Investigation of the contribution of RyR target-site mutations in diamide resistance by CRISPR/Cas9 genome modification in Drosophila

Diamide insecticides are used widely against lepidopteran pests, acting as potent activators of insect Ryanodine Receptors (RyRs) and thus inducing muscle contraction and eventually death. However, resistant phenotypes have recently evolved in the field, associated with the emergence of target site resistance mutations (G4946E/V and I4790M). We investigated the frequency of the mutations found in a resistant population of Tuta absoluta from Greece (G4946V ~79% and I4790M ~21%) and the associated diamide resistance profile: there are very high levels of resistance against chlorantraniliprole (9329-fold) and flubendiamide (4969-fold), but moderate levels against cyantraniliprole (191-fold). To further investigate functionally the contribution of each mutation in the resistant phenotype, we used CRISPR/Cas9 to generate genome modified Drosophila carrying alternative allele combinations, and performed toxicity bioassays against all three diamides. Genome modified flies bearing the G4946V mutation exhibited high resistance ratios to flubendiamide (91.3-fold) and chlorantraniliprole (194.7-fold) when compared to cyantraniliprole (5.4-fold). Flies naturally wildtype for the I4790M mutation were moderately resistant to flubendiamide (15.3-fold) but significantly less resistant to chlorantraniliprole (7.5-fold), and cyantraniliprole (2.3-fold). These findings provide in vivo functional genetic confirmation for the role and relative contribution of RyR mutations in diamide resistance and suggest that the mutations confer subtle differences on the relative binding affinities of the three diamides at an overlapping binding site on the RyR protein.     

Functional characterization of SlitPBP3 in Spodoptera litura by CRISPR/Cas9 mediated genome editing

Functional gene analysis by using genome editing techniques is limited only in few model insects. Here, we reported an efficient and heritable gene mutagenesis analysis in an important lepidopteran pest, Spodoptera litura, using the CRISPR/Cas9 system. By using this system, we successfully obtained the homozygous S. litura strain by targeting the pheromone binding protein 3 gene (SlitPBP3), which allowed us to elucidate the role of this gene in the olfaction of the female sex pheromones. By co-injection of Cas9 mRNA and sgRNA into S. litura eggs, highly efficient chimera mutation in SlitPBP3 loci was detected both in injected eggs (39.1%) and in the resulting individual moths (87.5%). We used the mutant moths as parents to obtain the G1 offspring and the homozygous mutant strain in G2. The function of SlitPBP3 was explored by Electroantennogram (EAG) recordings with a homozygous mutant strain. The result showed that the EAG responses were significantly decreased in mutant males than in control males when treated with the major sex pheromone component (Z9,E11-14:Ac) and a minor component (Z9-14:Ac) at higher dosages. The results demonstrate that s SlitPBP3 gene plays a minor role in the perception of the female sex pheromones. Furthermore, our study provides a useful methodology with the CRISPR/Cas9 system for gene in vivo functional study, particular for lepidopteran species in which the RNAi approach is not efficient.     

CRISPR/Cas9‐mediated ebony knockout results in puparium melanism in Spodoptera litura

Insect body pigmentation and coloration are critical to adaption to the environment. To explore the mechanisms that drive pigmentation, we used the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) genome editing system to target the ebony gene in the non-model insect Spodoptera litura. Ebony is crucial to melanin synthesis in insects. By directly injecting Cas9 messenger RNA and ebony-specific guide RNAs into S. litura embryos, we successfully induced a typical ebony-deficient phenotype of deep coloration of the puparium and induction of melanin formation during the pupal stage. Polymerase chain reaction-based genotype analysis demonstrated that various mutations had occurred at the sites targeted in ebony. Our study clearly demonstrates the function of ebony in the puparium coloration and also provides a potentially useful marker gene for functional studies in S. litura as well as other lepidopteran pests.     

