Introduction

Mosaic animals containing genetically distinct populations of cells in the same organism are useful for in vivo studies of complex biological processes. For this reason, techniques that can generate genetically labeled mosaic clones have been utilized in both vertebrates and invertebrates to study tissue-specific functions of pleiotropic genes, developmental timing, cell lineages, cell proliferation, neural wiring, and many other biological phenomena (Xu and Rubin 2012, Griffin, Binari et al. 2014, Germani, Bergantinos et al. 2018). The most popular mosaic techniques rely on site-specific recombination systems, such as FRT/Flp (Golic and Lindquist 1989, Xu and Rubin 1993) and LoxP/Cre (Zong, Espinosa et al. 2005, Henner, Ventura et al. 2013), to induce somatic recombination between homologous chromosomes. Such techniques require the introduction of recombination sites to specific locations in the genome and thus cannot be applied to unmodified chromosomes. To overcome this limitation, we recently developed a recombinase-independent mosaic technique called mosaic analysis by gRNA-induced crossing-over (MAGIC) (Allen, Koreman et al. 2021). In MAGIC, the CRISPR/Cas9 system generates double-strand breaks (DSBs) at a predefined genome location to induce homologous recombination in precursor cells during S/G2 phase. Subsequent chromosomal segregation during mitosis can result in clones homozygous for the chromosomal segments distal to the crossover site (Figure 1 - figure supplement 1A). Two variants of this technique in Drosophila, positive MAGIC (pMAGIC) and negative MAGIC (nMAGIC), label the resulting homozygous clones by the presence and absence of fluorescent markers, respectively (Figure 1 - figure supplement 1B) (Allen, Koreman et al. 2021). Like FRT/Flp-based techniques, MAGIC enables characterization of homozygous clones of lethal mutations in otherwise heterozygous animals (Allen, Koreman et al. 2021) (e.g. Figure 1 - figure supplement 1C).

Figure 1.

However, unlike FRT/Flp-based techniques, MAGIC does not require prior genetic modification of the test chromosome. Thus, it can potentially be used on any chromosome and have much wider applications. Foremost, mutations of diverse natures have been established for most Drosophila genes (Hacker, Nystedt et al. 2003, Bellen, Levis et al. 2004, Thibault, Singer et al. 2004, Staudt, Molitor et al. 2005, Bellen, Levis et al. 2011, Yamamoto, Jaiswal et al. 2014), and thousands of deficiency strains harbor deletions that collectively uncover 98.4% of the Drosophila genome (Cook, Christensen et al. 2012). However, most of these mutant chromosomes cannot be analyzed by traditional mosaic techniques due to the lack of FRT sites or incompatibility with the FRT/Flp system. Although FRT sites can be introduced onto mutant chromosomes through genetic recombination, this process is labor-intensive and time-consuming and thus is impractical at a large scale. In contrast, MAGIC can theoretically be applied to any existing stock, including those from classical mutagenesis screens and deficiency libraries, allowing convenient genome-wide mosaic screens. In addition, genes located more proximal to centromeres than existing FRT sites cannot be analyzed by FRT/Flp techniques. In comparison, MAGIC can potentially be used to study these genes because the crossover site in MAGIC can be flexibly defined by users. Lastly, given that MAGIC is compatible with wild-derived chromosomes (Allen, Koreman et al. 2021), it may be able to generate homozygous clones of a chromosome derived from a single species in an interspecific hybrid animal, allowing the study of species-related cell-cell interactions.

Despite these potentials, MAGIC has not been widely adopted by the Drosophila community. A major barrier is the lack of gRNA-marker transgenes on most chromosomal arms, which are necessary for DSB induction and fluorescent clone labeling (Allen, Koreman et al. 2021). In addition, the existing gRNA-markers suffer from several limitations, including low frequency of clone induction, weak labeling of pMAGIC clones, and suboptimal visualization of nMAGIC clones. Because of these reasons, MAGIC has only been successfully applied to a few genes in a limited number of Drosophila tissues (Allen, Koreman et al. 2021, Chen, Wang et al. 2025).

To overcome these limitations, here we first optimized gRNA-marker designs to improve clonal induction, the brightness of pMAGIC clones, and visualization of nMAGIC clones. Then we generated pMAGIC and nMAGIC gRNA-markers for all chromosomal arms and characterized their ability to generate clones. Using this kit, we demonstrate mosaic analysis of centromere-proximal genes, deficiency chromosomes, chromosomes derived from different Drosophila species in interspecific hybrid animals. This kit allows optimal clone induction in diverse cell and tissue types and should be useful for studying a wide range of biological processes.

