实验方案 生物化学 生物信息学与计算生物学 生物物理学 癌症生物学 细胞生物学 发育生物学 免疫学 微生物学 分子生物学 神经科学 植物科学 干细胞 系统生物学 文章和过刊查询 现期刊物 全部刊物 Articles In Press 实验方案合集 Articles In Press 作者指南 提交流程 稿件书写指南 提交稿件 稿件评审 评审标准 AI使用指南 出版伦理 利益冲突 文章处理费 关于 关于我们 目标与定位 顾问 编辑委员会 评审委员会 领导与管理团队 开放获取政策 内容可用性和索引 期刊合作伙伴 专业组织会员 联系我们 Homogeneous Time-Resolved Fluorescence-Based Assay to Screen ADP-Ribosyl Hydrolase Inhibitors 基于均相时间分辨荧光的 ADP-核糖水解酶抑制剂筛选方法 The John Cooper School, The Woodlands, TX, USA SynRx Therapeutics, Houston, TX, USASchool of Life Sciences, Westlake University, Hangzhou, Zhejiang, China ADP ribosylation (ADPr) is a crucial post-translational modification that plays a vital role in DNA damage repair. Catalyzed by ADP ribose polymerases using NAD + as a substrate, ADPr activates DNA repair pathways rapidly, thereby maintaining genomic integrity. The involvement of ADP ribose hydrolases in this process is significant, as they hydrolyze PAR chains, facilitating the release of ADPr-modified proteins from DNA or other proteins, which is essential for subsequent DNA repair steps. This protocol outlines a high-throughput screening method for identifying inhibitors of ADP ribose hydrolases, utilizing His-Tb-conjugated and ADPr-modified His-ADP ribose polymerase as the signal donor, and GST-d2-conjugated GST-XRCC1 as the signal receptor. The detection of time-resolved fluorescence signals enables efficient evaluation of compounds with potential therapeutic activity against cancer. Protecting Against Cytoplasmic Protein Aggregates With Cytoplasmic PML Variants 利用胞质 PML 变体清除胞质蛋白聚集体 State Key Laboratory of Genetic Engineering, Department of Cell and Developmental Biology at School of Life Sciences, Fudan University, Shanghai, ChinaDepartment of Neurology, Shanghai Sixth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China State Key Laboratory of Genetic Engineering, Department of Cell and Developmental Biology at School of Life Sciences, Fudan University, Shanghai, China State Key Laboratory of Genetic Engineering, Department of Cell and Developmental Biology at School of Life Sciences, Fudan University, Shanghai, China Department of Neurology, Shanghai Sixth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China State Key Laboratory of Genetic Engineering, Department of Cell and Developmental Biology at School of Life Sciences, Fudan University, Shanghai, China State Key Laboratory of Genetic Engineering, Department of Cell and Developmental Biology at School of Life Sciences, Fudan University, Shanghai, China Cytoplasmic protein aggregation is a defining feature of multiple neurodegenerative diseases, including amyotrophic lateral sclerosis, frontotemporal dementia, Huntington’s disease, and certain forms of motor neuron disease. Recent evidence indicates that promyelocytic leukemia protein (PML) and engineered PML-derived variants can act as versatile aggregate-remodeling factors. In particular, cytoplasmically redirected PML variants recognize pathological cytoplasmic inclusions and promote their clearance. Here, we describe a protocol to generate and validate two engineered cytoplasmic PML variants: full-length mPML, which is redirected to the cytoplasm by disruption of its nuclear localization sequence, and the truncated mPML ΔRBC variant, which lacks the RING, B-box, and coiled-coil domain but retains aggregate-reducing activity. The protocol integrates fluorescence-based imaging, bimolecular fluorescence complementation, detergent-soluble/insoluble fractionation, and validation in primary rat cortical neurons. This workflow provides a practical platform for assessing cytoplasmic aggregate burden and for comparing the aggregate-remodeling activities of PML-derived constructs. It can also be adapted to other disease-associated aggregation-prone proteins, including TDP-43, SOD1, FUS, tau, polyGA, and polyQ-expanded proteins. How to Perform a Tracer Displacement BRET Assay for the TRPML1 Ion Channel TRPML1 离子通道示踪剂置换 BRET 检测方法 Micael R. Cunha Centro de Química Medicinal (CQMED), Centro de Biologia Molecular e Engenharia Genética (CBMEG), Universidade Estadual de Campinas, Campinas, Brasil Carolina M. C. Catta-Preta Centro de Química Medicinal (CQMED), Centro de Biologia Molecular e Engenharia Genética (CBMEG), Universidade Estadual de Campinas, Campinas, Brasil Katlin B. Massirer Centro de Química Medicinal (CQMED), Centro de Biologia Molecular e Engenharia Genética (CBMEG), Universidade Estadual de Campinas, Campinas, Brasil Rafael M. Couñago Centro de Química Medicinal (CQMED), Centro de Biologia Molecular e Engenharia Genética (CBMEG), Universidade Estadual de Campinas, Campinas, BrasilEshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA The transient receptor mucolipin subtype 1 (TRPML1) is a ubiquitously expressed ion channel involved in lysosomal homeostasis. Recent pharmaceutical interest in developing agonist ligands has emerged due to beneficial effects in neurodegenerative diseases. The major high-throughput screening techniques to investigate this ion channel involve fluorescent calcium imaging and electrophysiology. Despite their high capacity for screening compounds, it is well known that both methods face hurdles, such as the need for expensive, specialized equipment. Here, we present a novel technique to screen for ligands of TRPML1 using a bioluminescence resonance energy transfer (BRET) assay. This assay consists of a target engagement assay in live cells, which permits the determination of binding constants between ligands and the target of interest in equilibrium or time-dependently. We employ a full-length TRPML1 C-terminally tagged with the small bioluminescent protein nanoluciferase. This ensures the correct localization of the ion channel in the lysosomal membrane and an optimal placement of the luciferase in the cytoplasm. We also developed a cell- and lysosome-permeable fluorescent BRET tracer that gives a BRET signal only when bound to the ion channel. This new protocol allows researchers worldwide to screen compounds that would interact with TRPML1 by using any plate reader with luminescent and fluorescence filters. A SPAAC-Based Bioorthogonal Method for Verifying Protein Palmitoylation 基于 SPAAC 生物正交反应的蛋白质棕榈酰化验证方法 School of Life Sciences and Medical Engineering, Anhui University, Hefei, China School of Life Sciences and Medical Engineering, Anhui University, Hefei, China School of Life Sciences and Medical Engineering, Anhui University, Hefei, China School of Life Sciences and Medical Engineering, Anhui University, Hefei, ChinaFuyang Tianxiang Food Technology, Co., Ltd., Fuyang, Anhui, China Department of Oncology, The Third Affiliated Hospital of Anhui Medical University, Hefei, China School of Life Sciences and Medical Engineering, Anhui University, Hefei, China School of Life Sciences and Medical Engineering, Anhui University, Hefei, China Palmitoylation is a crucial post-translational modification, and bioorthogonal chemistry based on azide-alkyne cycloaddition is typically used to verify protein palmitoylation. Traditional copper-catalyzed click chemistry (CuAAC) proceeds with fast kinetics and is widely used, but it requires a copper catalyst and suffers from copper-induced toxicity and nonspecific labeling. By contrast, strain-promoted click chemistry (SPAAC) has slower kinetics but is catalyst-free, offering high specificity, low cytotoxicity, and simple operation. However, SPAAC is mostly applied to live-cell labeling and imaging of known palmitoylated proteins in the field of palmitoylation, and its use in identifying novel palmitoylated proteins is still limited. Here, we present a SPAAC-based method for detecting endogenous protein palmitoylation. Compared with CuAAC, this method eliminates the need for copper catalysts and reducing agents, thereby simplifying the procedure and reducing reagent usage. Identifying D-Group Mitogen-Activated Protein Kinases as Substrates of Arabidopsis Tyrosine Phosphatase RLPH2 Using Phospho-Tyrosine Peptide Enrichment 利用磷酸化酪氨酸肽富集技术鉴定 D 组丝裂原活化蛋白激酶为拟南芥酪氨酸磷酸酶 RLPH2 的底物 Anne-Marie Labandera INRAE, ONF, UMR BioForA, Orléans, France Brooklyn Kurucz Department of Biological Sciences, University of Calgary, Calgary, AB, Canada Department of Biological Sciences, University of Alberta, Edmonton, AB, CanadaDepartment of Biochemistry, University of Alberta, Edmonton, AB, Canada Greg B. Moorhead Department of Biological Sciences, University of Calgary, Calgary, AB, Canada Identifying substrates of protein phosphatases has been technically challenging and has hampered progress in the field of plant sciences. Small molecule inhibitors of protein phosphatases have aided in uncovering classes of phosphatases that target substrates, but that too has severe limitations. Here, we describe a method that enriches phosphorylated substrates using TiO 2 and phospho-tyrosine antibodies in phosphatase knockout lines of Arabidopsis thaliana . When compared to wild-type plants, this approach permits identification of putative substrates and