Mosaicism in CRISPR/Cas9-mediated genome editing

The CRISPR/Cas9 system is a rapid, simple, and often extremely efficient gene editing method. This method has been used in a variety of organisms and cell types over the past several years. However, using this technology for generating gene-edited animals involves a number of obstacles. One such obstacle is mosaicism, which is common in founder animals. This is especially the case when the CRISPR/Cas9 system is used in embryos. Here we review the pros and cons of mosaic mutations of gene-edited animals caused by using the CRISPR/Cas9 system in embryos. Furthermore, we will discuss the mechanisms underlying mosaic mutations resulting from the CRISPR/Cas9 system, as well as the possible strategies for reducing mosaicism. By developing ways to overcome mosaic mutations when using CRISPR/Cas9, genotyping for germline gene disruptions should become more reliable. This achievement will pave the way for using the CRISPR technology in the research and clinical applications where mosaicism is an issue.     

CRISPR/Cas9介导的高产谷胱甘肽原养型酵母工程菌的构建

谷胱甘肽(Glutathione,GSH)是具有多种生理功能的非蛋白质类巯基化合物,已广泛应用于药品、食品等行业,且市场需求量逐年增加。遗传工程育种是提高细胞内GSH含量的重要策略,但在遗传操作过程中使用到的营养缺陷型遗传标记可能会影响菌株的正常生长,且不利于高密度发酵的进行。为回复工程菌株的营养缺陷型,利用g RNA转录表达框和靶基因同源DNA片段直接共转化酵母细胞,由细胞内表达的Ⅱ型CRISPR/Cas9(Clustered regularly interspaced short palindromic repeats(CRISPR)-Cas9)介导的基因组编辑技术将营养缺陷型GSH工程菌株W303-1b/FGP回复为原养型菌株。结果显示,与营养缺陷型菌株相比,原养型菌株生长周期缩短,且可以利用简单的合成培养基进行培养,方便菌株的大规模培养。     

金针菇G蛋白偶联受体基因的CRISPR/Cas9基因组编辑载体构建及转化研究

通过氨基酸同源比对（Blast P）以及金针菇冷诱导前后菌丝阶段和原基阶段的转录组数据分析,获得了金针菇中的两个假定G蛋白偶联受体基因Fvgpcr1、Fvgpcr2。对获得的金针菇假定G蛋白偶联受体基因Fvgpcr1、Fvgpcr2构建了基因组编辑（CRISPR/Cas9）的pCAMBIA0390-hph-Fvcas9-Fvgpcr1- sgRNA1/sgRNA2、pCAMBIA0390-hph-Fvcas9-Fvgpcr2-sgRNA1/sgRNA2等4个表达载体。通过农杆菌介导（ATMT）将表达载体pCAMBIA0390-hph-Fvcas9-Fvgpcr-sgRNA转化金针菇菌丝体,采用潮霉素和头孢毒素低浓度初筛和高浓度复筛,经两段筛选获得金针菇拟转化子。经对拟转化子进行PCR鉴定、RT-qPCR检测和Western杂交验证,结果显示表达载体pCAMBIA0390-hph-Fvcas9-Fvgpcr-sgRNA成功整合进金针菇基因组中,FvCas9蛋白正常表达,但未得到Fvgpcr基因敲除突变体。本研究利用农杆菌介导转化法在金针菇中构建了CRISPR/Cas9敲除体系,对后续目标基因的敲除有着重要意义。     

CRISPR/Cas9基因组编辑技术在农业动物中的应用

CRISPR(Clustered regularly interspaced short palindromic repeats)/Cas(CRISPR associated proteins)是在细菌和古细菌中发现的一种用来抵御病毒或质粒入侵的获得性免疫系统.目前已发现的CRISPR/Cas系统包括Ⅰ,Ⅱ和Ⅲ型,其中Ⅱ型系统的组成较简单,由其改造成的CRISPR/Cas9技术已成为一种高效的基因组编辑工具.自2013年CRISPR/Cas9技术成功用于哺乳动物基因组定点编辑以来,应用该技术进行基因组编辑的报道呈现出爆发式的增长.农业动物不仅是重要的经济动物,也是人类疾病和生物医药研究的重要模式动物.本文综述了CRISPR/Cas9技术在农业动物中的研究和应用进展,简述了该技术的脱靶效应及减少脱靶的主要方法,并展望了该技术的应用前景.     