Results

New gRNA-marker designs improve pMAGIC and nMAGIC

MAGIC relies on a gRNA-marker dual-transgene (Figure 1A and Figure 1 - figure supplement 1B) inserted in a specific chromosomal arm to both induce and visualize clones homozygous for this arm (Allen, Koreman et al. 2021). The gRNA part of the construct expresses two gRNAs ubiquitously to target a pericentromeric location on this arm. Two gRNAs targeting two sequences that are close to each other, instead of a single gRNA, are used to increase the probability of DSBs and thus the clone frequency. The marker part in pMAGIC utilizes a ubiquitously expressed Gal80 to prevent Gal4-dependent labeling of heterozygous and homozygous cells for the gRNA-marker, while allowing labeling of homozygous cells that lose the gRNA-marker. In contrast, nMAGIC uses ubiquitously expressed BFP, which results in brighter labeling of gRNA-marker homozygous cells, intermediate labeling of gRNA-marker heterozygous cells, and lack of labeling of gRNA-marker-negative homozygous cells (Figure 1 - figure supplement 1B).

We have previously developed gRNA-marker vectors for both pMAGIC and nMAGIC. However, gRNA-markers made with these vectors exhibit some limitations. First, the clone frequency can be low for certain gRNAs (Allen, Koreman et al. 2021). Second, the ubi-Gal80 in pMAGIC gRNA-markers does not completely suppress Gal4 activity in certain tissues, such as hemocytes. Third, pMAGIC clones are sometimes too dim to visualize cell morphology, such as thin dendrite and axon projections of neurons. Lastly, nMAGIC utilizes a nuclear BFP marker (nlsBFP), which does not show the cell shape and sometimes cannot mark clones effectively. Thus, to optimize MAGIC, we first sought to improve the gRNA-marker designs.

Since clone induction in MAGIC depends on gRNA-induced DNA DSBs, we asked whether a more efficient gRNA design enhances clone frequency in somatic tissues. The previous gRNA-marker vectors used a tgFE design, which contains a flip of A-U positions and a stem-loop extension (F+E) of the original gRNA scaffold and a tRNA^Gly before each gRNA targeting sequence (Figure 1A) (Allen, Koreman et al. 2021). However, we have shown that an improved Qtg2.1 design, which contains an additional extension of the second stem loop in the gRNA scaffold (gRNA2.1) and a single tRNA^Gln spacer between the two gRNAs (Figure 1A), is much more mutagenic than tgFE in somatic tissues (Koreman, Xu et al. 2021). We thus compared two pMAGIC gRNA-markers that are based on these two gRNA designs but target the same genomic sequences at cytological band 40D2 to assess their ability to induce clones in peripheral sensory neurons. When used with the same neuronal/epidermal precursor Cas9, zk-Cas9 (Allen, Koreman et al. 2021), Qtg2.1 resulted in three times more neuronal clones as compared to tgFE (Figure 1B), confirming a positive correlation between gRNA efficiency and clone frequency.

To ensure more complete suppression of Gal4 activity by Gal80 in pMAGIC, we replaced the ubi enhancer driving Gal80 expression with an αTub84B (tub) enhancer that was used in tub-Gal80 in the MARCM system (Lee and Luo 1999). When combined with the larval hemocyte marker pxn-Gal4 UAS-CD4-tdTom (Han, Song et al. 2014) (Figure 1C), ubi-Gal80 did not completely suppress the labeling of hemocytes (Figure 1D). In contrast, no labeled hemocytes could be detected in the presence of tub-Gal80 (Figure 1E), suggesting that tub-Gal80 is a better marker for pMAGIC.

To improve clone brightness in pMAGIC, we sought to destabilize Gal80 and reduce its expression, reasoning that dim clones are due to prolonged Gal80 activity after clone induction. In addition to replacing the ubi enhancer with the tub enhancer, we also introduced protein and mRNA destabilization sequences (DE) (Zubiaga, Belasco et al. 1995, Li, Zhao et al. 1998) at the 3’ end of the Gal80 coding sequence, and replaced the His2Av polyA by SV40 polyA (Figure 1F), as the latter reduces transgene expression (Han, Jan et al. 2011). By measuring the brightness of epidermal (Figure 1G) and neuronal (Figure 1H) clones induced by gRNA-40D2 and gRNA-42A4, we found that each of the three changes improved clone brightness.

Finally, to visualize cell shape in nMAGIC, we replaced nlsBFP with cytosolic BFP tagged by 3X Hemagglutinin (HA), in addition to utilizing the tub enhancer. This new design allowed us to better discern clone shapes in both the wing imaginal disc and the epidermis (Figure 1J, 1J’, and 1L), in contrast to the previous ubi-nlsBFP design (Figure 1I and 1K). To make nMAGIC compatible with more fluorescent reagents, we generated an additional vector that contains miRFP680, a far-red/infrared fluorescent protein (IFP) (Matlashov, Shcherbakova et al. 2020), in place of BFP. Wing-disc clones labeled with this marker were readily detectable in unstained tissues (Figure 1M). By measuring the sizes of homozygous gRNA-marker clones (BFP/BFP) and homozygous wildtype (WT) clones (+/+) in wing discs, we found that these two cell populations in the twin spots showed no noticeable bias in growth or viability (Figure 1O).