specific phosphorylation sites by mass spectrometry, allowing for further in vitro or functional validation. The key to the approach described here is the use of phosphatase knockout lines to maintain substrates in a phosphorylated state and using phospho-tyrosine antibodies to enrich for tyrosine phosphorylated peptides. Endoscopic Collection and Analysis of Gastric Fluid DNA: A Liquid Biopsy Methodology for Tumor Biomarker Discovery 胃液 DNA 的内镜下采集与分析：用于发现肿瘤生物标志物的液体活检方法 Francine Carla Cadoná Masters in Health & Life Sciences, Franciscana University, Santa Maria, RS, Brazil Adriane G. Pelosof A. C. Camargo Cancer Center, São Paulo, SP, Brazil Thais F. Bartelli Department of Genetics, The University of Texas, M. D. Anderson Cancer Center, Houston, TX, USA Division of Surgical Oncology, Rutgers Cancer Institute, New Brunswick, NJ, USA Early Cancer Institute, Department of Oncology, University of Cambridge, Cambridge, MA, UK Israel Tojal da Silva A. C. Camargo Cancer Center, São Paulo, SP, Brazil Renata Pasqualini Rutgers Cancer Institute, Newark, NJ, USADivision of Cancer Biology, Department of Radiation Oncology, Rutgers New Jersey Medical School, Newark, NJ, USA Rutgers Cancer Institute, Newark, NJ, USADivision of Hematology/Oncology, Department of Medicine, Rutgers New Jersey Medical School, Newark, NJ, USA Diana Noronha Nunes Rutgers Cancer Institute, Newark, NJ, USADivision of Cancer Biology, Department of Radiation Oncology, Rutgers New Jersey Medical School, Newark, NJ, USA Emmanuel Dias-Neto Rutgers Cancer Institute, Newark, NJ, USADivision of Cancer Biology, Department of Radiation Oncology, Rutgers New Jersey Medical School, Newark, NJ, USA

Gastric cancer remains a major global health challenge, and reliable prognostic biomarkers are urgently needed to guide treatment decisions. Here, we present a simple and efficient protocol for a novel liquid biopsy approach based on quantifying gastric fluid DNA (gfDNA) collected during routine esophagogastroduodenoscopy (EGD). We have previously shown that gfDNA carries gastric cancer–derived mutations; moreover, its concentration increases with tumor progression and varies according to cancer prognosis. This empirically observed increase in gfDNA may mechanistically stem from enhanced cellular turnover, tissue disorganization, dysbiosis of the local microbiota, and/or fluctuations in immune cell infiltrates. Surprisingly, however, in patients diagnosed with gastric cancer, elevated gfDNA levels were also associated with improved survival. This paradoxical finding may be reconciled by an increased anti-tumor immune cell response in treatment-responsive gastric cancers, as well as by the contribution of non-tumoral DNA from inflammatory processes within the microenvironment of the stomach. Here, we detail a standardized protocol for gastric fluid collection and processing, designed to support downstream gfDNA quantification among other potential molecular applications. ChromID: A Protocol for Mapping Protein Chromatin Interactions in Living Cells ChromID：活细胞中蛋白质—染色质相互作用图谱的绘制方法 Richard Cardoso da Silva Genome Biology and Epigenetics, Institute of Biodynamics and Biocomplexity, Department of Biology, Utrecht University, Utrecht, The Netherlands Douwe ten Bulte Genome Biology and Epigenetics, Institute of Biodynamics and Biocomplexity, Department of Biology, Utrecht University, Utrecht, The Netherlands Genome Biology and Epigenetics, Institute of Biodynamics and Biocomplexity, Department of Biology, Utrecht University, Utrecht, The Netherlands Chromatin modifications regulate genome function by recruiting proteins that control transcription, genome organization, and DNA repair. Identifying the proteins associated with specific chromatin modifications is therefore essential for understanding how these regulatory processes operate. Traditional approaches, including chromatin immunoprecipitation and affinity purification coupled to mass spectrometry, have uncovered many chromatin-associated proteins. However, they often rely on crosslinking and chromatin fragmentation, which can disrupt native chromatin architecture and limit the detection of transient interactions. Here, we describe a proximity-labeling protocol for identifying the chromatin-dependent protein interactome associated with specific chromatin marks, termed ChromID. ChromID uses engineered chromatin readers (eCRs) fused to a promiscuous biotin ligase, which labels proteins in the immediate vicinity of the targeted chromatin