Name: one-step crispr-based strategy for endogenous gene tagging in drosophila melanogaster
Uploaded: 2024-01-26T12:30:00
Description: Watch this Scientific Journal Video about One-step CRISPR-based Strategy for Endogenous Gene Tagging in Drosophila melanogaster at JoVE.com

We thank George Watase and Josie Clowney for discussions during the initial stages of the protocol development. The work was supported by funds from the NIH (grant no. R35GM133737 to S.Y.), Alfred P. Sloan Fellowship (to S. Y.) and McKnight Scholar Award (to S. Y.).

1. Design and construction of gene editing reagents
NOTE: To carry out endogenous tagging, it is essential to identify the various isoforms of the desired gene. Depending on the specific goals of the experiment, a fluorescent tag can be incorporated either internally or at the N- or C-termini of the selected protein isoform(s). For optimal CRISPR-mediated editing, it&#39;s crucial to position the two sgRNAs suitably close to the intended knock-in site. Subsequently, to enable editing through hom...

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The results demonstrate a streamlined, simple one-step CRISPR-based strategy for tagging endogenous genes in Drosophila. In the protocol, we use two gRNAs to induce DNA double-strand break and homologous recombination more efficiently. The gRNAs and the donor plasmid are injected into fruit fly embryos, which express Cas9 enzyme in their germline. These embryos are subsequently cultivated under standard conditions until adulthood, at which point they are crossed with balancer flies to produce the first generatio...

Fluorescent proteins have emerged as powerful tools for visualizing protein localization, dynamics, and protein-protein interactions in cells within living organisms1,2. Tagging endogenous genes with fluorescent proteins enables long-term live imaging of cellular processes with subcellular resolution. Furthermore, these endogenous gene-labeling techniques have several advantages compared to overexpression and immunostaining approaches. For example, overexpression of proteins might lead to protein misfolding, nonspecific protein-protein interactions, and mislocalization3. To date, researchers have been able to create vast libraries of endogenous genes fused to fluorescent proteins for a few model organisms, such as budding yeast, which can undergo efficient homologous recombination4. In the case of Drosophila melanogaster, various tools such as MiMICs5, transposon-based enhancer traps6, and fosmids7 have been devised to fluorescently tag genes.
The development of CRISPR-Cas9-based tools has made it possible to efficiently perform genome editing, including tagging endogenous proteins, in a wide range of model organism8,9. Cas9 is an RNA-guided enzyme that cleaves double-stranded DNA in a highly efficient and specific manner8, which can then be repaired by a nonhomologous end-joining (NHEJ) pathway leading to point mutations or deletions. Alternatively, the homology-directed repair (HDR) pathway allows the integration of heterologous DNA into the chromosome.
Past methods for CRISPR-based gene tagging in the model organism, Drosophila melanogaster, have relied on a two-step process10,11,12. This involves using a discernible eye-color marker, Dsred, to detect successful HDR events. Afterward, the Dsred marker would be excised using the Cre/loxP recombination system. To streamline and expedite this process, here, we present a simpler and more efficient method. This approach circumvents the need for lethal genotyping and allows for the direct screening of individual flies. Specifically, we employ a PCR-based approach to extract DNA from a small segment of the fly&#39;s middle leg, allowing us to genotype individual flies. Notably, this procedure does not compromise the vitality, movement, or reproductive capabilities of the sampled fly. Only the appropriate individuals, identified through this method, are further developed into stable stocks.
Here, we present a detailed protocol for generating fluorescent protein tagged flies in which endogenous genes are labeled with fluorescent reporters. This technique uses CRISPR-Cas9 genome editing to fuse any desired fluorophore tag to the N- or C- terminal of an endogenous gene. Below, we describe the design and construction of gRNA plasmids and a donor plasmid, one-step screening strategy to select CRISPR-positive flies, and steps to establish stable lines. Specifically, we describe strategies to tag an endogenous core clock Drosophila gene, period, with a fluorophore at the C-terminal. We also briefly describe the control experiments that need to be performed to confirm that the tagged protein is functional and live-imaging techniques to visualize the Period protein in live clock neurons within intact Drosophila brains13. The protocol described here is also broadly applicable to screen candidates for deletions and insertions of specific bits of DNA by CRISPR-Cas9 genome editing.

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one-step crispr-based strategy for endogenous gene tagging in drosophila melanogaster

The study of protein subcellular localization, dynamics, and regulation in live cells has been profoundly transformed by the advent of techniques that allow the tagging of endogenous genes to produce fluorescent fusion proteins. These methods enable researchers to visualize protein behavior in real time, providing valuable insights into their functions and interactions within the cellular environment. Many current gene tagging studies employ a two-step process where visible markers, such as eye color changes, are used to identify genetically modified organisms in the first step, and the visible marker is excised in the second step. Here, we present a one-step protocol to perform precise and rapid endogenous gene tagging in Drosophila melanogaster, which enables screening for engineered lines without the visible eye marker, offering a significant advantage over past methods. To screen for successful gene-tagging events, we employ a PCR-based technique to genotype individual flies by analyzing a small segment from their middle leg. Flies that pass the screening criteria are then used to produce stable stocks. Here, we detail the design and construction of CRISPR editing plasmids and methods for screening and confirmation of engineered lines. Together, this protocol improves the efficiency of endogenous gene tagging in Drosophila significantly and enables studies of cellular processes in vivo.