Thus, by altering the designs of the gRNAs and the Gal80 and BFP markers, we created new vectors optimized for more robust applications of nMAGIC and pMAGIC.

A gRNA-marker kit is established for all four chromosomes of Drosophila

To enhance the utility of MAGIC in Drosophila, we generated complete sets of pMAGIC and nMAGIC (BFP version) gRNA-markers for all four chromosomes (Figure 2A). To identify suitable gRNA target sites, we analyzed the pericentromeric sequences of X, 2L, 2R, 3L, 3R, and 4, based on three criteria (Allen, Koreman et al. 2021): conserved in closely related Drosophila species to minimize the chance of single nucleotide polymorphism, (2) located away from functionally critical regions to avoid disrupting essential processes, and (3) unique within the genome to minimize off-target effects. For each MAGIC construct, we selected a pair of non-repetitive gRNA target sequences in intergenic regions to maximize the chances of DSBs. These two sequences are closely linked to minimize the risk of large deletions. Given the variable efficiency of gRNA target sequences, we selected three pairs of gRNAs targeting three chromosomal locations for each chromosomal arm and named them according to the corresponding cytoband (Table 1).

Figure 2.

Table 1.

To evaluate the clone-induction properties of these gRNAs, we combined the pMAGIC set with zk-Cas9 and counted the number of neuronal clones in A1-A7 larval hemi-segments (Figure 2B). As expected, the clone frequency varied from gRNA to gRNA, but we were able to identify efficient gRNAs (>= 10 clones per larva) for every chromosomal arm. We previously found that different gRNAs follow the same trend of relative efficiency in different tissues (Allen, Koreman et al. 2021). Here we additionally tested nMAGIC gRNAs for 2R in the wing disc (Figure 2C) and noticed a similar trend of clone induction to that of their pMAGIC counterparts in sensory neurons (Figure 2B), suggesting that the results in sensory neurons are transferrable to other tissues.

Certain gRNAs (e.g. gRNA-40E1) exhibited very low clone frequency. Such gRNAs can be useful for inducing sparse clones in highly packed tissues such as the brain. For example, using the same ey-Cas9 (Ji, Sapar et al. 2022), the highly efficient gRNA-40D2 induced too many neuronal clones in the adult brain for morphological analysis (Figure 2D), while gRNA-40E1 gave rise to few clones, whose projection patterns were much easier to analyze (Figure 2E).

Together, the pMAGIC and nMAGIC gRNA-marker lines constitute a complete kit (Table 1) for genome-wide MAGIC applications in Drosophila.

MAGIC allows clonal analysis in diverse tissues and cell types

To determine if MAGIC can be applied to diverse tissues in Drosophila, we conducted clonal analysis in the larva using gRNA-40D2(Gal80) and several tissue-specific Cas9s. With the ubiquitous vas-cas9 (Lopez Del Amo, Juste et al. 2022) and tub-Gal4>UAS-mCD8-GFP, we readily detected clones in the larval brain (Figure 3A), proliferating tissues like eye and leg discs (Figure 3B-C), and polyploid tissues like the fat body, gut, and trachea (Figure 3D-F). Clone induction in polyploid tissues suggests that crossing-over events occurred before the last cell division. Using zk-cas9 and R38F11-Gal4>UAS-tdTomato (tdTom), we observed frequent epidermal clones (Figure 3G). Using the glia precursor gcm-Cas9 and repo-Gal4>UAS-mCD8-GFP, we detected individual glial clones in the brain (Figure 3H). The new pMAGIC gRNA-marker design allowed us to reliably induce hemocyte clones (Figure 3I).

Figure 3.

The ability to control the timing of clone induction has been instrumental for neuronal birth dating and modulation of clone frequency in traditional MARCM analysis of the Drosophila adult brain. To explore the potential of pMAGIC to serve similar purposes, we used heat-shock (hs) Cas9 (Garcia-Marques, Espinosa-Medina et al. 2020), along with gRNA-40D(Gal80) and a pan-neuronal Gal4/UAS-membrane marker combination (RabX4-Gal4>UAS-MApHS), to induce neuronal clones in the adult brain. Heat shock at 120 h after egg laying (AEL) induced too many clones for separating individual neurons (Figure 3J-J’), while later heat shock at 48 h after pupal formation (APF) produced many fewer, spatially separated clones in the central brain. These results collectively demonstrate MAGIC’s efficacy and flexibility in generating clones in diverse Drosophila tissues, indicating its value in studying gene function and cell lineage in various tissue contexts.

