New gRNA-marker designs improve pMAGIC and nMAGIC.

(A) Original and new designs of gRNA-markers for pMAGIC and nMAGIC. (B) Comparison of clone frequency in larval sensory neurons between two gRNA designs. The number represents clones between A1 and A7 segments on one side of each larva. n = larvae number: tgFE (n=10), Qtg2.1 (n=10). (C-E) Labeling of hemocytes in whole 3rd instar larvae by pxnGal4>CD4-tdTom alone (C) or together with ubi-Gal80 (D) or tub-Gal80 (E). The panels on the right show enlarged views of the boxed regions. (F) Designs of Gal80 variants tested in pMAGIC gRNA-markers. (G) The brightness of epidermal clones labeled by pMAGIC gRNA-markers. n = image numbers: gRNA-40D2-uH (n = 32), gRNA-40D2-uDEH (n = 31), gRNA-42A4-uDEH (n = 52), gRNA-42A4-tDEH (n = 39), gRNA-42A4-tDES (n = 38). (H) The brightness of neuronal clones labeled by pMAGIC gRNA-markers. n = neuron numbers: gRNA-40D2-uH (n = 16), gRNA-40D2-uDEH (n = 16), gRNA-42A4-uDEH (n = 16), gRNA-42A4-tDEH (n = 15), gRNA-42A4-tDES (n = 16). (I) Portion of a larval wing disc containing nMAGIC clones visualized by nlsBFP. (J and J’) Portion of a wing disc containing nMAGIC clones labeled by cytosolic BFP (J) and HA staining (J’). (K) Epidermal clones on the larva body wall labeled by nlsBFP. (L) Epidermal clones visualized by cytosolic BFP. In all plots, black bar, mean; red bar, SD; AU, arbitrary unit. Student’s t-test in (B); one-way analysis of variance (ANOVA) and Tukey’s honest significant difference (HSD) test in (G) and (H). *p≤0.05, **p≤0.01, ***p≤0.001, ns, not significance. For (C-E), scale bar, 300 µm. For (I-M), scale bar, 100 µm.

A genome-wide gRNA-marker kit suits diverse needs of clone frequency.

(A) Scheme of gRNA-marker insertion sites and target sites on Drosophila chromosomes. (B) Comparison of clone frequencies of all pMAGIC gRNA-markers in larval sensory neurons, clones are labeled using RabX4-Gal4 UAS-MApHs (for Chromosome X, II and IV) or 21-7-Gal4 UAS-MApHs (for Chromosome III). n = larvae number: X2 (n = 10), 20F2 (n = 10), 20F1(n = 10), 40D2 (n = 20), 40D4 (n = 10), 40E1 (n = 10), 41F9 (n = 20), 41F11 (n = 10), 42A4 (n = 10), 80C1 (n = 20), 80C2 (n = 14), 80F5 (n = 15), 81F (n = 10), 82A4 (n = 10), 82C3 (n = 10), 101F1a (n = 10), 101F1b (n = 10), 101F1c (n = 10). (C) Comparison of clone areas in larval wing discs labeled by nMAGIC gRNA-markers on 2R. n = wing disc number: 41F9 (n = 14), 41F11 (n = 16), 42A4 (n = 15). (D and E) Neuronal clones in the central part of the adult brain induced by pMAGIC gRNA-markers gRNA-40D2 (D) and gRNA-40E1 (E). In all plots, black bar, mean; red bar, SD. One-way ANOVA and Tukey’s HSD test. *p≤0.05, **p≤0.01, ***p≤0.001, ns, not significance. For (D) and (E), scale bar 100 µm.

gRNA-marker collection

MAGIC allows clonal analysis in diverse tissues and cell types.

(A-F) pMAGIC clones induced in different tissues by vas-Cas9 gRNA-40D2(Gal80) and labeled by tub-Gal4 UAS-CD8-GFP (green). DAPI staining (white) shows all nuclei. (G) A pMAGIC epidermal clone on the larval body wall induced by zk-Cas9 gRNA-40D2(Gal80) and labeled by R38F11-Gal4 UAS-tdTom (green). Epidermal junctions are labeled by α-Catenin-GFP (white). (H) pMAGIC glia clones in the larval brain induced by gcm-Cas9 gRNA-40D2(Gal80) and labeled by repo-Gal4 UAS-CD8-GFP (green). Glial nuclei are labeled by Repo staining (white). (I) pMAGIC hemocyte clones induced by Act-Cas9 gRNA-40D2(Gal80) and labeled by pxn-Gal4 UAS-tdTom. For figure A, D-F, H, scale bar 100 µm. For figure B-C, G, scale bar 50 µm. For Figure I, scale bar 25 µm.

MAGIC facilitates clonal analysis at the NMJ.

(A-A”) pMAGIC clones of VGlut1 mutation in motor neurons at the neuromuscular junction. Clones were induced by zk-Cas9 gRNA-40D2(Gal80) and labeled by tub-Gal4 UAS-CD8-GFP., The loss of VGlut is confirmed by VGlut staining. The mutant clones are outlined in (A”). (B-B”) A pMAGIC clone of brpd09839 mutation in a motor neuron at the neuromuscular junction. Clones were induced by zk-Cas9 gRNA-42A4(Gal80) and labeled by tub-Gal4 UAS-CD8-GFP. The loss of Brp is confirmed by Brp staining. The mutant clone is outlined in (B”). In both experiments, HRP staining shows all axons. Scale bars, 10 µm.

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

(A) A WT pMAGIC class IV da neuron clone exhibiting complete dendrite pruning at 16 hours APF. (B-D) pMAGIC clones of EcRM554fs mutation in da neurons imaged at 16 hours APF, exhibiting the lack of pruning (B and D) or apoptosis (C). In (A-D), the clones were induced by zk-cas9 with gRNA-41F9(Gal80) and labeled by RabX4-Gal4 UAS-MApHS. Neuronal cell bodies are indicated by arrows. MApHS contains pHluorin and tdTom [19], but only tdTom signals are shown. The signals in epidermal cells (A) were due to engulfment of pruned dendrites by epidermal cells [19]. (E and F) WT (E) and Df(4)ED6380 (F) pMAGIC clones in C4da neurons induced by zk-cas9 gRNA-101Fc(Gal80) and labeled by RabX4-Gal4 UAS-MApHS. Only tdTom signals are shown. (G) Normalized dendrite length of WT clones and deficiency clones. Black bar, mean; red bar, SD. Student’s t-test. ***p≤0.001. (H) Scheme for interspecific crosses between D. melanogaster (D.m) and D. simulans (D.s). (I and J) Wing discs from male (I) and female (J) progeny carrying clones. Scale bars, 50 µm.

Transgenic markers on the 4th chromosome show uneven expression.

(A-B) Representative epidermal (A) and wing disc (B) images showing uneven expression of gRNA-101F1c(BFP) inserted at attP102D on the 4th chromosome. Yellow arrowheads point to cells lacking BFP expression. Scale bars, 50 µm. (C) Frequency of labeled neurons by indicated gRNA(Gal80) in the absence and presence of Cas9. The zk-Cas9 dataset is the same as that for chromosome 4 in Figure 2B. Black bar, mean; red bar, SD. One-way ANOVA and HSD test. ***p≤0.001.