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Cloning scheme and sgRNA activity tests. (A) Single-step Golden Gate assembly scheme of the STOPdsRed donor vector cloned into a modified pBluescript backbone. (B) Scheme of the "generic" attB plasmids used in our study. A simple cloning step is sufficient to generate any gene-specific attB plasmid that can be used to replace an excised exon. (C) sgRNA synthesis scheme. (D) sgRNA activity assay of 12 different sgRNAs in S2 cells. PCR result with (bottom) or without (middle) T7-Endonuclease I treatment are shown. Digested products are marked by arrow heads and effective sgRNAs are marked by a red asterisk. C are controls without sgRNA.
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The development of clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated (Cas) technologies promises a quantum leap in genome-engineering of model organisms. However, CRISPR-mediated gene targeting reports in Drosophila melanogaster are still restricted to a few genes, use variable experimental conditions and vary i...
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... generate pJET1.2-STOP-dsRed. Because this STOP-dsRed cassette is flanked by two BsmBI sites, it can be easily assembled with both homology arms ( Figure 3A): each homology arm of approximately 1 kb was amplified from genomic DNA of the target genotype with Phusion polymerase (NEB) and blunt-end cloned into pJET1.2 (CloneJET PCR Cloning Kit, Thermo Scientific). ...
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... sgRNA dsDNA template was produced using overlap PCR with a small amount of a common sgRNA scaffold primer, a shorter sgRNA amplification primer, and a sgRNA gene-specific primer that includes the T7 promoter ( Figure 3C) (Böttcher et al. 2014). All sgRNA primer sequences are listed in Table S1. ...
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... the same strategy should be applicable to any gene, we established a single-step Golden Gate protocol to assemble the STOP-dsRed donor plasmid containing approximately 1-kb homology arms on each side, which has been shown to be of sufficient length for efficient HDR (Beumer et al. 2013b) and can be easily amplified by PCR. Cloning of the ho- mology arms into the donor vector is thus very straightforward and takes only a few days for the gene of choice ( Figure 3A, see Materials and Methods). This donor vector is the template for the HDR in step 1. ...
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... plasmids are fully compatible with our step 2 de- sign. We have generated additional "generic" attB plasmids that can be used to tag any gene with a 2xTY1-V5 tag or to engineer a conditional allele using an FRT flanked 2xTY1 cassette followed by a V5 tag ( Figure 3B). Flp-mediated deletion of the 2xTY1 cassette will lead to a frame shift and thus can be used to create loss-of-function clones at very high efficiency, as the flip-out will occur in cis ( Hadjieconomou et al. 2011). ...
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... developed such a selection assay to be able to only inject effective sgRNAs into fly embryos. We designed 12 different sgRNAs targeting different regions in the salm gene and synthesized the sgRNAs by a standard PCR and in vitro transcription reaction ( Figure 3C). These sgRNAs were then individually transfected into Cas9 expressing S2 cells (Böttcher et al. 2014), and their cleavage efficiency was determined with a simple T7-Endonuclease I assay (see Materials and Methods) ( Zhang et al. 2014). ...
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... sgRNAs were then individually transfected into Cas9 expressing S2 cells (Böttcher et al. 2014), and their cleavage efficiency was determined with a simple T7-Endonuclease I assay (see Materials and Methods) ( Zhang et al. 2014). On average, approximately half of the tested sgRNAs work efficiently in this assay ( Figure 3D), strongly suggesting that such a pre- selection test is useful to improve the in vivo success rates. ...
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Citations
... ) Zhang et al. work 417 using two-step (CRISPR-and RMCE-based) strategy for efficient genome engineering in 418Drosophila(Zhang et al. 2014), and (2) DICE experiment mentioned earlier, TALEN-419 assisted homologous recombination was first used to direct a gene cassette to human 420 chromosome locus H11 before the subsequent RMCE approach was carried out(Zhu et 421 al. 2014). 422Neither the DICE method nor the 'CRISPR-assisted SSR-mediated' RMCE 423 approach has been reported in plants although designer nucleases seem to have readily 424 edited specific genes in plant genomes(Puchta 2017). ...