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.     

Chromosome engineering in zygotes with CRISPR/Cas9

Deletions, duplications, and inversions of large genomic regions covering several genes are an important class of disease causing variants in humans. Modeling these structural variants in mice requires multistep processes in ES cells, which has limited their availability. Mutant mice containing small insertions, deletions, and single nucleotide polymorphisms can be reliably generated using CRISPR/Cas9 directly in mouse zygotes. Large structural variants can be generated using CRISPR/Cas9 in ES cells, but it has not been possible to generate these directly in zygotes. We now demonstrate the direct generation of deletions, duplications and inversions of up to one million base pairs by zygote injection. genesis 54:78–85, 2016. © 2016 The Authors. genesis Published by Wiley Periodicals, Inc.     

Functional validation of cadherin as a receptor of Bt toxin Cry1Ac in Helicoverpa armigera utilizing the CRISPR/Cas9 system

Cadherins have been identified as receptors of Bacillus thuringiensis (Bt) Cry1A toxins in several lepidopteran insects including the cotton bollworm, Helicoverpa armigera. Disruption of the cadherin gene HaCad has been genetically linked to resistance to Bt toxin Cry1Ac in H. armigera. By using the CRISPR/Cas9 genome editing system (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9), HaCad from the Cry1Ac-susceptible SCD strain of H. armigera was successfully knocked out. A single positive CRISPR event with a frame shift deletion of 4 nucleotides was identified and made homozygous to create a knockout line named SCD-Cad. Western blotting confirmed that HaCad was no longer expressed in the SCD-Cad line while an intact HaCad of 210 kDa was present in the parental SCD strain. Insecticide bioassays were used to show that SCD-Cad exhibited 549-fold resistance to Cry1Ac compared with SCD, but no significant change in susceptibility to Cry2Ab. Our results not only provide strong reverse genetics evidence for HaCad as a functional receptor of Cry1Ac, but also demonstrate that the CRISPR/Cas9 technique can act as a powerful and efficient genome editing tool to study gene function in a global agricultural pest, H. armigera.     

Chromosomal deletions mediated by CRISPR/Cas9 in Helicoverpa armigera

Helicoverpa armigera, cotton bollworm, is one of the most disastrous pests worldwide, threatening various food and economic crops. Functional genomic tools may provide efficient approaches for its management. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system, dependent on a single guide RNA (sgRNA), has been used to induce indels for targeted mutagenesis in cotton bollworm. However, genomic deletions may be more desirable to disrupt the function of noncoding genes or regulatory sequences. By injecting two sgRNAs with Cas9 protein targeting different exons, we obtained predictable genomic deletions of several hundred bases. We achieved this type of modification with different combinations of sgRNA pairs, including HaCad and HaABCC2. Our finding indicated that CRISPR/Cas9 can be used as an efficient tool to engineer genomes with chromosomal deletion in H. armigera.     

CRISPR/Cas9系统在昆虫中的应用

CRISPR/Cas9(clustered regularly interspaced short palindromic repeat/CRISPR-associated nuclease 9)技术是一种RNA引导的基因组靶向编辑技术,能对基因组序列进行精确编辑,在探究基因功能、修复受损基因、沉默有害基因、改良品质性状等方面具有广阔的应用前景。近年来,随着对CRISPR/Cas9系统研究的不断深入和改造,该系统以其操作简易、省时、高效等优点在生物学研究的众多领域中得以推广和应用,特别是在果蝇(Bombyx mori)、家蚕(silkworm)、埃及伊蚊(Aedes aegypti)和蝴蝶(butterfly)等多种昆虫中。本文概述了CRISPR/Cas9的结构、作用原理及发展优化,总结了CRISPR/Cas9导入昆虫的策略和在昆虫中的应用,以及对CRISPR/Cas9系统产生脱靶问题的应对策略,以期对经济昆虫和有益昆虫的分子育种、害虫的生物技术防控等研究提供参考。     

