediated clearance in activated macrophages. ROP5 was required for the catalytic activity of the active S/T kinase ROP18, which phosphorylates host immunity related GTPases (IRGs) and protects the parasite from clearance. ROP5 directly regulated activity of ROP18 in vitro, and both proteins were necessary to avoid IRG recruitment and clearance in macrophages. Clearance of both the Δrop5 and Δrop18 mutants was reversed in macrophages lacking Irgm3, which is required for IRG function, and the virulence defect was fully restored in Irgm3^−/− mice. Our findings establish that the pseudokinase ROP5 controls the activity of ROP18, thereby blocking IRG mediated clearance in macrophages. Additionally, ROP5 has other functions that are also Irgm3 and IFN-γ dependent, indicting it plays a general role in governing virulence factors that block immunity.     

Production of a mutant of large-scale loach Paramisgurnus dabryanus with skin pigmentation loss by genome editing with CRISPR/Cas9 system

CRISPR/Cas9 system has been developed as a highly efficient genome editing technology to specifically induce mutations in a few aquaculture species. In this study, we described induction of targeted gene (namely tyrosinase, tyr) mutations in large-scale loach Paramisgurnus dabryanus, an important aquaculture fish species and a potential model organism for studies of intestinal air-breathing function, using the CRISPR/Cas9 system. Tyr gene in large-scale loach was firstly cloned and then its expressions were investigated. Two guide RNAs (gRNAs) were designed and separately transformed with Cas9 in the loach. 89.4% and 96.1% of injected loach juveniles respectively displayed a graded loss of pigmentation for the two gRNAs, in other words, for target 1 and target 2. We classified the injected loach juveniles into five groups according to their skin color phenotypes, including four albino groups and one wild-type-like group. And one of them was clear albino group, which was of high ornamental and commercial value. More than 50 clones for each albino transformant with a visible phenotype in each target were randomly selected and sequenced. Results obtained here showed that along with the increase of pigmentation, wild-type alleles appeared in the injected loach juveniles more often and insertion/deletion alleles less frequently. This study demonstrated that CRISPR/Cas9 system could be practically performed to modify large-scale loach tyr to produce an albino mutant of high ornamental and commercial value, and for the first time showed successful use of the CRISPR/Cas9 system for genome editing in a Cobitidae species.

     

CRISPR/Cas9‐mediated gene knockout in the ascidian Ciona intestinalis

Knockout of genes with CRISPR/Cas9 is a newly emerged approach to investigate functions of genes in various organisms. We demonstrate that CRISPR/Cas9 can mutate endogenous genes of the ascidian Ciona intestinalis, a splendid model for elucidating molecular mechanisms for constructing the chordate body plan. Short guide RNA (sgRNA) and Cas9 mRNA, when they are expressed in Ciona embryos by means of microinjection or electroporation of their expression vectors, introduced mutations in the target genes. The specificity of target choice by sgRNA is relatively high compared to the reports from some other organisms, and a single nucleotide mutation at the sgRNA dramatically reduced mutation efficiency at the on‐target site. CRISPR/Cas9‐mediated mutagenesis will be a powerful method to study gene functions in Ciona along with another genome editing approach using TALE nucleases.     

一种新型的CRISPR/Cas9-hLacI双链DNA供体适配基因编辑系统

在CRISPR/Cas9系统介导的基因编辑中,借助于双链DNA （double-stranded DNA,dsDNA）供体模板的重组效应能够实现对目标基因组靶位点的精确编辑和基因敲入,然而高等真核生物细胞中同源重组的低效性限制了该基因编辑策略的发展和应用。为提高CRISPR/Cas9系统介导dsDNA供体模板的同源重组效率,本研究利用大肠杆菌（Escherichia coli）乳糖操纵子阻遏蛋白LacI与操纵序列LacO特异性结合的特点,通过重组DNA技术将密码子人源化优化的阻遏蛋白基因LacI分别与脓链球菌（Streptococcus pyogenes）源的SpCas9和路邓葡萄球菌（Staphylococcus lugdunensis）源的SlugCas9-HF融合表达,通过PCR将操纵序列LacO与dsDNA供体嵌合,构建了新型的CRISPR/Cas9-hLacI供体适配系统（donor adapting system,DAS）。首先在报告载体水平上对Cas9核酸酶活性、DAS介导的同源引导修复（homology-directed repair,HDR）效率进行了验证和优化,其次在基因组水平对其介导的基因精确编辑进行了检测,并最终利用CRISPR/SlugCas9-hLacI DAS在HEK293T细胞中实现了VEGFA位点的精确编辑,效率高达30.5%,显著高于野生型。综上所述,本研究开发了新型的CRISPR/Cas9-hLacI供体适配基因编辑系统,丰富了CRISPR/Cas9基因编辑技术种类,为以后的基因编辑及分子设计育种研究提供了新的工具。     