The neuromuscular junction (NMJ) in Drosophila larvae has been a valuable model for elucidating synaptic biology, owing to its simplicity, accessibility, and conserved features shared with mammalian excitatory synapses (Menon, Carrillo et al. 2013, Charng, Yamamoto et al. 2014, Chou, Johnson et al. 2020). Despite its popularity, the NMJ has rarely been analyzed by the MARCM technique in the literature, likely due to the difficulty of inducing clones in motor neurons. To test the effectiveness of MAGIC in analyzing gene function at the NMJ, we selected two genes crucial for synaptic function and construction: Vesicular glutamate transporter (VGlut) (Daniels, Collins et al. 2004) and bruchpilot (brp) (Kittel, Wichmann et al. 2006, Wagh, Rasse et al. 2006), null mutations of each of which exhibit recessive lethality in larvae. Combined with appropriate gRNA(Gal80) lines (42A4 for VGlut on 2R and 40D2 for brp on 2L) and tub-Gal4 UAS-CD8-GFP, zk-Cas9 induced frequent clones in type Ib boutons (Figure 4A-B”). The loss of VGlut or Brp specifically in GFP-labeled NMJs was confirmed by immunostaining. These results exemplify the value of pMAGIC for dissecting gene function in NMJ biology at the single cell level.

Figure 4.

MAGIC enables clonal analysis of pericentromeric genes, 4^th chromosome-associated mutations, and in interspecific hybrid animals

Because all FRT sites in the existing FRT/Flp mosaic system are located at some distances away from centromeres, it has not been possible to study genes located between the FRT sites and the corresponding centromeres by clonal analysis. In contrast, the gRNA target site in a MAGIC experiment can be user-selected to enable clonal analysis of any given gene. To illustrate this potential, we used gRNA-41F9 to induce homozygous mutant clones of Ecdysone receptor (EcR), which is located at 42A10 and is inaccessible to all FRT sites on 2R. EcR is a transcription factor required for neuronal remodeling during metamorphosis (Brown, Cherbas et al. 2006). In peripheral sensory neurons, EcR activation at the beginning of pupariation causes apoptosis of some dendritic arborization (da) neurons while triggering dendrite pruning of other da neurons (Williams and Truman 2005). As expected, a wildtype (WT) pMAGIC clone of class IV da (C4da) neuron exhibited complete dendrite pruning at 16 h after puparium formation (APF), accompanied by dendrite debris phagocytosed into epidermal cells (Han, Song et al. 2014) (Figure 5A). In contrast, EcR mutant da neuron clones still maintained larval dendritic arbors at 16 h APF (Figures 5B-D), instead of dying (in the case of C3da) or undergoing dendrite pruning (in the cases of C1da and C4da).

Figure 5.

Mosaic analysis of genes located on the 4^th chromosome had not been possible until recent introduction of FRT sites onto this chromosome by CRISPR-mediated knock-in (Goldsmith, Shimell et al. 2022). Despite these advances, existing mutations on FRT-lacking 4^th chromosomes still cannot be analyzed by the FRT/Flp system, given that meiotic recombination is exceedingly rare on the 4^th chromosome, preventing introduction of FRT sites onto mutant chromosomes. A valuable gene-disruption resource in Drosophila is the deficiency library consisting of strains harboring chromosomal deletions. MAGIC can potentially be used in conjunction with the deficiency library to screen for genes important in a particular biological process. To test the potential of MAGIC for analyzing mutations on the 4^th chromosome and for gene discovery with deficiencies, we generated pMAGIC C4da clones of Df(4)ED6380, a deficiency that deletes a segment between cytological bands 102B7 and 102D5. These clones show dramatically reduced dendrites compared to the WT controls (Figure 5E-5G), indicating the existence of genes important for dendrite growth in this region.

When examining nMAGIC gRNA-markers for the 4^th chromosome, we noticed uneven expression of tub-3xHA-BFP in epidermal cells and some imaginal tissues, exemplified by cells lacking detectable BFP signal (Figure 5 – figure supplement 1A and B). This variegated expression is likely due to transgenes residing in heterochromatin and is common for transgenes located on the 4^th chromosome (Riddle, Minoda et al. 2011). While this uneven expression limits the usefulness of our gRNA-markers in the corresponding epithelial tissues, Gal80 in pMAGIC gRNA-markers efficiently suppressed Gal4 activities in most neurons (Figure 5 – figure supplement 1C), conforming their effectiveness in neuronal MAGIC analysis.

Lastly, we wondered if the MAGIC system can generate clones in interspecific hybrid animals derived from D. melanogaster and D. simulans parents, given that the two species show a large degree of synteny (Chakraborty, Chang et al. 2021). The clones in these animals would contain homozygous chromosomal arms derived from a single species. To test this idea, we crossed D. melanogaster females containing gRNA-42A4(BFP); hh-cas9 to D. simulans males carrying a loss of function mutation of Lethal hybrid rescue mutation (Lhr), which ensures the viability of the hybrid male progeny (Barbash 2010). Indeed, we observed twin spots consisting of dark clones, containing only D. simulans 2R, and brighter clones, containing only D. melanogaster 2R, in wing discs of both female and male progeny (Figure 5G and 5H), demonstrating the feasibility of studying species-specific alleles in cell-cell interactions in interspecific hybrids.

