Heritable Genome Editing with CRISPR/Cas9 in the Silkworm,Bombyx mori

We report the establishment of an efficient and heritable gene mutagenesis method in the silkworm Bombyx mori using modified type II clustered regularly interspaced short palindromic repeats (CRISPR) with an associated protein (Cas9) system. Using four loci Bm-ok, BmKMO, BmTH, and Bmtan as candidates, we proved that genome alterations at specific sites could be induced by direct microinjection of specific guide RNA and Cas9-mRNA into silkworm embryos. Mutation frequencies of 16.7–35.0% were observed in the injected generation, and DNA fragments deletions were also noted. Bm-ok mosaic mutants were used to test for mutant heritability due to the easily determined translucent epidermal phenotype of Bm-ok-disrupted cells. Two crossing strategies were used. In the first, injected Bm-ok moths were crossed with wild-type moths, and a 28.6% frequency of germline mutation transmission was observed. In the second strategy, two Bm-ok mosaic mutant moths were crossed with each other, and 93.6% of the offsprings appeared mutations in both alleles of Bm-ok gene (compound heterozygous). In summary, the CRISPR/Cas9 system can act as a highly specific and heritable gene-editing tool in Bombyx mori.     

CRISPR/Cas9-Mediated Genome Editing in Soybean Hairy Roots

As a new technology for gene editing, the CRISPR (clustered regularly interspaced short palindromic repeat)/Cas (CRISPR-associated) system has been rapidly and widely used for genome engineering in various organisms. In the present study, we successfully applied type II CRISPR/Cas9 system to generate and estimate genome editing in the desired target genes in soybean (Glycine max (L.) Merrill.). The single-guide RNA (sgRNA) and Cas9 cassettes were assembled on one vector to improve transformation efficiency, and we designed a sgRNA that targeted a transgene (bar) and six sgRNAs that targeted different sites of two endogenous soybean genes (GmFEI2 and GmSHR). The targeted DNA mutations were detected in soybean hairy roots. The results demonstrated that this customized CRISPR/Cas9 system shared the same efficiency for both endogenous and exogenous genes in soybean hairy roots. We also performed experiments to detect the potential of CRISPR/Cas9 system to simultaneously edit two endogenous soybean genes using only one customized sgRNA. Overall, generating and detecting the CRISPR/Cas9-mediated genome modifications in target genes of soybean hairy roots could rapidly assess the efficiency of each target loci. The target sites with higher efficiencies can be used for regular soybean transformation. Furthermore, this method provides a powerful tool for root-specific functional genomics studies in soybean.     

Guide RNA Design for CRISPR/Cas9-Mediated Potato Genome Editing

The activity of the pool of sgRNA molecules designed for different regions of potato coilin and phytoene desaturase genes was compared in vitro. Due to the presence of nucleotides unpaired with DNA, sgRNA is able not only to inhibit but also to stimulate the activity of the Cas9–sgRNA complex in vitro. Although the first six nucleotides located in the DNA substrate proximally to the PAM site at the 3' end are the binding sites for cas9, they had no significant effect on the activity of the Cas9–sgRNA complex.     

Characterization of an Enantioselective Odorant Receptor in the Yellow Fever Mosquito Aedes aegypti

Enantiomers differ only in the left or right handedness (chirality) of their orientations and exhibit identical chemical and physical properties. In chemical communication systems, enantiomers can be differentially active at the physiological and behavioral levels. Only recently were enantioselective odorant receptors demonstrated in mammals while their existence in insects has remained hypothetical. Using the two-microelectrode voltage clamp of Xenopus oocytes, we show that the yellow fever mosquito, Aedes aegypti, odorant receptor 8 (AaOR8) acts as a chiral selective receptor for the (R)-(—)-enantiomer of 1-octen-3-ol, which in the presence of other kairomones is an attractant used by blood-sucking insects to locate their hosts. In addition to steric constraints, chain length and degree of unsaturation play important roles in this recognition process. This is the first characterization of an enantioselective odorant receptor in insects and the results demonstrate that an OR alone, without helper proteins, can account for chiral specificity exhibited by olfactory sensory neurons (OSNs).     

The CRISPR/Cas9 Genome Editing Revolution

正Genomes encode the genetic information that controls the development and physiological functions of all living organisms on our planet,and are therefore of central interest in all aspects of biomedical research.To understand the blueprint of life,scientists have long aimed to read and manipulate the genome using a rapidly expanding toolbox.To read the genome,novel state-of-the-art sequencing technologies have made it possible to sequence any single genome rapidly and cheaply.However,methods for introducing targeted modifications of the genome have lagged behind,and though     

CRISPR/Cas9 and Genome Editing in Drosophila

Recent advances in our ability to design DNA binding factors with specificity for desired sequences have resulted in a revolution in genetic engineering, enabling directed changes to the genome to be made relatively easily. Traditional techniques for generating genetic mutations in most organisms have relied on selection from large pools of randomly induced mutations for those of particular interest, or time-consuming gene targeting by homologous recombination. Drosophila melanogaster has always been at the forefront of genetic analysis, and application of these new genome editing techniques to this organism will revolutionise our approach to performing analysis of gene function in the future. We discuss the recent techniques that apply the CRISPR/Cas9 system to Drosophila, highlight potential uses for this technology and speculate upon the future of genome engineering in this model organism.     

