2C-Cas9: a versatile tool for clonal analysis of gene function

Labeling of mutant single cells by combining the 2C-Cas9 and the Brainbow methodologies. (A) Microinjection of the pUAS:Cas9T2ACre;U6sgRNA1;U6sgRNA2 construct in the double transgenic embryos Tg(promoter:Gal4) × Tg(UAS:Brb1.0L)s1997t leads to synthesis of both Cas9 and Cre. In Gal4-expressing cells, Cas9 promotes targeted cleavage of the chosen genomic locus. At the same time, Cre activity induces stochastic recombination of the UAS:Brb1.0L cassette, thus promoting a switch from the default tdTomato to YFP or Cerulean fluorescence in the same cells. (B) Recombination of the UAS:Brb1.0L cassette in a tissue-specific pattern induced by injection of the pUAS:Cas9T2ACre;U6sgRNA1;U6sgRNA2 plasmid. No target sequence was inserted in the vector prior to injection. (Left panel) Multicolor labeling of muscle cells in the Tg(rpl5b:Gal4) transgenic line. (Right panel) Single YFP motor neuron labeled by expression of UAS:Cas9T2ACre;U6sgRNA1;U6sgRNA2 transgene in the double transgenic embryos Tg(mnx1:Gal4) × Tg(UAS:Brb1.0L)s1997t. tdTomato marks motor neurons that do not express the UAS transgenic cassette. Scale bar = 50 µm. (C) Double transgenic Tg(isl2b:Gal4; kif5aa*^162/+-) × Tg(UAS:Brb1.0L)s1997t larva, injected with pUAS:Cas9T2ACre;U6:Kif5aasgRNA1;U6:Kif5aasgRNA2. isl2b:Gal4 drives the expression of UAS:Brb1.0L transgene in RGC axons in the optic tectum. Note that the axon marked by cerulean expression, and therefore by Cre recombinase activity, shows reduced arbor size as expected from the phenotype of kif5aa^−/− RGCs transplanted in wild-type host (Auer et al. 2015). Scale bar = 30 µm. (D) Quantification of total branch length in tdTomato-positive wild-type and potentially mutant Cerulean- or YFP-positive RGC axons (analyzed in kif5aa^+/+: [*] P-value < 0.05 following Wilcoxon Mann–Whitney test, and kif5aa*^162/+: [***] P-value < 0.001 following Wilcoxon Mann–Whitney test). Data are represented as mean ± SEM. (E) Table describing the percentage of RGCs displaying a wild-type-like (axonal length > 200 µm) and a mutant-like (axonal length < 200 µm) phenotype in the tectum of double transgenic Tg(isl2b:Gal4; kif5aa*^162/+-) × Tg(UAS:Brb1.0L)s1997t larva, injected with pUAS:Cas9T2ACre;U6:Kif5aasgRNA1;U6:Kif5aasgRNA2. (First row) tdTomato-positive cells (UAS:Cas9T2ACre;U6:Kif5aasgRNA1;U6:Kif5aasgRNA2-negative). (Second row) Cerulean- or YFP-positive neurons (UAS:Cas9T2ACre;U6:Kif5aasgRNA1;U6:Kif5aasgRNA2-positive) in kif5aa^+/+fish. (Third row) Cerulean- or YFP-positive neurons (UAS:Cas9T2ACre;U6:Kif5aasgRNA1;U6:Kif5aasgRNA2-positive) in kif5aa*^162/+fish.