Discussion

Conceptually, MAGIC is a simpler and more convenient mosaic technique compared to traditional recombinase-dependent methods. Theoretically, it can be applied directly to any existing stock, including those that are not currently compatible with the FRT/Flp system. However, the lack of gRNA-marker transgenes has prevented its wide application in Drosophila. In this study, we present a complete gRNA-marker kit that enables genome-wide MAGIC in Drosophila, removing this bottleneck. The new gRNA-markers incorporate optimized designs that improve clone frequency and labeling in both pMAGIC and nMAGIC. We further demonstrate the compatibility of MAGIC with broad tissues and cell types. More importantly, MAGIC enables mosaic analyses that could not be accomplished by existing FRT/Flp systems, such as those of pericentromeric genes, deficiency chromosomes, and interspecific homologous chromosomes. Thus, this MAGIC kit provides Drosophila researchers with greater flexibility for conducting mosaic analyses of diverse purposes.

To implement MAGIC, one needs to first choose a proper Cas9 that is expressed in the precursor cells of the targeted cell population. We show that ubiquitously expressed Cas9 lines, such as vas-Cas9 (Lopez Del Amo, Juste et al. 2022) and Act5C-Cas9 (Port, Chen et al. 2014), are sufficient to induce clones in broad tissues. Alternatively, heat shock (HS)-induced Cas9 (Garcia-Marques, Espinosa-Medina et al. 2020) can offer temporal control of clone induction in most tissues, akin to the HS-Flp commonly used in FRT-based mosaic analysis. The third option is a Cas9 driven by a tissue-specific enhancer specifically in the precursor cells of the target tissues. Examples shown in this study include ey-Cas9 expressed in many neuronal lineages (Ji, Sapar et al. 2022), gcm-Cas9 expressed in glial progenitor cells (Chen, Perry et al. 2024), zk-Cas9 expressed in precursor cells of epidermal cells, motor neurons, and somatosensory neurons (Koreman, Xu et al. 2021), and hh-Cas9 expressed in imaginal tissues (Poe, Wang et al. 2019). These Cas9 lines have the advantages of being more specific and efficient, and more convenient to use than ubiquitous or inducible ones. Multiple strategies, including enhancer fusion (Port, Chen et al. 2014, Poe, Wang et al. 2019), Gal4-to-Cas9 conversion (Chen, Yao et al. 2020, Koreman, Xu et al. 2021), and insertion of Cas9 in specific gene loci (Chen, Perry et al. 2024), have been developed to ease the generation of such tissue-specific Cas9 lines. As an ongoing effort, we have been converting Gal4 lines known to be expressed in progenitor cells into Cas9 (available at https://bdsc.indiana.edu/stocks/genome_editing/crispr_cas9.html), in the hope of making MAGIC accessible to more Drosophila tissues. It is worth noting that many Cas9 lines show leaky activity in the germline, which could mutate and inactivate the target sequence in the presence of a gRNA. Thus, it is not recommended to combine Cas9 and gRNA transgenes in the same strain as a long-term stock, unless a Cas9 inhibitor can also be introduced into this stock to suppress the germline activity.

The second component of MAGIC is a gRNA-marker line that resides on the appropriate chromosome arm and targets Cas9 to cut a pericentromeric site on the same arm. In the gRNA-marker kit, we generated three lines targeting different sites for each chromosome arm. These lines exhibit a broad range of clone frequencies in larval sensory neurons. As different gRNA-markers maintain similar relative efficiencies of clone induction across tissues (Allen, Koreman et al. 2021), one can choose a gRNA-marker more appropriate for their applications based on the target gene location and the desired clone frequency. Notably, a higher clone frequency may not always be beneficial, such as when studying the projection patterns of individual neurons in the brain. A low efficiency gRNA-marker in this kit may be more desirable in such applications.

