Figure 1. Conditional CRISPR mutagenesis with pCFD6 is robust across target genes and tissues.

(A) Schematic overview of the workflow. To perform tissue-specific targeted mutagenesis flies transgenic for a specific Gal4 driver (X-Gal4) and UAS-Cas9 are crossed to flies with a UAS-sgRNA transgene. Offspring from this cross express Cas9 and sgRNAs in Gal4 expressing cells, leading to mutagenesis of the target gene. (B) Schematic of gene editing outcomes typically observed with a single, ubiquitous sgRNA (lower left) or a conditional array of several sgRNAs (lower right). Leaky expression, that is expression in the absence of Gal4, from conditional Cas9 transgenes gives rise to ectopic mutagenesis in combination with ubiquitous, but not conditional, sgRNAs. Gene editing in tissues typically results in genetic mosaics, which can be enriched for bi-allelic knock-out cells through sgRNA multiplexing. (C) Conditional CRISPR mutagenesis in wing imaginal discs with nub-Gal4 in the wing pouch. Gene editing with pCFD6-arm^2x results in loss of Arm protein exclusively in the Gal4 expression domain in nearly all cells. Control animals express the nub-Gal4 driver and UAS-cas9.P2. Scale bar = 50 µm. (D) Conditional CRISPR mutagenesis of Notch in intestinal stem cells drives tumor formation in the midgut. esg^ts (esg-Gal4 tub-Gal80^ts) was used to repress expression of UAS-cas9.P2 and pCFD6-N^2x until adult stages. Mutagenesis was induced for 5 days at 29°C and flies were returned to 18°C to avoid Cas9.P2 mediated toxicity. Posterior midguts 15 days after induction of mutagenesis are shown. esg^ts UAS-cas9.P2 pCFD6-N^2x tissue shows an accumulation of stem cells (DNA marked in cyan) and an increase in mitotic cells (pHistone3 in magenta). Quantification of phenotypes are shown in Figure 1—figure supplement 2. Control genotype is esg^ts UAS-cas9.P2 pCFD6-se^2x. Scale bar = 50 µm. (E) Mutagenesis of neur in pnr-Gal4 UAS-cas9.P2 pCFD6-neur^2x animals results in loss of thoracic bristles along the dorsal midline, where pnr-Gal4 is expressed. Note the tissue patch that retains bristles, reflecting mosaic mutagenesis. (F) Mutagenesis of the pigmentation gene se in the eye. GMR-Gal4 UAS-casp.P2 pCFD6-se^2x animals develop a uniform dark eye coloration. Control animals in (E) and (F) express the respective Gal4 driver and UAS-cas9.P2 pCFD6-Sfp24C1^2x. (G) pCFD6 mediated mutagenesis in the germline. Shown is a summary of the mutational status at each sgRNA target site in individual F1 flies. nos-Gal4VP16 UAS-cas9.P1 pCFD6 flies expressing sgRNAs targeting the indicated essential genes are viable, demonstrating germline restricted mutagenesis, and transmit mutant alleles to their offspring. Shown is a summary of the mutational status at each sgRNA target site in individual flies. All lines, except the one targeting Dpp (asterisk), transmit mutant alleles to the majority of offspring. Flies expressing sgRNAs targeting Dpp in the germline produce few viable offspring and transmitted only a single, in-frame, mutation out of 11 analysed offspring. The same sgRNA construct results in highly efficient mutagenesis in somatic tissues (see Figure 4), consistent with haploinsufficiency of Dpp in the Drosophila embryo.

Figure 1—figure supplement 1. Efficient conditional CRISPR mutagenesis in various Drosophila tissues.

(A) CRISPR mutagenesis of smo in the posterior compartment of the wing imaginal disc. Smo protein was detected by immunohistochemistry. Smo is normally expressed in all wing disc cells, but protein levels are higher in the posterior compartment (see Control (hh-Gal4 UAS-cas9.P2)). In hh-Gal4 UAS-cas9.P2 pCFD6-smo^2x wing disc cells in the posterior compartment express no or reduced levels of Smo, presumably reflecting cells containing only one or no functional smo alleles. (B) CRISPR mutagenesis of sens in the dorsal compartment of wing imaginal discs with ap-Gal4 leads to a loss of Sens expression in most, but not all cells. (C) Mutagenesis of y in the dorsal abdomen. In pnr-Gal4 UAS-cas9.P2 pCFD6-y^2x animals, cuticle coloration is uniformly changed in a broad stripe centred around the dorsal midline, compared to control animals (pnr-Gal4 UAS-cas9.P2 pCFD6-Sfp24C1^2x). Note that the strong phenotype mediated by pCFD6-y^2x is in line with the high levels of mutagenesis with this construct reported in Figure 3—figure supplement 1B.

Figure 1—figure supplement 2. Qualitative differences between CRISPR mutagenesis and RNAi knock-down of Notch in the Drosophila midgut.

(A–H) Representative images of the posterior midgut of male or female esg^ts > w1118 (A, E), esg^ts > N RNAi^KK (B, F), esg^ts > N RNAi^Trip (C, G) and esg^ts > UAS N^DN animals, stained with pH3 antibody in red to mark mitotic cells. esg-GFP is shown in green, nuclei are stained with DAPI (blue). Animals were raised at 18°C to adulthood and then incubated at 29°C for 15 days. (I) Quantification of pH3-positive cells per adult midgut of the indicated genotypes after 15 days at 29°C. (J–O) Representative images of the posterior midgut of male or female esg^ts UAS-Cas9.P2 pCFD6-se^2x (J, M), esg^ts UAS-Cas9.P2 pCFD3-N (K, N) and esg^ts UAS-Cas9.P2 pCFD6-N^2x (L, O) flies stained with pH3 antibody in red and nuclei are stained with DAPI (blue). Animals were raised at 18°C, after eclosion mutagenesis was induced for 5 days at 29°C, followed by 18°C for 15 days. Unlike in the RNAi condition, no GFP is visible due to repression by Gal80 at 18°C. (P) Quantification of pH3-positive cells per adult midgut of the indicated genotypes at 18°C for 30 days after inducing CRISPR/Cas9 mediated mutagenesis. Significant differences in the number of pH3-positive cells between Notch RNAi or knock-out group (esg^ts > N RNAi^KK, esg^ts > N RNAi^trip, esg^ts;UAS-Cas9 >N gRNA ^PCFD3 or esg^ts;UAS-Cas9 >N-gRNA¹¹⁸⁴) and the control groups. (esg^ts > w1118 or esg^ts;UAS-Cas9 >sepia gRNA) are indicated with asterisks (*p<0.05; **p<0.01; ***p<0.001; two-tailed T-test) Scale bars: 30 µm (A–H, J–O). Note that the total number of mitotic cells in the RNAi and CRISPR conditions cannot be directly compared, as animals were raised at different temperatures.