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。     

sgRNA Sequence Motifs Blocking Efficient CRISPR/Cas9-Mediated Gene Editing

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CRISPR/Cas9:一种新型的基因组定点编辑技术

CRISPR/Cas9系统是原核生物抵御病毒或质粒等外来遗传物质入侵的一种获得性免疫系统,主要由非特异性的Cas9核酸酶和起识别作用的cr RNA所组成。相较于传统的基因组编辑技术,基于CRISPR/Cas9系统的基因组定点编辑技术具有快速、简单、高效等优点,并且几乎可以用于任何物种的基因编辑。尽管CRISPR/Cas9系统的基因组特异性还有待进一步确认,但该系统在基因组编辑方面的简便性和有效性必将促进生物学的研究和人类疾病基因治疗方面的发展。     

Pharmacological and Genetic Evidence for Gap Junctions as Potential New Insecticide Targets in the Yellow Fever Mosquito,Aedes aegypti

The yellow fever mosquito Aedes aegypti is an important vector of viral diseases that impact global health. Insecticides are typically used to manage mosquito populations, but the evolution of insecticide resistance is limiting their effectiveness. Thus, identifying new molecular and physiological targets in mosquitoes is needed to facilitate insecticide discovery and development. Here we test the hypothesis that gap junctions are valid molecular and physiological targets for new insecticides. Gap junctions are intercellular channels that mediate direct communication between neighboring cells and consist of evolutionarily distinct proteins in vertebrate (connexins) and invertebrate (innexins) animals. We show that the injection of pharmacological inhibitors of gap junctions (i.e., carbenoxolone, meclofenamic acid, or mefloquine) into the hemolymph of adult female mosquitoes elicits dose-dependent toxic effects, with mefloquine showing the greatest potency. In contrast, when applied topically to the cuticle, carbenoxolone was the only inhibitor to exhibit full efficacy. In vivo urine excretion assays demonstrate that both carbenoxolone and mefloquine inhibit the diuretic output of adult female mosquitoes, suggesting inhibition of excretory functions as part of their mechanism of action. When added to the rearing water of 1^st instar larvae, carbenoxolone and meclofenamic acid both elicit dose-dependent toxic effects, with meclofenamic acid showing the greatest potency. Injecting a double-stranded RNA cocktail against innexins into the hemolymph of adult female mosquitoes knock down whole-animal innexin mRNA expression and decreases survival of the mosquitoes. Taken together these data indicate that gap junctions may provide novel molecular and physiological targets for the development of insecticides.     

Wolbachia-Associated Bacterial Protection in the Mosquito Aedes aegypti

Background

Wolbachia infections confer protection for their insect hosts against a range of pathogens including bacteria, viruses, nematodes and the malaria parasite. A single mechanism that might explain this broad-based pathogen protection is immune priming, in which the presence of the symbiont upregulates the basal immune response, preparing the insect to defend against subsequent pathogen infection. A study that compared natural Wolbachia infections in Drosophila melanogaster with the mosquito vector Aedes aegypti artificially transinfected with the same strains has suggested that innate immune priming may only occur in recent host-Wolbachia associations. This same study also revealed that while immune priming may play a role in viral protection it cannot explain the entirety of the effect.

Methodology/Findings

Here we assess whether the level of innate immune priming induced by different Wolbachia strains in A. aegypti is correlated with the degree of protection conferred against bacterial pathogens. We show that Wolbachia strains wMel and wMelPop, currently being tested for field release for dengue biocontrol, differ in their protective abilities. The wMelPop strain provides stronger, more broad-based protection than wMel, and this is likely explained by both the higher induction of immune gene expression and the strain-specific activation of particular genes. We also show that Wolbachia densities themselves decline during pathogen infection, likely as a result of the immune induction.

Conclusions/Significance

This work shows a correlation between innate immune priming and bacterial protection phenotypes. The ability of the Toll pathway, melanisation and antimicrobial peptides to enhance viral protection or to provide the basis of malaria protection should be further explored in the context of this two-strain comparison. This work raises the questions of whether Wolbachia may improve the ability of wild mosquitoes to survive pathogen infection or alter the natural composition of gut flora, and thus have broader consequences for host fitness.     