Besides commonly conducted mosaic analysis in Drosophila, MAGIC also enables many novel analyses that are difficult or impossible to accomplish with traditional systems. One example is to analyze interactions among species-specific cells in an interspecific hybrid animal for understanding the cell biological basis of hybrid incompatibility. Interspecific crosses between WT D. melanogaster and WT D. simulans result in sex-specific lethality of F1 progeny (Barbash 2010). The cellular basis of this lethality is poorly understood. With MAGIC, one may generate mixed cell populations that are homozygous or heterozygous for species-specific alleles in the same hybrid embryo or larva, in which the relative fitness of the three cell populations can be compared. Similarly, MAGIC could complement genome-wide association studies (GWAS) using wild-derived isogenic strains and provide much more mechanistic detail. Such strains as those in the Drosophila Genetic Reference Panel (DGRP) (MacKay, Richards et al. 2012) have been very useful for discovering loci that are responsible for phenotypic variations. MAGIC can be combined with these strains to analyze the effects of homozygosity of specific variants at the cell biology level (Allen, Koreman et al. 2021). In addition, MAGIC can be combined with the Drosophila deficiency kit for rapid genome-wide mosaic screens. A deficiency exhibiting a desired phenotype in such screens can be further dissected with smaller deficiencies within the deleted region or existing mutations in candidate genes. In this way, the convenience of MAGIC could significantly accelerate phenotype-based gene discovery. Lastly, because the crossover site in MAGIC can be user-defined, one can easily generate gRNA-markers that induce somatic recombination at specific genome locations. With this feature, it is possible to induce crossover between two mutant alleles on the same chromosomal arm to generate clones homozygous for the distal mutation but heterozygous for the proximal mutation. This flexibility could also be useful for mapping undefined genetic loci that are responsible for certain phenotypes, especially in non-traditional model organisms.

Materials and methods

Fly Stocks and Husbandry

See the Key Resource Table (Supplementary File 1) for details of fly stocks used in this study. Most fly lines were either generated in the Han lab or obtained from the Bloomington Drosophila Stock Center. lethal hybrid rescue (lhr) mutant D. Simulans was a gift from Dr. Dan Barbash. All flies were grown on standard yeast-glucose medium, in a 12:12 light/dark cycle, at 25°C unless otherwise noted. Virgin males and females for mating experiments were aged for 3-5 days.

To generate and label pMAGIC clones in larval peripheral sensory neurons, we used either RabX4-Gal4 UAS-MApHS (for gRNA-markers on chromosomes X, II, and IV) or 21-7-Gal4 UAS-MApHS (for gRNA-markers on chromosome III) combined with zk-cas9. To count peripheral sensory neuronal clones on the larval body wall, third instar larvae were mounted laterally on slides and then counted in segment A1-A7 under a Nikon SMZ18 stereomicroscope. pMAGIC clones were induced and labeled by RabX4-Gal4 UAS-MApHS combined with ey-cas9 or hs-cas9 in the fly adult brain, by tub-Gal4 UAS-mCD8-GFP combined with vas-cas9 in larval brains, imaginal discs, fat bodies, guts, and trachea, by repo-Gal4 UAS-mCD8-GFP combined with gcm-cas9 in glia, by pxn-Gal4 UAS-tdTom combined with Act-cas9 in hemocytes, by tub-Gal4 UAS-mCD8-GFP combined with zk-cas9 in larval motor neurons, by R38F11-Gal4 UAS-tdTom combined with zk-cas9 in the larval epidermis. To induce nMAGIC clones in wing imaginal discs of interspecific hybrid animals, we use gRNA-42A4(BFP); hh-cas9 virgin females of D. melanogaster to cross with Lhr¹ (Brideau, Flores et al. 2006) D. simulans males.

Molecular Cloning

MAGIC gRNA cloning vectors

pMAGIC gRNA-marker vectors constructed in this study include pAC-U63-gRNA2.1-ubiGal80(DE)-His2Av, pAC-U63-gRNA2.1-tubGal80(DE)-His2Av and pAC-U63-gRNA2.1-tubGal80(DE)-SV40. To make pAC-U63-gRNA2.1-ubiGal80(DE)-His2Av, a fragment containing gRNA2.1 scaffold (gRNA2.1) and U6 3’ flanking sequence (U63fl) was first used to replace gRNA2.1-QtRNA-Gal80(TS)-gRNA2.1-U63fl in pAC-U63-QtgRNA2.1-8R (Addgene 170514) (Koreman, Xu et al. 2021). Then a destabilization sequence (DE) containing a GS linker, amino acids (AAs) 422-461 of mouse ornithine decarboxylase (Zubiaga, Belasco et al. 1995), 2X RNA destabilizing nonamer (TTATTTATTgatccTTATTTATT) (Zubiaga, Belasco et al. 1995) was added to the C-terminus of Gal80 in frame. To make pAC-U63-gRNA2.1-tubGal80(DE)-His2Av, a 2.6 kb tub enhancer was amplified from pENTR221-tubP (Chen, Wang et al. 2025) by PCR and used to replace the ubi enhancer in pAC-U63-gRNA2.1-ubiGal80(DE)-His2Av. To make pAC-U63-gRNA2.1-tubGal80(DE)-SV40, a SV40 polyA sequence was amplified from pAPIC-PHCS (Han, Jan et al. 2011) and used to replace the His2Av polyA in pAC-U63-gRNA2.1-ubiGal80(DE)-His2Av. To make the nMAGIC gRNA-marker vector pAC-U63-gRNA2.1-tubBFP(HA), the gRNA2.1-U63fl fragment was first used to replace gRNA2.1-QtRNA-BFP(TS)-gRNA2.1-U63fl in pAC-U63-QtgRNA2.1-BR (Addgene 170513) (Koreman, Xu et al. 2021). The tub enhancer was then used to replace the ubi enhancer. Lastly, a synthetic DNA fragment (GenScript) encoding 3X HA was used to replace the nuclear localization signal at the N-terminus of mTagBFP. To make nMAGIC gRNA-marker vector pAC-U63-gRNA2.1-tub-miRFP680-T2A-HO1(HA), an miRFP680-T2A-HO1 coding sequence was used to replace BFP in pAC-U63-gRNA2.1-tubBFP(HA). HO1 encodes haeme oxygenase 1 and is necessary for generating the chromophore of miRFP680. Cloning was carried out by ligation with T4 ligase or NEBuilder DNA Assembly reactions (New England Biolabs Inc.).