Site-specific recombination (SSR) systems have been extensively used for controlled genome modification in eukaryotic cells, especially for DNA excision and DNA site-specific integration. Unidirectional SSR systems are particularly attractive for efficient site-specific gene integration because of their irreversible recombination activities. Bxb1-att is one such unidirectional SSR system, which has emerged as a molecular tool for eukaryotic genome manipulation. We will review the discovery, development and current applications of the Bxb1-att system with an emphasis on utility in plants. We provide perspectives on future applications using dual integrase cassette exchange for precise gene integration at a specific locus. Finally, potential applications of combining unidirectional SSR systems with newly developed genome-editing tools (e.g., CRISPR-Cas9) will be explored to enhance capabilities for precise locus-specific gene integration into plant genomes.
... Initially, researchers used plasmid donor templates containing long (>1 kb) homology arms for knock-in Drosophila [1,3,4,13]. Constructing these large plasmid donor templates required extensive molecular cloning [14].As a result, there have been approaches that have looked towards decreasing the amount of time towards molecular cloning in preparation for CRISPR/Cas9-mediated knock-in Drosophila. One way to bypass cloning the donor template is to use single-stranded oligonucleotide donors -which can be outsourced by a commercial entity [1,12,[14][15][16][17]. ...
... Constructing these large plasmid donor templates required extensive molecular cloning [14].As a result, there have been approaches that have looked towards decreasing the amount of time towards molecular cloning in preparation for CRISPR/Cas9-mediated knock-in Drosophila. One way to bypass cloning the donor template is to use single-stranded oligonucleotide donors -which can be outsourced by a commercial entity [1,12,[14][15][16][17]. For example, Port et al. successfully introduced an 11 bp mutation containing a restriction enzyme site into the ebony genomic locus, using a 50 nt single-stranded oligonucleotide donor and thus, demonstrating a single-stranded oligonucleotide as a cloning-free donor format for CRISPR/Cas9-mediated insertion [1]. ...
CRISPR/Cas9 technology has been a powerful tool for gene editing in Drosophila, particularly for knocking in base-pair mutations or a variety of gene cassettes into endogenous gene loci. Among the Drosophila community, there has been a concerted effort to establish CRISPR/Cas9-mediated knock-in protocols that decrease the amount of time spent on molecular cloning. Here, we report the CRISPR/Cas9-mediated insertion of a ∼50 base-pair sequence into the ebony gene locus, using a linear double-stranded DNA (PCR product) donor template. By circumventing the cloning step of the donor template, our approach suggests the PCR product as a useful, alternative knock-in donor format.
... To generate both mmt knock-out line and the Gal4 driver line, we adapted protocols from two-step CRISPR-and RMCEbased strategies for genome editing in Drosophila (Port & Bullock, 2016;Zhang et al., 2014). To generate the knockout line (Suppl . ...
Neuropeptides play a key role in regulating physiology and behavior, including feeding. While animals modify their food choices to respond to the lack of specific nutrients, the mechanisms mediating nutrient-specific appetites remain unclear. Here, we identified marmite (mmt), a previously uncharacterized Drosophila melanogaster gene encoding a secreted peptide that controls feeding decisions. We show that both mmt mutants and neuronal knockdown of mmt specifically increased the intake of proteinaceous food, whereas neuronal mmt overexpression reduced protein appetite. mmt expression is also higher in animals maintained on amino acid rich food, suggesting that mmt encodes a protein-specific satiety signal. Mmt is expressed in a small number of neurons in the adult nervous system, with a single pair of neurons modulating protein appetite. Finally, sequence and phylogenetic analysis showed that mmt is part of an ancient and conserved family of neuropeptides, including the poorly understood vertebrate neuropeptides B and W genes. Functional experiments showed that mmt and vertebrate NPB and NPW modulate food intake in both flies and mice. Therefore, we discovered an ancient family of neuropeptides involved in controlling feeding across phyla.