In our experiments, we typically found a success rate of ~20%-30% in screening for various endogenous tagged genes on all chromosomes (X, II, III). The efficiency of this process can vary based on several factors, such as gRNA selection, the nature of the gene, and injection quality. Here, we illustrate the results from our strategy to tag the endogenous period gene with the mNeonGreen fluorophore13. The period gene, positioned on the X chromosome...

Watch this Scientific Journal Video about One-step CRISPR-based Strategy for Endogenous Gene Tagging in Drosophila melanogaster at JoVE.com

One-step CRISPR-based Strategy for Endogenous Gene Tagging in Drosophila melanogaster

Here, we present a simplified endogenous gene tagging protocol for Drosophila, which utilizes a PCR-based technique for marker-free identification of successful genetic modifications, facilitating the development of stable knock-in lines.

One-step CRISPR-based Strategy for Endogenous Gene Tagging in Drosophila melanogaster

The study of protein subcellular localization, dynamics, and regulation in live cells has been profoundly transformed by the advent of techniques that ...

The purpose of this protocol is to generate an endogenous edited fly line. We show a fluorescent protein knock-in as an example, but it could also be applied to other knock-in, knockout, as well as mutation generation. Our protocol is a CRISPR homologous recombination based method, so there's nearly no restriction on mutation sites.Moreover, our protocol greatly reduced the time and effort involved in genotyping steps. To begin, open the fly CRISPR website to design sgRNAs for Drosophila, and enter approximately 100 base pair sequences surrounding the desired knock-in location. Select CRISPR targets with the five prime guanine option.Click the find CRISPR targets button to generate a list of possible sgRNA sequences. Then click the evaluate button for each sequence, and choose two sgRNAs that have zero off-targets. Since the natural protein expression levels are typically much lower, select fluorescent tags that are bright and photostable, and fit the experimental setup.Next, design a linker to link epitope tags to the fluorescent marker for biochemical analysis. Use a linker peptide ranging from two to 20 amino acids, comprising amino acids like glycine and serine. To design the donor vector for Drosophila, organize the DNA sequence of the linker peptide and the fluorescent tag, and ensure homologous sequences flanking both ends.Modify any PAM sequences within the donor plasmid to maintain the resulting proteins amino acid sequence. Next, position the start and stop codons depending on the tag location. For internal tagging, ensure the fluorescent tag is in frame with the coding sequence of the target gene.For N-terminus tagging, position the fluorescent tag after the endogenous start codon. For C-terminus tagging, position the fluorescent tag before the natural stop codon. Ensure to retain only a single start codon to avoid potential translation issues.Use the alternative restriction enzyme based cloning approach to insert the sgRNA sequence into the plasmid U6 Bbs1-chiRNA plasmid. After creating the sgRNA plasmid, transform the plasmid in DH5-Alpha Escherichia coli as per the manufacturer's instructions. Verify the resulting clones through Sanger sequencing, using either T3 or T7 standard sequencing primers.To create the donor plasmid, use double-stranded DNA with the desired sequence, and integrate it into the plasmid Bluesript 2SK negative multiple cloning site. Sequence the donor plasmid to ensure sgRNA or PAM sequences are correctly modified and there are no SNPs present in the plasmid. Next, take Cas9 flies injected with the constructed gRNA and donor plasmids.Anesthetize the flies with carbon dioxide, and separate male flies and virgin females. To establish fly crosses, pair each G zero male with at least two FM7L virgin females in individual narrow CT food vials. Place crossed vials in a vented incubator maintained at 25 degrees Celsius and 60%humidity until F1 flies eclose.Then anesthetize and collect at least 10 virgin female offspring from both G zero male and female crosses. Ensure each F1 fly is tagged with the details of its parentage and stored separately. To screen flies for PCR, design primers with a melting temperature of around 55 degrees Celsius that encompass the editing site of the target gene.Prepare lysis buffer for each fly sample, and dispense 10 microliters into each well of a 96 well PCR plate. Maintain the buffer at a cold temperature during the leg dissection process. Next, prepare glass capillary tubes, filling one end with sucrose agar food.Place these tubes in a 96 well deep well plate, referred to as the fly hotel. For leg dissection, position the carbon dioxide anesthetization pad under a stereo microscope. Anesthetize a candidate fly, and sever one of its middle legs.After that, immerse the leg into the lysis buffer. Hold the fly wing with a pair of fine metal forceps. Move the fly to the fly hotel, and seal the tube with yarn immediately.House the fly hotel in a vented incubator maintained at 25 degrees Celsius and 60%humidity. Submerge the severed legs in the lysis buffer solution in a thermal cycler. Organize the PCR reaction as instructed by the manufacturer, using 1.5 microliters of the fly leg lysate as the DNA source.Subject the PCR products to agarose gel electrophoresis, and interpret the results based on band size. Based on the gel results, select the positive flies from the fly hotel and individually cross them with balancer flies to produce the F2 generation. Use homozygous stocks for Sanger sequencing of the editing site to ensure accuracy.Back-cross validated fly lines with wild type flies to remove background mutations. Screen each generation using the single leg PCR protocol. Confocal microscope images of adult Drosophila brains showed period protein organized into discreet nuclear foci throughout the late night and early morning time points.Overall, this protocol is robust if your genotyping PCR primers are specific.

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