MAGIC gRNA expression vectors

34 gRNA expression vectors were constructed with the corresponding gRNA cloning vectors as listed in Table 1 according to published protocols (Koreman, Xu et al. 2021). Briefly, for each expression vector, two primers containing appropriate gRNA target sequences (Supplementary File 2) were used to amplify a fragment consisting of 3’ end of U6:3 promotor, the first target sequence (TS1), gRNA2.1, tRNA^Q, the second target sequence (TS2), and the beginning sequence of gRNA2.1 with pAC-U63-QtgRNA2.1-BR as the PCR template. The PCR product was then assembled with SapI-digested gRNA cloning vectors using NEBuilder DNA Assembly.

Injections were carried out by Rainbow Transgenic Flies (Camarillo, CA 93012 USA) or Genetivision (Stafford, TX 77477) to transform flies through φC31 integrase-mediated integration into attP docker sites. The 3xP3-RFP selection marker in gRNA-markers inserted at the attP[ZH-102D] site were removed by crossing to Cre.

Live Imaging

Live imaging of larval epidermal cells, sensory neurons, and hemocytes was performed as previously described (Poe, Tang et al. 2017). Animals were collected at 96 (for late third larvae) or 120 h (for wandering third instar larvae) AEL and mounted in glycerol on a slide with vacuum grease as a spacer. Animals were imaged using a Leica SP8 confocal microscope with a 40X NA1.3 oil objective, pinhole size 2 airy units, and a z-step size of 1 µm. For epidermis, images were taken at the dorsal midline of A2 and A3 segments. For dendritic arborization neurons, images were taken from A1 to A7 hemi-segments.

For imaging sensory neurons in pupae, newly formed pupae were collected and incubated at 25 °C. After 16 hours, the pupal cases were carefully removed, and the pupae were mounted dorsal side up on slides with halocarbon oil beneath a coverslip. Double-sided tape was used as a spacer. The mounted pupae were imaged using a Leica SP8 confocal microscope with a 40X NA1.3 oil objective.

Heat shock induction of neuronal clones in the adult brain

To induce heat shock, vials containing animals at the appropriate developmental stages were submerged in a 37°C water bath for 1 h. Subsequently, the vials were transferred back to a 25°C incubator until adults emerged.

Imaging of wing discs and other larval tissues

Larval dissections were performed as described previously (Poe, Wang et al. 2019). Briefly, wandering third instar larvae were dissected in a small petri dish filled with cold phosphate-buffered saline (PBS). The anterior half of the larva was inverted. To prepare imaginal discs, trachea and gut were removed. Samples were then transferred to 4% formaldehyde in PBS and fixed for 20 minutes at room temperature. After washing with PBS, the tissues were stained in DAPI (1:1000) in 0.2% PBST (PBS with 0.2% Triton X-100) for 5 minutes. The tissues were washed again in PBST and mounted in SlowFade Diamond Antifade Mountant (Thermo Fisher Scientific) on a glass slide. A coverslip was lightly pressed on top. Imaginal discs were imaged using a Leica SP8 confocal microscope with a 20X NA0.8 oil objective.

Adult brain imaging

Flies were aged for 1 day after eclosion. Brains were dissected in PBS at room temperature and then fixed in 4% paraformaldehyde in PBS with constant circular rotation for 20 minutes at room temperature. The brains were subsequently washed in 0.2% PBST and mounted on a glass slide under a glass coverslip. Vacuum grease was used as a spacer between the coverslip and the slide. Brains were imaged using a Leica SP8 confocal microscope with a 40X NA1.3 oil objective.

