(A) Schematic of the Cas9/sgRNA-targeting sites in Tet1, 2, and 3. The sgRNA-targeting sequence is underlined, and the protospacer-adjacent motif (PAM) sequence is labeled in green. The restriction sites at the target regions are bold and capitalized. Restriction enzymes used for RFLP and Southern blot analysis are shown, and the Southern blot probes are shown as orange boxes.
(B) Surveyor assay for Cas9-mediated cleavage at Tet1, 2, and 3 loci in ES cells.
(C) Genotyping of triple-targeted ES cells, clones 51, 52, and 53 are shown. Upper: RFLP analysis. Tet1 PCR products were digested with SacI, Tet2 PCR products were digested with EcoRV, and Tet3 PCR products were digested with XhoI. Lower: Southern blot analysis. For the Tet1 locus, SacI digested genomic DNA was hybridized with a 5′ probe. Expected fragment size: WT = 5.8 kb, TM (targeted mutation) = 6.4 kb. For the Tet2 locus, SacI, and EcoRV double-digested genomic DNA was hybridized with a 3′ probe. Expected fragment size: WT = 4.3 kb, TM = 5.6 kb. For the Tet3 locus, BamHI and XhoI double-digested genomic DNA was hybridized with a 5′ probe. Expected fragment size: WT = 3.2 kb, TM = 8.1 kb.
(D) The sequence of six mutant alleles in triple-targeted ES cell clone 14 and 41. PAM sequence is labeled in red.
(E) Analysis of 5hmC levels in DNA isolated from triple-targeted ES cell clones by dot blot assay using anti-5hmC antibody. A previously characterized DKO clone derived using traditional method is used as a control. See also .

(A) Genotyping of Tet1 single-targeted mice.
(B) Upper: genotyping of Tet2 single-targeted mice. RFLP analysis; lower: Southern blot analysis.
(C) The sequence of both alleles of targeted gene in Tet1 biallelic mutant mouse 2 and Tet2 biallelic mutant mouse 4.
(D) Genotyping of Tet1/Tet2 double-mutant mice. Analysis of mice 1 to 12 is shown. Upper: RFLP analysis; lower: southern blot analysis. The Tet1 locus is displayed on the left and the Tet2 locus on the right.
(E) The sequence of four mutant alleles from double-mutant mouse 9 and 10. PAM sequences are labeled in red.
(F) Three-week-old double-mutant mice. All RFLP and Southern digestions and probes are the same as those used in . See also .

(A) Schematic of the oligo-targeting sites at Tet1 and Tet2 loci. The sgRNA-targeting sequence is underlined, and the PAM sequence is labeled in green. Oligo targeting each gene is shown under the target site, with 2 bp changes labeled in red. Restriction enzyme sites used for RFLP analysis are bold and capitalized.
(B) RFLP analysis of double oligo injection mice with HDR-mediated targeting at the Tet1 and Tet2 loci.
(C) The sequences of both alleles of Tet1 and Tet2 in mouse 5 and 7 show simultaneously HDR-mediated targeting at one allele or two alleles of each gene, and NHEJ-mediated disruption at the other alleles. See also .

(A) Multiple gene targeting in ES cells.
(B) One-step generation of mice with multiple mutations. Upper: multiple targeted mutations with random indels introduced through NHEJ. Lower: multiple predefined mutations introduced through HDR-mediated repair.