... Different approaches have been tried to achieve the induction of HDR in flies for example, a gRNA plasmid and donor repair template were injected into transgenic Cas9 embryos [37], while the transgenic embryos containing a sgRNA and Cas9 were injected with a donor template plasmid. All of these three components containing the Cas9 gene, sgRNA and donor repair template in a plasmid form were delivered into non-transgenic flies [38,39]. ...
Insect pests impose a serious threat to agricultural productivity. Initially, for pest management, several breeding approaches were applied which have now been gradually replaced by genome editing (GE) strategies as they are more efficient and less laborious. CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeat/CRISPR-associated system) was discovered as an adaptive immune system of bacteria and with the scientific advancements, it has been improvised into a revolutionary genome editing technique. Due to its specificity and easy handling, CRISPR/Cas9-based genome editing has been applied to a wide range of organisms for various research purposes. For pest control, diverse approaches have been applied utilizing CRISPR/Cas9-like systems, thereby making the pests susceptible to various insecticides, compromising the reproductive fitness of the pest, hindering the metamorphosis of the pest, and there have been many other benefits. This article reviews the efficiency of CRISPR/Cas9 and proposes potential research ideas for CRISPR/Cas9-based integrated pest management. CRISPR/Cas9 technology has been successfully applied to several insect pest species. However, there is no review available which thoroughly summarizes the application of the technique in insect genome editing for pest control. Further, authors have highlighted the advancements in CRISPR/Cas9 research and have discussed its future possibilities in pest management.
... Frame-shift mutant alleles for CG2678 and rhino were generated in isogenised white embryos after co-injection of plasmids pBS-Hsp70-Cas9 (Addgene #46294) and pU6-BbsI-chiRNA (Addgene #45946) modified to express sgRNAs. Whole locus CG2678 deletion for RMCE was achieved following co-injection into ZH-2A(Act5C-Cas9) embryos (derived from Bloomington stock #58492) of plasmids pXZ13 (Zhang et al., 2014a) containing 1 kb homology arms around a 3xP3-dsRed marker flanked by attP sites, together with pCFD4 (Addgene 49411) (Port et al., 2014) expressing two sgRNAs. sgRNA sequences are given in the Key Resource Table. ...
... Generation of dMegf8 mutants. To generate a targeted deletion in the dMegf8 locus that would produce a dMegf8 null allele, we used CRISPR/ Cas9 methodology and procedures established at the GenetiVision company based on the previously reported strategies (Zhang et al., 2014). The targeting construct contained 59 and 39 homology arms flanking a GFP expression cassette that replaced the deleted sequences in the dMegf8 locus. ...
... The dMegf8 locus (Fig. 1B) is composed of 6 exons with the ATG codon in exon 1 and the termination codon in exon 6. We used the CRISPR-Cas9 gene editing strategy (Zhang et al., 2014) and inserted a GFP cassette to replace ;4062 bp starting from nucleotide sequence 59-AAGCTATGGGGTCTG-39 in exon 3 and ending in nucleotide sequence 59-GATCGAAT GCCTCTT-39 in exon 4 (for details, see Fig. 1B). We conducted PCR amplification using fly genomic DNA with a primer combination that distinguished the WT allele (primers 1 1 2 = 407 bp) ( Fig. 1B,C) from the dMegf8 mutant allele (primers 1 1 3 = 505 bp) (Fig. 1B,D). ...