Larval fillet preparation

Larval fillet dissection was performed on a petri dish half-filled with PMDS gel. Wandering third instar larvae were pinned on the dish in PBS dorsal-side up and then dissected to expand the body wall. PBS was then removed and 4% formaldehyde in PBS was added to fix larvae for 15 minutes at room temperature. For VGlut staining, the fillets were fixed in Bouin’s solution for 5 minutes at room temperature. Fillets were rinsed and then washed at room temperature in PBS for 20 minutes or until the yellow color from Bouin’s solution faded. After immunostaining, the head and tail of fillets were removed, and the remaining fillets were placed in SlowFade Diamond Antifade Mountant on a glass slide. A coverslip was lightly pressed on top. Larval fillets were imaged using a Leica SP8 confocal microscope with a 40X NA1.3 oil objective.

Immunohistochemistry

Larval brains and larval fillets were rinsed and washed at room temperature in 0.2% PBST after fixation. The samples were then blocked in PBST with 5% normal donkey serum (NDS) for 1 hour before incubating with appropriate primary antibodies in the blocking solution. Brains were stained for 2 hours at room temperature, and fillets were stained overnight at 4°C. After additional rinsing and washing, the samples were incubated with secondary antibodies for 2 hours at room temperature. The samples were then rinsed and washed again before mounting and imaging. Primary antibodies used in this study are mouse anti-Repo antibody 8D12 (1:50 dilution), mouse anti-Brp antibody nc82 (1:100 dilution), rabbit anti-VGlut (1:200 dilution) (Chen, Perry et al. 2024), mouse anti-HA antibody (12CA5, 1:100), and goat anti-HRP conjugated with Cy3 (1:200). Secondary antibodies include donkey anti-mouse antibody conjugated with Cy5 (1:400) and donkey anti-rabbit antibody conjugated with Cy5 (1:400).

Image Analysis and Quantification

ImageJ analyses were conducted in Fiji/ImageJ. To compare brightness of clones in sensory neurons and epidermal cells, clones were detected based on thresholds to generate masks. The clone brightness within the masks was then measured as mean pixel intensity. To quantify clones in wing discs, 2-3 slices of optical sections in the middle of the disc were projected into a 2-dimensional (2D) image. Clones were detected based on a fixed threshold to generate masks, and the total area of clones in the wing pouch of each disc was measured.

The tracing and measurement of da neuron dendrites were done as previously described in detail (Poe, Tang et al. 2017). Briefly, dendrites were segmented using local thresholding. The segments were then converted into single-pixel-width skeletons. The total length of skeletons was calculated based on pixel distance. Normalized dendrite length was calculated as dendritic length (μm)/segment width (μm).

Statistical Analysis

One-way analysis of variance (ANOVA) with Tukey’s honest significant difference (HSD) test was used when dependent variable was normally distributed and there was approximately equal variance across groups. Student’s t-test or paired t-test was used when two groups are compared. For additional information on the number of samples, see figure legends. R studio was used for all statistical analyses.

Figure supplements

Figure 1 figure supplement 1.

Figure 5 figure supplement 1.

Data availability

All data generated or analyzed during this study are included in the manuscript and supporting files. The MAGIC gRNA-marker fly stocks generated in this study have been deposited to the Bloomington Drosophila Stock Center. Plasmids have been deposited to Addgene.

Acknowledgements

We thank Dion Dickman, Marcus Smolka, and Developmental Studies Hybridoma Bank (DSHB) for antibodies; Dan Barbash, Ethan Bier, and Bloomington Drosophila Stock Center for fly stocks; Dan Barbash and Tianzhu Xiong for advice on interspecific crosses; Claire Ho and Christina Breneman for helping to establish and test transgenes; Dan Barbash and Mariana Wolfner for feedback on the manuscript.

Additional information

Author contributions

Conceptualization: Chun Han, Yifan Shen, Ann Yeung

Data curation: Yifan Shen, Ann Yeung, Elizabeth Korn, Rhiannon Clements, Parker Jarman

Formal analysis: Yifan Shen, Ann Yeung, Rhiannon Clements

Funding acquisition: Chun Han

Investigation: Yifan Shen, Ann Yeung, Elizabeth Korn, Rhiannon Clements, Xinchen Chen, Zixian Huang, Michael Sheen, Parker Jarman

Methodology: Chun Han, Yifan Shen, Ann Yeung

Supervision: Chun Han, Yifan Shen, Ann Yeung, Rhiannon Clements, Elizabeth Korn

Resources: Yifan Shen, Payton Ditchfield, Bei Wang

Validation: Yifan Shen

Visualization: Yifan Shen, Ann Yeung, Rhiannon Clements

Writing – original draft: Chun Han, Yifan Shen

Writing – review & editing: Chun Han, Yifan Shen

Funding

HHS | NIH | NIH Office of the Director (OD) (R24OD031953)

• Chun Han

• Ann T Yeung

• Parker A Jarman

• Bei Wang

• Rhiannon Clements

• Xinchen Chen

• Zixian Huang

• Yifan Shen

• Payton Ditchfield

• Michael Sheen

• Elizabeth Korn

Additional files

Supplementary Table 1.

Supplementary Table 2.