mark. The protocol includes in vivo biotin labeling, nuclear extract preparation, streptavidin-based enrichment, and tryptic digestion for downstream LC-MS/MS analysis. The protocol has been validated across multiple cell types and chromatin contexts and can be extended to other chromatin-associated proteins, providing a versatile approach to profile chromatin-associated proteomes within their native cellular environment. R-Loop Modification and Quantification by Dot Blot R-loop 修饰的斑点印迹检测与定量分析 Department of Biochemistry and Winship Cancer Institute, Emory University School of Medicine, Atlanta, GA, USA Department of Biochemistry and Winship Cancer Institute, Emory University School of Medicine, Atlanta, GA, USA Blerta Xhemalçe Department of Biochemistry and Winship Cancer Institute, Emory University School of Medicine, Atlanta, GA, USA RNA modifications and their “writer,” “eraser,” and “reader” proteins are emerging as key regulators of gene expression and DNA repair through dynamically regulating RNA:DNA hybrids, or R-loops, during transcription. Therefore, it is paramount to develop rigorous techniques for accurate analysis of R-loop modifications. A convenient method for analyzing RNA modifications within total RNA is by dot blot with specific RNA modification antibodies; however, analysis of the modification of the RNA moiety within R-loops presents specific challenges. Here, we provide a detailed protocol for the production or purification of DNA containing R-loops in vitro and from cells, and the analysis of the RNA moiety modifications by dot blot. The DNA containing R-loops is treated with either mock or RNase H, which specifically degrades the RNA within RNA:DNA hybrids, to control for the specificity of the signal as originating from R-loops. Known quantities of the mock or RNase H–treated DNA are then spotted on three membranes, each blotted with antibodies that recognize double-stranded DNA, RNA:DNA hybrids, or the specific RNA modification antibodies of interest, such as m 6 A or ac 4 C. Thus, this protocol is useful to both biochemists and cell biologists with scientific interests at the intersection of R-loops and epitranscriptomics. Massively Parallel In Vitro Functional Analysis of Evolution-Derived Transcriptional Riboswitch Sequences 进化筛选获得的转录型核糖开关序列的大规模并行体外功能分析 Interdisciplinary Biological Sciences Graduate Program, Northwestern University, Evanston, IL, USADepartment of Chemical and Biological Engineering, Northwestern University, Evanston, IL, USA Julius B. Lucks Interdisciplinary Biological Sciences Graduate Program, Northwestern University, Evanston, IL, USADepartment of Chemical and Biological Engineering, Northwestern University, Evanston, IL, USA Riboswitches are structured non-coding RNA elements that regulate gene expression in response to small molecules; they serve as valuable systems in both public health and biophysical research by elucidating principles around RNA–ligand interactions, structure, and cellular function. Traditional approaches to studying riboswitches have relied on low-throughput techniques such as reporter assays or gel electrophoresis analysis of transcriptional products, which are limited in scalability. In this study, we present a high-throughput protocol to characterize the transcriptional activity of nearly 2,000 natural variants of the fluoride riboswitch in in vitro transcription. Starting with bioinformatics, we compiled a comprehensive dataset of riboswitch variants and then employed massive parallel oligonucleotide synthesis to generate an oligo pool of the riboswitch library. This pool was transcribed in vitro, converted into an Illumina-compatible next-generation sequencing (NGS) library, and analyzed to identify transcriptionally active riboswitch candidates. The workflow integrates natural riboswitch bioinformatic acquisition into a quantitative readout in a single streamlined pipeline, enabling large-scale exploration of transcriptional riboswitch function. This protocol offers a scalable method for mapping genotype-to-function relationships across transcriptional riboswitch families, accelerating the identification of functional variants for desired applications. Engineering MRI-Based Programmable Genetic Sensors Using the MAPPER Platform 利用 MAPPER 平台构建基于磁共振成像的可编程遗传传感器 Asish N. Chacko Department of Chemistry and Biochemistry, University of California, Santa Barbara, CA, USA Department of Chemistry and Biochemistry, University of California, Santa Barbara, CA, USA