Drosophila Multiple Epidermal Growth Factor-like Domains 8 (dMegf8) is a homolog of human MEGF8. MEGF8 encodes a multi-domain transmembrane protein which is highly conserved across species. In humans, MEGF8 mutations cause a rare genetic disorder called Carpenter syndrome, which is frequently associated with abnormal left-right patterning, cardiac defects and learning disabilities. MEGF8 is also associated with psychiatric disorders. Despite its clinical relevance MEGF8 remains poorly characterized, and though it is highly conserved, studies on animal models of Megf8 are also very limited. The presence of intellectual disabilities in Carpenter syndrome patients and association of MEGF8 with psychiatric disorders indicate that mutations in MEGF8 cause underlying defects in synaptic structure and functions. In this study, we investigated the role of Drosophila dMegf8 in glutamatergic synapses of the larval neuromuscular junctions (NMJ) in both males and females. We show that dMegf8 localizes to NMJ synapses and is required for proper synaptic growth. dMegf8 mutant larvae and adults show severe motor coordination deficits. At the NMJ, dMegf8 mutants show altered localization of pre- and post-synaptic proteins, defects in synaptic ultrastructure and neurotransmission. Interestingly, dMegf8 mutants have reduced levels of the type II BMP receptor Wishful thinking (Wit). dMegf8 displays genetic interactions with neurexin-1 (dnrx) and wit, and in association with Dnrx and Wit plays an essential role in synapse organization. Our studies provide insights into human MEGF8 functions and potentially into mechanisms that may underlie intellectual disabilities observed in Carpenter syndrome as well as MEGF8-related synaptic structural and/or functional deficits in psychiatric disorders.SIGNIFICANCE STATEMENT:Carpenter Syndrome, known for over a century now, is a genetic disorder linked to mutations in Multiple Epidermal Growth Factor-like Domains 8 (MEGF8) gene and associated with intellectual disabilities among other symptoms. MEGF8 is also associated with psychiatric disorders. Despite the high genetic conservation and clinical relevance, the functions of MEGF8 remain largely uncharacterized. Patients with intellectual disabilities and psychiatric diseases often have an underlying defect in synaptic structure and function. This work defines the role of the fly homolog of human MEGF8, dMegf8, in glutamatergic synapse growth, organization and function and provide insights into potential functions of MEGF8 in human central synapses and synaptic mechanisms that may underlie psychiatric disorders and intellectual disabilities seen in Carpenter Syndrome.
... Next, using recombinases, a genomic sequence between two different recognition sites is replaced by a donor DNA sequence carrying the same set of recognition sites (figure 2b). In Drosophila, RMCE has been successfully used for gene replacement [114,115]. These studies suggest that a combination of the genome editing and the RMCE will facilitate replacement of larger genomic regions at desired target loci, although loci involved in speciation and adaptation have not yet been analysed. ...
Understanding the genetic basis of reproductive isolation and adaptive traits in natural populations is one of the fundamental goals in evolutionary biology. Genome editing technologies based on CRISPR-Cas systems and site-specific recombinases have enabled us to modify a targeted genomic region as desired and thus to conduct functional analyses of target loci, genes and mutations even in non-conventional model organisms. Here, we review the technical properties of genome editing techniques by classifying them into the following applications: targeted gene knock-out for investigating causative gene functions, targeted gene knock-in of marker genes for visualizing expression patterns and protein functions, precise gene replacement for identifying causative alleles and mutations, and targeted chromosomal rearrangement for investigating the functional roles of chromosomal structural variations. We describe examples of their application to demonstrate functional analysis of naturally occurring genetic variations and discuss how these technologies can be applied to speciation and adaptation research.
This article is part of the theme issue ‘Genetic basis of adaptation and speciation: from loci to causative mutations’.
... We used CRISPR-Cas9 to disrupt genomic dAux gene by injecting vas-cas9 (X chr) flies (Norrander et al, 1983) with pCFD3-dU6:3gRNA (Addgene #49410; (Port et al, 2014)) carrying guide RNA sequences and a pWhiteStar donor plasmid carrying an integrase mediated cassette exchange (IMCE) and a white gene. Knock-ins were created by injecting pBS-KS-attB1-2 (Addgene#61255; (Zhang et al, 2014)) containing HA-tagged dAux WT or HA-tagged dAux RG (the R1119G mutation) into recombinant vas-cas9:Phi31 flies. Transgenic fly lines were produced by injecting plasmids into VK37 for PhiC31 integrase-mediated site-specific transgenesis. ...