Locus-specific integration of extrachromosomal transgenes in C. elegans with the CRISPR/Cas9 system

We established a method to generate integration from extrachromosomal arrays with the CRISPR/Cas9 system. Multi-copy transgenes were integrated into the defined loci of chromosomes by this method, while a multi-copy transgene is integrated into random loci by previous methods, such as UV- and gamma-irradiation. The effects of a combination of sgRNAs, which define the cleavage sites in extrachromosomes and chromosomes, and the copy number of potential cleavable sequences were examined. The relative copy number of cleavable sequences in extrachromosomes affects the frequency of fertile F1 transgenic animals. The expression levels of the reporter gene were almost proportional to the copy numbers of the integrated sequences at the same integration site. The technique is applicable to the transgenic strains abundantly stored and shared among the C. elegans community, particularly when researchers use sgRNAs against common plasmid sequences such as β-lactamase.     

CRISPR/Cas9技术在小麦育种中的应用进展

小麦（Triticum aestivum L.）是世界上主要的农作物之一,在粮食安全供应中发挥重要作用。在过去的几十年,由于小麦基因组复杂和遗传转化困难,导致小麦的基础和应用研究落后于其他谷类作物。2014年小麦基因组编辑取得了显著进展,进而促进了小麦生物技术的发展。综述了CRISPR/Cas9技术在小麦育种中的研究进展,简单介绍了CRISPR/Cas9基因编辑技术的发现、原理和优缺点,指出小麦基因编辑过程中农杆菌介导的遗传转化较粒子轰击法可降低转基因沉默频率,未来将成为基因编辑过程中主流的遗传转化方式;优化sgRNA的启动子、选择同源保守序列做为靶点可以提高基因编辑效率;新开发的碱基编辑器和prime editor需引入更多突变类型。展望了进一步提高小麦基因编辑效率和安全性的可行性,以期为未来小麦育种工作提供参考。     

CRISPR/Cas9技术在微生物研究中的应用进展

CRISPR(Clustered regularly interspaced short palindromic repeats)/Cas(CRISPR associated proteins)系统是在细菌和古生菌中发现的一种RNA指导的降解入侵病毒或质粒DNA的适应性免疫系统。由II型CRISPR/Cas系统改造而成的CRISPR/Cas9技术已经被开发成一种强大的基因组编辑和表达调控工具,并且广泛应用于基因功能研究、代谢工程和合成生物学等领域。本文从CRISPR/Cas9系统的发现过程、分类、作用原理、在微生物研究中的应用进展等方面进行总结,并展望了该技术的应用前景。     

CRISPR/Cas9基因组定点编辑中脱靶现象的研究进展

近年来, CRISPR定点编辑技术发展迅猛, 在动物、植物和微生物中均得到广泛应用。其中, 备受关注的脱靶现象也是研究的热点, 迄今已取得了重要进展。该文介绍了脱靶现象的产生原理及体内和体外检测脱靶现象的方法, 评价了通过改进sgRNA设计和优化CRISPR系统等来降低脱靶率的方法。在植物基因组定点编辑过程中, 应适时检测脱靶现象, 提高脱靶检测的精确度和准确度。     

CRISPR/Cas9 Gene editing of RyR2 in human stem cell-derived cardiomyocytes provides a novel approach in investigating dysfunctional Ca2+ signaling