Raymond E. Borg Department of Chemistry, University of Hawaii Maui College, Kahului, HI, USA Department of Chemistry and Biochemistry, University of California, Santa Barbara, CA, USA Arnab Mukherjee Department of Chemistry and Biochemistry, University of California, Santa Barbara, CA, USADepartment of Chemical Engineering, University of California, Santa Barbara, CA, USA Genetically encodable reporters that produce signals detectable in deep tissues offer a powerful tool for noninvasive monitoring of molecular events in vivo. Although magnetic resonance imaging (MRI) is a standard technique for noninvasive clinical imaging, its wider application in detecting molecular activities has been constrained by the lack of programmable sensors. This limitation is in stark contrast to the widespread use of fluorescent reporter–derived sensors in cultured cells and in transparent specimens. To overcome this limitation, we recently developed the modular aquaporin-based protease-activatable probe for enhanced reporting (MAPPER) platform. This sensor engineering framework integrates a metal-free MRI reporter derived from human aquaporin-1 (hAqp1) with synthetic protease-based circuits. This integration facilitates the modular and scalable creation of a wide range of sensors by regulating protease activity through precise molecular events, such as protein–protein interactions, pharmacological inhibition, and second messenger signaling. In this paper, we present a detailed protocol for constructing and deploying sensors using the MAPPER paradigm. The protocol encompasses genetic design, lentiviral production, stable cell line generation, biochemical and microscopic validation of sensor function, diffusion-weighted MRI, and MR image analysis to quantify sensor signals in terms of the apparent diffusion coefficient. We describe two distinct MAPPER architectures: DD-MAPPER, which leverages protease-controlled protein degradation, and ER-MAPPER, which utilizes protease-controlled, subcellular trafficking. The MAPPER framework allows adaptation to various molecular targets without the need to redesign the core MRI reporter mechanism, making MAPPER a versatile platform for noninvasive biosensing in living cells and tissues. Identification of DNA-Binding Factor Enrichment in Chromatin Accessibility Data to Define a Persister Cell Signature 通过染色质可及性数据中的 DNA 结合因子富集分析界定持留细胞特征 Division of Experimental Pathology, Department of Lab Medicine and Pathology, Mayo Clinic, Rochester, MN, USA Mihai G. Dumbrava Division of Experimental Pathology, Department of Lab Medicine and Pathology, Mayo Clinic, Rochester, MN, USAMayo Medical Scientist Training Program, Mayo Clinic, Rochester, MN, USA Alexandre Gaspar-Maia Division of Experimental Pathology, Department of Lab Medicine and Pathology, Mayo Clinic, Rochester, MN, USA Wazim Mohammed Ismail Division of Experimental Pathology, Department of Lab Medicine and Pathology, Mayo Clinic, Rochester, MN, USA Chemotherapy-resistant persister cells are a major driver of cancer recurrence, yet their epigenetic basis remains poorly characterized. This protocol describes a computational pipeline for identifying DNA-binding factors (DBFs) that are enriched in accessible chromatin that collectively define a persister cell signature (PCS). Starting from single-nucleus ATAC-seq (snATAC-seq) data processed through the 10x Genomics CellRanger ARC pipeline, this protocol covers (1) the creation of a Seurat/Signac object with ATAC peaks, (2) the optional integration of DNA-binding data from the ReMap2022 database as a per-cell chromatin module assay, (3) differential accessibility analysis across clinically defined comparison groups, and (4) identifying and defining the top enriched DBFs as the PCS. This approach is applicable to any snATAC-seq dataset in which cells can be grouped by clinical response, treatment status, or resistance phenotype. Efficient and Fast Site-Directed Mutagenesis via Partially or Completely Overlapping Primer Pairs 利用部分或完全重叠引物对实现快速高效的定点突变 Paulina Varela-Castillo Rosalind and Morris Goodman Cancer Institute, McGill University, Montreal, QC, CanadaDepartment of Medicine, McGill University, Montreal, QC, Canada Arezousadat Razavi Rosalind and Morris Goodman Cancer Institute, McGill University, Montreal, QC, CanadaDepartment of Medicine, McGill University, Montreal, QC, Canada Changsheng Zhao Rosalind and Morris Goodman Cancer Institute, McGill University, Montreal, QC, CanadaDepartment of Medicine, McGill