Recent evidence links dysfunctional lipid metabolism to the pathogenesis of Parkinson's disease, but the mechanisms are not resolved. Here, we created a new Drosophila knock-in model of DNAJC6/Auxilin and find that the pathogenic mutation causes synaptic dysfunction, neurological defects and neurodegeneration, as well as specific lipid metabolism alterations. In these mutants membrane lipids containing long-chain polyunsaturated fatty acids, including phosphatidylinositol lipid species that are key for synaptic vesicle recycling and organelle function are reduced. Overexpression of another protein mutated in Parkinson's disease, Synaptojanin-1, known to bind and synthesize specific phosphoinositides, strongly rescues the DNAJC6/Auxilin neuronal defects and neurodegeneration. Our work reveals a functional relation between two proteins mutated in Parkinson's disease and implicates deregulated phosphoinositide metabolism in the maintenance of neuronal integrity and neuronal survival in Parkinsonism.
... A second UAS-Bru1-PA line was generated by cloning the full-length bru1-RA cDNA (obtained by RT-PCR from w 1118 ) into the pUAS-TattB transformation vector (Bischof et al, 2007) and integrating into the attP-86Fb landing site. The bru1 M2 and bru1 M3 alleles were generated using a CRISPR approach (Zhang et al, 2014). In the bru1 M2 mutant, a selectable 3xP3-DsRed cassette was inserted upstream of bru1 exon 12. ...
Protein isoform transitions confer muscle fibers with distinct properties and are regulated by differential transcription and alternative splicing. RNA-binding Fox protein 1 (Rbfox1) can affect both transcript levels and splicing, and is known to contribute to normal muscle development and physiology in vertebrates, although the detailed mechanisms remain obscure. In this study, we report that Rbfox1 contributes to the generation of adult muscle diversity in Drosophila . Rbfox1 is differentially expressed among muscle fiber types, and RNAi knockdown causes a hypercontraction phenotype that leads to behavioral and eclosion defects. Misregulation of fiber type–specific gene and splice isoform expression, notably loss of an indirect flight muscle–specific isoform of Troponin-I that is critical for regulating myosin activity, leads to structural defects. We further show that Rbfox1 directly binds the 3′-UTR of target transcripts, regulates the expression level of myogenic transcription factors myocyte enhancer factor 2 and Salm, and both modulates expression of and genetically interacts with the CELF family RNA-binding protein Bruno1 (Bru1). Rbfox1 and Bru1 co-regulate fiber type–specific alternative splicing of structural genes, indicating that regulatory interactions between FOX and CELF family RNA-binding proteins are conserved in fly muscle. Rbfox1 thus affects muscle development by regulating fiber type–specific splicing and expression dynamics of identity genes and structural proteins.
... Generation of KI Strains. Four different KI strains were generated using CRISPR-Cas9 technology (61). The WT hPNPO cDNA was amplified from the human brain cDNA library (TaKaRa; catalog no. ...
Significance
Both genetic and environmental factors contribute to epilepsy. Understanding their contributions and interactions helps disease management. However, it is often challenging to study gene–environment interaction in humans due to their heterogeneous genetic background and less controllable environmental factors. The fruit fly, Drosophila melanogaster , has been proven to be a powerful model to study human diseases, including epilepsy. We generated knock-in flies carrying different epilepsy-associated pyridox(am)ine 5 ′ -phosphate oxidase ( PNPO ) alleles and studied the developmental, behavioral, electrophysiological, and fitness effects of each mutant allele under different dietary conditions. We showed that phenotypes in knock-in flies are allele and diet dependent, providing clues for timely and specific diet interventions. Our results offer biological insights into mechanisms underlying phenotypic variations and specific therapeutic strategies.