Type-2 ryanodine receptors (RyR2s) play a pivotal role in cardiac excitation-contraction coupling by releasing Ca²⁺ from sarcoplasmic reticulum (SR) via a Ca²⁺ -induced Ca²⁺ release (CICR) mechanism. Two strategies have been used to study the structure-function characteristics of RyR2 and its disease associated mutations: (1) heterologous cell expression of the recombinant mutant RyR2s, and (2) knock-in mouse models harboring RyR2 point mutations. Here, we establish an alternative approach where Ca²⁺ signaling aberrancy caused by the RyR2 mutation is studied in human cardiomyocytes with robust CICR mechanism. Specifically, we introduce point mutations in wild-type RYR2 of human induced pluripotent stem cells (hiPSCs) by CRISPR/Cas9 gene editing, and then differentiate them into cardiomyocytes. To verify the reliability of this approach, we introduced the same disease-associated RyR2 mutation, F2483I, which was studied by us in hiPSC-derived cardiomyocytes (hiPSC-CMs) from a patient biopsy. The gene-edited F2483I hiPSC-CMs exhibited longer and wandering Ca²⁺ sparks, elevated diastolic Ca²⁺ leaks, and smaller SR Ca²⁺ stores, like those of patient-derived cells. Our CRISPR/Cas9 gene editing approach validated the feasibility of creating myocytes expressing the various RyR2 mutants, making comparative mechanistic analysis and pharmacotherapeutic approaches for RyR2 pathologies possible.     

CRISPR/Cas9基因组编辑技术在癌症研究中的应用

CRISPR/cas9基因组编辑技术因其设计简单以及操作容易,使其在基因编辑的研究中越来越受到欢迎。利用该技术,科研人员可以实现在碱基的水平对基因组进行定点修饰。CRISPR系统现已经被广泛地应用到多个物种的基因组编辑以及癌症的相关研究中。本文在最新研究进展的基础上,结合对癌症研究及基因组编辑技术的理解,对CRISPR/Cas9技术在癌症研究中的应用进行了综述。     

CRISPR/Cas9在肿瘤治疗中的研究进展

CRISPR/Cas9技术自从出现以来便迅速应用于肿瘤研究。在肿瘤发生的机理研究中,CRISPR/Cas9可用于研究单核苷酸突变、染色体异位等因素在肿瘤发生中的作用机制,同时也可以用于肿瘤细胞中功能缺陷基因的筛选。在肿瘤治疗方法的研究中,CRISPR/Cas9主要用于诱发机制比较清晰且诱因为病毒的肿瘤类型,例如鼻咽癌、宫颈癌等,通过对相应病毒的基因进行编辑从而抑制其致癌作用。利用CRISPR/Cas9技术还可以加速新肿瘤治疗靶点基因的发现。尽管发展和应用十分迅速,但是CRISPR/Cas9在肿瘤研究和治疗中的作用仍然受多种因素的限制,包括Cas9和sgRNA的输送效率、脱靶效应以及安全性和成本等。对CRISPR/Cas9在肿瘤研究中的应用进展进行了综述,以期为肿瘤发生、转移机制和肿瘤治疗等方面的研究提供参考。     

CRISPR/Cas9‐mediated genome modification in the mollusc,Crepidula fornicata

The discovery and application of the CRISPR/Cas9 genome editing method has greatly enhanced the ease with which transgenic manipulation can occur. We applied this technology to the mollusc, Crepidula fornicata, and have successfully created transgenic embryos expressing mCherry fused to endogenous β‐catenin. Specific integration of the fluorescent reporter was achieved by homologous recombination with a β‐catenin‐specific donor DNA containing the mCherry coding sequence. This fluorescent gene knock‐in strategy permits in vivo observations of β‐catenin expression during embryonic development and represents the first demonstration of CRISPR/Cas9‐mediated transgenesis in the Lophotrochozoa superphylum. The CRISPR/Cas9 method is a powerful and economical tool for genome modification and presents an option for analysis of gene expression in not only major model systems, but also in those more diverse species that may not have been amenable to the classic methods of transgenesis. This approach will allow one to generate transgenic lines of snails for future studies. genesis 53:237–244, 2015. © 2014 Wiley Periodicals, Inc.