Generation of Mouse Strains using CRISPR/Cas9

GD^Δ and SV-Enh^Δ mouse strains were generated by microinjection or electroporation of CRISPR/Cas9 components into fertilized mouse eggs. Single guide (sg) RNAs located 5’ and 3’ of the genomic sequence of interest were designed using CHOPCHOP¹²¹ or CRISPOR¹²² (http://crispor.tefor.net/), respectively. The GD^Δ allele was engineered as previously described¹²³. Briefly, a mix containing Cas9 mRNA (100 ng/μl) and two single guide RNAs (sgRNAs) (25 ng/μl each) in injection buffer (10 mM Tris, pH 7.5; 0.1 mM EDTA) was microinjected into the cytoplasm of fertilized FVB/NJ (Jackson Laboratory; Strain#:001800) strain oocytes obtained from the oviducts of super-ovulated 7–8 weeks old FVB/NJ females mated to 7–8 weeks old FVB/NJ males. The injected embryos were cultured in M16 medium supplemented with amino acids at 37 °C under 5% CO2 and transferred into the uteri of pseudo-pregnant CD-1 (Charles River Laboratories; Strain Code: 022) surrogate mothers on the same day. The SV-Enh^Δ allele was engineered using CRISPR-EZ⁷⁰ at the Center of Transgenic Models (CTM) of the University of Basel. HiFi Cas9 Nuclease V3 (16 μM) enzyme was incubated with cr:tracrRNA (8 μM each) in a 1:1 molar ratio (IDT) in Hepes-KCl buffer. Minimal Essential Medium (MEM) was added to get a final concentration of 8uM for the electroporation. Following incubation in M16 (Sigma/Merck M7292) with sodium bicarbonate and lactic acid at 37 °C 5%CO2, FVB/NRj (Janvier Labs) strain mouse oocytes obtained from the oviducts of super-ovulated FVB/NRj females (8 weeks) mated to FVB/NRj males (8 weeks or older) were electroporated with the RNP mix. Subsequently, embryos were cultured again in supplemented M16 medium until transferred into the oviduct of pseudo-pregnant Swiss Albino (Janvier Labs; Strain Name: RjOrl:SWISS) females on the same day. CRISPR-derived founder mice (F0) were genotyped using PCR with High Fidelity Platinum Taq Polymerase (Thermo Fisher Scientific) (GD^Δ line) or conventional Taq Polymerase (SV-Enh^Δ) to identify non-homologous end-joining (NHEJ)-generated deletion breakpoints. Sanger sequencing was used to identify and confirm deletion breakpoints in F0 and F1 mice (see Supplementary Figs. 4 and 5 for genotyping strategy, primers, genotyping PCR and Sanger sequencing).