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 authors declare no competing interests.

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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			<td>5-alpha Competent&#160;E. coli&#160;(High Efficiency)</td>
			<td>NEB</td>
			<td>C2987H</td>
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			<td>96-well deep well plate</td>
			<td>BRAND</td>
			<td>701354</td>
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			<td>Agar</td>
			<td>Fisher Scientific</td>
			<td>BP1423-500</td>
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			<td>BbsI-HF</td>
			<td>NEB</td>
			<td>R3539S</td>
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			<td>DreamTaq Green PCR Master Mix (2X)</td>
			<td>Thermo Scientific</td>
			<td>K1081</td>
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			<td>EcoRI-HF</td>
			<td>NEB</td>
			<td>R3101S</td>
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			<td>Fisher Scientific</td>
			<td>A142-212</td>
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			<td>Prolong Glass Antifade Mounting Medium</td>
			<td>Invitrogen</td>
			<td>P36982</td>
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			<td>Proteinase K</td>
			<td>QIAGEN</td>
			<td>19131</td>
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			<td>Schneider&#39;s Drosophila Medium</td>
			<td>Gibco</td>
			<td>21720001</td>
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			<td>Fisher Scientific</td>
			<td>S271-500</td>
			<td></td>
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		<tr>
			<td>T4 DNA ligase</td>
			<td>NEB</td>
			<td>M0202S</td>
			<td></td>
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			<td>Tris Base</td>
			<td>Fisher Scientific</td>
			<td>BP152-500</td>
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			<td>XbaI</td>
			<td>NEB</td>
			<td>R0145S</td>
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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 ...

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

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Neuroscience

476 次观看.  University of Michigan. 该协议的目的是生成内源性编辑的飞线。我们以荧光蛋白敲入为例，但它也可以应用于其他敲入、敲除以及突变生成。我们的方案是基于CRISPR同源重组的方法，因此对突变位点几乎没有限制。此外，我们的方案大大减少了基因分型步骤所涉及的时间和精力。首先，打开苍蝇CRISPR网站，为果蝇设计sgRNA，并输入围绕所需敲入位置的大约100个碱基对序列。使用五个主要鸟嘌?...