University, Montreal, QC, Canada Rosalind and Morris Goodman Cancer Institute, McGill University, Montreal, QC, CanadaDepartment of Medicine, McGill University, Montreal, QC, Canada Rosalind and Morris Goodman Cancer Institute, McGill University, Montreal, QC, Canada Xiang-Jiao Yang Rosalind and Morris Goodman Cancer Institute, McGill University, Montreal, QC, CanadaDepartment of Medicine, McGill University, Montreal, QC, Canada Site-directed mutagenesis is an indispensable molecular biology tool, but traditional methods often suffer from extended reaction time, structural limitations, and variable success rates. This article details three optimized protocols: P3a (primer pairs with 3′-overhangs, version a), P3b, and QuickChange 2.0, which rely on two highly processive DNA polymerases (Platinum SuperFi II and Q5) to accelerate and standardize plasmid engineering. The P3a method utilizes partially complementary primer pairs with distinct 3′-overhangs, achieving ~100% efficiency and enabling seamless cassette mutagenesis (insertion, deletion, and replacement). Building on this, the P3b method introduces specific thermal cycling modifications and a pre-denaturation step to overcome structural barriers resulting from GC-rich sequences. QuickChange 2.0 applies these two advanced polymerases to completely complementary primer pairs, even though the average efficiency decreases to 50%–60%. Replacing Pfu with the highly processive DNA polymerases also reduces PCR time to approximately 2 h. Thus, these new methods are more efficient and rapid than classical QuickChange mutagenesis based on Pfu polymerase. A Luciferase-Based Assay for Assessing Cap-Independent Translation in Wheat Germ Extract 基于荧光素酶检测小麦胚芽提取物中的非帽依赖性翻译活性 Department of Plant Cell Biology, Biochemistry, and Biotechnology, University of Regensburg, Regensburg, Germany Thorsten Stehlik Promega GmbH, Walldorf, Germany Department of Plant Cell Biology, Biochemistry, and Biotechnology, University of Regensburg, Regensburg, Germany Department of Plant Cell Biology, Biochemistry, and Biotechnology, University of Regensburg, Regensburg, Germany Efficient protein synthesis in eukaryotic cells typically requires a 5′ cap structure on messenger RNAs (mRNAs). However, under stress conditions or in viral infection, translation can also occur independently of the cap via internal ribosomal entry sites (IRES). IRES elements are therefore key regulators of protein expression in both viral and cellular contexts. Here, we describe a cell-free protocol to quantitatively assess cap-independent translation using wheat germ extract (WGE) and a firefly luciferase (FLuc) reporter. The protocol includes template preparation, RNA synthesis, and luminescence measurement following in vitro translation in WGE. This method enables rapid and robust comparison of translation activity under controlled conditions and can additionally be applied to evaluate mRNA modifications designed to enhance translation efficiency. Clonal Analysis in Drosophila Tissues With an Enhanced MAGIC Transgenesis Method 利用改进的 MAGIC 转基因方法开展果蝇组织克隆分析 Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, USA Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, USAWeill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY, USA Mosaic animals are highly valuable for investigating complex biological processes and cell lineages in vivo. Traditional mosaic techniques in Drosophila , such as the FRT/Flp system, rely on exogenous site-specific recombination sequences, preventing their application to unmodified mutant chromosomes or wild-derived strains. Mosaic analysis by gRNA-induced crossing-over (MAGIC) overcomes this limitation by utilizing the CRISPR/Cas9 system to generate targeted double-strand breaks (DSBs) that induce somatic homologous recombination in precursor cells. Here, we describe a comprehensive protocol for applying MAGIC with a newly developed, genome-wide MAGIC kit. This protocol utilizes optimized gRNA-markers with the Qtg2.1 scaffold for high-efficiency clone induction, alongside improved fluorescent labeling strategies for both positive MAGIC (pMAGIC) and negative MAGIC (nMAGIC). The procedure details the genetic crossing schemes, temporal induction of clones, and tissue processing for diverse Drosophila cell types. This method enables convenient mosaic analysis across all chromosomes and allows for the study of pericentromeric genes, deficiency chromosomes, and species-specific alleles in interspecific hybrids.