Efficient Editing of an Adenoviral Vector Genome with CRISPR/Cas9

Immunotherapy based on genetic modification of T cells has played an important role in the treatment of tumors and viral infections. Moreover, adenoviral vectors engineered with improved safety due to their inability to integrate into the host genome have been key in the clinical application of T cell therapy. However, the commonly used adenoviral vector Ad5 exhibits low efficiency of infection of human T cells and the details of the intracellular trafficking pathway of adenoviral vectors in human primary T cells remains unclear. Resolution of these issues will depend on successful modification of the adenoviral vector. To this end, here we describe the successful establishment of a simple and efficient method for editing adenoviral vectors in vitro using the CRISPR-Cas9 gene editing system to target the adenoviral fiber gene. Electronic supplementary materialThe online version of this article (10.1007/s12088-020-00905-3) contains supplementary material, which is available to authorized users.     

A Simple CRISPR/Cas9 System for Multiplex Genome Editing in Rice

<正>Generating mutants bearing multiple gene modifications is essential for determining the functions of gene family members with redundant functions,or for analyzing epistatic relationships in genetic pathways.Using conventional methods,mutants with multiple gene mutations are generated by several rounds of intercrossing plants carrying a single mutation and identification of the offspring.This process is both time-     

QTL Mapping of Genome Regions Controlling Temephos Resistance in Larvae of the Mosquito Aedes aegypti

Introduction

The mosquito Aedes aegypti is the principal vector of dengue and yellow fever flaviviruses. Temephos is an organophosphate insecticide used globally to suppress Ae. aegypti larval populations but resistance has evolved in many locations.

Methodology/Principal Findings

Quantitative Trait Loci (QTL) controlling temephos survival in Ae. aegypti larvae were mapped in a pair of F₃ advanced intercross lines arising from temephos resistant parents from Solidaridad, México and temephos susceptible parents from Iquitos, Peru. Two sets of 200 F₃ larvae were exposed to a discriminating dose of temephos and then dead larvae were collected and preserved for DNA isolation every two hours up to 16 hours. Larvae surviving longer than 16 hours were considered resistant. For QTL mapping, single nucleotide polymorphisms (SNPs) were identified at 23 single copy genes and 26 microsatellite loci of known physical positions in the Ae. aegypti genome. In both reciprocal crosses, Multiple Interval Mapping identified eleven QTL associated with time until death. In the Solidaridad×Iquitos (SLD×Iq) cross twelve were associated with survival but in the reciprocal IqxSLD cross, only six QTL were survival associated. Polymorphisms at acetylcholine esterase (AchE) loci 1 and 2 were not associated with either resistance phenotype suggesting that target site insensitivity is not an organophosphate resistance mechanism in this region of México.

Conclusions/Significance

Temephos resistance is under the control of many metabolic genes of small effect and dispersed throughout the Ae. aegypti genome.     

Precise and Heritable Genome Editing in Evolutionarily Diverse Nematodes Using TALENs and CRISPR/Cas9 to Engineer Insertions and Deletions

Exploitation of custom-designed nucleases to induce DNA double-strand breaks (DSBs) at genomic locations of choice has transformed our ability to edit genomes, regardless of their complexity. DSBs can trigger either error-prone repair pathways that induce random mutations at the break sites or precise homology-directed repair pathways that generate specific insertions or deletions guided by exogenously supplied DNA. Prior editing strategies using site-specific nucleases to modify the Caenorhabditis elegans genome achieved only the heritable disruption of endogenous loci through random mutagenesis by error-prone repair. Here we report highly effective strategies using TALE nucleases and RNA-guided CRISPR/Cas9 nucleases to induce error-prone repair and homology-directed repair to create heritable, precise insertion, deletion, or substitution of specific DNA sequences at targeted endogenous loci. Our robust strategies are effective across nematode species diverged by 300 million years, including necromenic nematodes (Pristionchus pacificus), male/female species (Caenorhabditis species 9), and hermaphroditic species (C. elegans). Thus, genome-editing tools now exist to transform nonmodel nematode species into genetically tractable model organisms. We demonstrate the utility of our broadly applicable genome-editing strategies by creating reagents generally useful to the nematode community and reagents specifically designed to explore the mechanism and evolution of X chromosome dosage compensation. By developing an efficient pipeline involving germline injection of nuclease mRNAs and single-stranded DNA templates, we engineered precise, heritable nucleotide changes both close to and far from DSBs to gain or lose genetic function, to tag proteins made from endogenous genes, and to excise entire loci through targeted FLP-FRT recombination.     

CRISPR/Cas9基因组编辑技术的研究进展及其应用

随着测序技术的不断进步,获得了越来越多物种的全基因组序列。面对这些海量的基因组数据,基因定点编辑技术是高效捕获目标基因、迅速获得基因功能和应用信息的重要研究手段。CRISPR/Cas9是目前最有效的一种基因定点编辑技术。CRISPR/Cas9系统(clustered regularly interspaced short palindromic repeats/CRISPR-associated)是广泛存在于细菌及古生菌中的,由细菌体长期进化而形成,能够降解入侵病毒或噬菌体DNA的适应性免疫系统。因此,对CRISPR/Cas9系统的发展、应用,以其在相关研究中的应用前景进行阐述显得尤为必要。