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...

<ol>
	<li>Chudakov, D. M., Matz, M. V., Lukyanov, S., Lukyanov, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+proteins+and+their+applications+in+imaging+living+cells+and+tissues.">Fluorescent proteins and their applications in imaging living cells and tissues.</a> Physiol Rev. 90 (3), 1103-1163 (2010).</li><li>Costantini, L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+palette+of+fluorescent+proteins+optimized+for+diverse+cellular+environments.">A palette of fluorescent proteins optimized for diverse cellular environments.</a> Nat Commun. 6, 7670 (2015).</li><li>Gibson, T. J., Seiler, M., Veitia, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transience+of+transient+overexpression.">The transience of transient overexpression.</a> Nat Methods. 10 (8), 715-721 (2013).</li><li>Huh, W. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+analysis+of+protein+localization+in+budding+yeast.">Global analysis of protein localization in budding yeast.</a> Nature. 425 (6959), 686-691 (2003).</li><li>Venken, K. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MiMIC:+a+highly+versatile+transposon+insertion+resource+for+engineering+Drosophila+melanogaster+genes.">MiMIC: a highly versatile transposon insertion resource for engineering Drosophila melanogaster genes.</a> Nat Methods. 8 (9), 737-743 (2011).</li><li>Quinones-Coello, A. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+strategies+for+protein+trapping+in+Drosophila.">Exploring strategies for protein trapping in Drosophila.</a> Genetics. 175 (3), 1089-1104 (2007).</li><li>Sarov, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genome-wide+resource+for+the+analysis+of+protein+localisation+in+Drosophila.">A genome-wide resource for the analysis of protein localisation in Drosophila.</a> Elife. 5, e12068 (2016).</li><li>Ran, F. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+engineering+using+the+CRISPR-Cas9+system.">Genome engineering using the CRISPR-Cas9 system.</a> Nat Protoc. 8, 2281-2308 (2013).</li><li>Port, F., Chen, H. M., Lee, T., Bullock, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+CRISPR/Cas+tools+for+efficient+germline+and+somatic+genome+engineering+in+Drosophila.">Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila.</a> Proc Natl Acad Sci U S A. 111 (29), E2967-E2976 (2014).</li><li>Gratz, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+specific+and+efficient+CRISPR/Cas9-catalyzed+homology-directed+repair+in+Drosophila.">Highly specific and efficient CRISPR/Cas9-catalyzed homology-directed repair in Drosophila.</a> Genetics. 196 (4), 961-971 (2014).</li><li>Kanca, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+CRISPR-based+strategy+to+insert+small+and+large+fragments+of+DNA+using+short+homology+arms.">An efficient CRISPR-based strategy to insert small and large fragments of DNA using short homology arms.</a> Elife. 8, e51539 (2019).</li><li>Lamb, A. M., Walker, E. A., Wittkopp, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tools+and+strategies+for+scarless+allele+replacement+in+Drosophila+using+CRISPR/Cas9.">Tools and strategies for scarless allele replacement in Drosophila using CRISPR/Cas9.</a> Fly (Austin). 11 (1), 53-64 (2017).</li><li>Xiao, Y., Yuan, Y., Jimenez, M., Soni, N., Yadlapalli, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clock+proteins+regulate+spatiotemporal+organization+of+clock+genes+to+control+circadian+rhythms.">Clock proteins regulate spatiotemporal organization of clock genes to control circadian rhythms.</a> Proc Natl Acad Sci U S A. 118 (28), e2019756118 (2021).</li><li>Costantini, L. M., Fossati, M., Francolini, M., Snapp, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+the+tendency+of+fluorescent+proteins+to+oligomerize+under+physiologic+conditions.">Assessing the tendency of fluorescent proteins to oligomerize under physiologic conditions.</a> Traffic. 13 (5), 643-649 (2012).</li><li>Snapp, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+proteins:+a+cell+biologist's+user+guide.">Fluorescent proteins: a cell biologist's user guide.</a> Trends Cell Biol. 19 (11), 649-655 (2009).</li><li>Kanca, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+expanded+toolkit+for+Drosophila+gene+tagging+using+synthesized+homology+donor+constructs+for+CRISPR-mediated+homologous+recombination.">An expanded toolkit for Drosophila gene tagging using synthesized homology donor constructs for CRISPR-mediated homologous recombination.</a> Elife. 11, e76077 (2022).</li><li>Nitabach, M. N., Taghert, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+of+the+Drosophila+circadian+control+circuit.">Organization of the Drosophila circadian control circuit.</a> Curr Biol. 18 (2), R84-R93 (2008).</li><li>Carvalho, G. B., Ja, W. W., Benzer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-lethal+PCR+genotyping+of+single+Drosophila.">Non-lethal PCR genotyping of single Drosophila.</a> Biotechniques. 46 (4), 312-314 (2009).</li><li>Gummadova, J. O., Coutts, G. A., Glossop, N. R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+Drosophila+Clock+promoter+reveals+heterogeneity+in+expression+between+subgroups+of+central+oscillator+cells+and+identifies+a+novel+enhancer+region.">Analysis of the Drosophila Clock promoter reveals heterogeneity in expression between subgroups of central oscillator cells and identifies a novel enhancer region.</a> J Biol Rhythms. 24 (5), 353-367 (2009).</li></ol>

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>
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			<td>701354</td>
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			<td>Agar</td>
			<td>Fisher Scientific</td>
			<td>BP1423-500</td>
			<td></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>Fisher Scientific</td>
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			<td>EcoRI-HF</td>
			<td>NEB</td>
			<td>R3101S</td>
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			<td>Hydrochloric Acid</td>
			<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>Sodium Chloride</td>
			<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>
			<td></td>
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		<tr>
			<td>XbaI</td>
			<td>NEB</td>
			<td>R0145S</td>
			<td></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

458 조회수.  University of Michigan. 이 프로토콜의 목적은 내인성 편집 플라이 라인을 생성하는 것입니다. 형광 단백질 knock-in을 예로 들었지만, 다른 knock-in, knockout 및 돌연변이 생성에도 적용할 수 있습니다. 당사의 프로토콜은 CRISPR 상동 재조합 기반 방법이므로 돌연변이 부위에 대한 제한이 거의 없습니다.또한, 당사의 프로토콜은 유전형 분석 단계와 관련된 시간과 노력을 크게 줄였습니다. 먼저 fly CRI...