A ROP2‐RIC1 pathway fine‐tunes microtubule reorganization for salt tolerance in Arabidopsis

The reorganization of microtubules induced by salt stress is required for Arabidopsis survival under high salinity conditions. RIC1 is an effector of Rho‐related GTPase from plants (ROPs) and a known microtubule‐associated protein. In this study, we demonstrated that RIC1 expression decreased with long‐term NaCl treatment, and ric1‐1 seedlings exhibited a higher survival rate under salt stress. We found that RIC1 reduced the frequency of microtubule transition from shortening to growing status and knockout of RIC1 improved the reassembly of depolymerized microtubules caused by either oryzalin treatment or salt stress. Further investigation showed that constitutively active ROP2 promoted the reassembly of microtubules and the survival of seedlings under salt stress. A rop2‐1 ric1‐1 double mutant rescued the salt‐sensitive phenotype of rop2‐1, indicating that ROP2 functions in salt tolerance through RIC1. Although ROP2 did not regulate RIC1 expression upon salt stress, a quick but mild increase of ROP2 activity was induced, led to reduction of RIC1 on microtubules. Collectively, our study reveals an ROP2‐RIC1 pathway that fine‐tunes microtubule dynamics in response to salt stress in Arabidopsis. This finding not only reveals a new regulatory mechanism for microtubule reorganization under salt stress but also the importance of ROP signalling for salinity tolerance.     

空肠弯曲菌CRISPR/Cas系统的研究进展

多数细菌都存在发挥免疫防御机制作用的CRISPR/Cas系统,在不同种属间呈现多态性。空肠弯曲菌是全球范围内重要的食源性病原菌,所致疾病也是典型的自限性疾病,其复杂的致病机制并未得到明确的解析,而空肠弯曲菌CRISPR/Cas系统的结构呈现多态性,研究两者关系仍存在诸多限制。本文从CRISPR/Cas系统在空肠弯曲菌中的结构、机制及技术应用等方面的研究进展进行综述,为探索空肠弯曲菌致病机制提供新思路。     

嗜热厌氧杆菌热稳定CRISPR/Cas9基因组编辑技术及在细胞工厂构建中的应用研究进展

嗜热厌氧杆菌属(Thermoanaerobacter)来源的菌株作为一个高温发酵的细胞工厂,具有生产生物燃料和化学品的潜力,已引起了研究者的广泛兴趣.随着嗜热厌氧杆菌属来源的多个菌株全基因组测序的完成以及相关的生理生化实验的开展,建立一个简便、快捷的基因操作技术将有助于进一步深入理解和认识嗜热厌氧杆菌有关的代谢途径.最...     

An Archaeal Immune System Can Detect Multiple Protospacer Adjacent Motifs (PAMs) to Target Invader DNA

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system provides adaptive and heritable immunity against foreign genetic elements in most archaea and many bacteria. Although this system is widespread and diverse with many subtypes, only a few species have been investigated to elucidate the precise mechanisms for the defense of viruses or plasmids. Approximately 90% of all sequenced archaea encode CRISPR/Cas systems, but their molecular details have so far only been examined in three archaeal species: Sulfolobus solfataricus, Sulfolobus islandicus, and Pyrococcus furiosus. Here, we analyzed the CRISPR/Cas system of Haloferax volcanii using a plasmid-based invader assay. Haloferax encodes a type I-B CRISPR/Cas system with eight Cas proteins and three CRISPR loci for which the identity of protospacer adjacent motifs (PAMs) was unknown until now. We identified six different PAM sequences that are required upstream of the protospacer to permit target DNA recognition. This is only the second archaeon for which PAM sequences have been determined, and the first CRISPR group with such a high number of PAM sequences. Cells could survive the plasmid challenge if their CRISPR/Cas system was altered or defective, e.g. by deletion of the cas gene cassette. Experimental PAM data were supplemented with bioinformatics data on Haloferax and Haloquadratum.     

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.