Supporting InformationWefers et al. 10.1073/pnas.1218721110SI Materials and MethodsSoftware for Transcription Activator-Like Effector-Binding Site Detection.Software for the identification of transcription activator-like (TAL)effector-binding sites is written in Perl 5.8.8 programming languageandusesBioperl libraries for parsing inputDNAsequences (1).TheTAL effector nuclease (TALEN)–Designer software takes DNAsequences as input and searches for 5′-to-3′ oriented homo- andheteromeric potential binding motifs on opposite strands of theinput DNA sequences. The TAL effector-binding sites have eithera length of 15 nt or a variable length of 13–15 nt. TheWeb interface(www.talen-design.de) takes one or multiple FASTA-formattedDNA sequences as input. The Web interface is running on anApache Web Server 2.2.3 on SUSE Linux Enterprise Server 10Service Pack 2 (SUSE LINUX Products GmbH, Nürnberg, Ger-many). The user can select one of the search pattern as describedabove and can customize the length of the spacer between the TALeffector-binding sites in a range of 13–17 nt. Potential binding sitesare only detected, if they are preceded by a thymine in position -1of theDNA sequence on opposite strands. Potential bindingmotifson the reverse complement of the input DNA sequences are au-tomatically identified as well. Replied to the user are the identifierand length of the input DNA sequences, theDNA sequences of themotif identified with colored heteromeric binding sites and thespacer in between, the total length of the TAL effector-bindingmotif, start and end coordinates of the motif with regard tothe input sequence where the motif has been identified, and theguanine content of the TAL effector-binding motifs.The software for genome-wide searches for TAL effector-

binding sites uses theEnsembl application program interface (API)(Release 6; www.ensembl.org) to download genomic sequences ofeach exon of themouse (Mus musculus) genome including 40 nt ofintronic sequence flanking each of the exons. Input sequences aresearched with two patterns consisting of 15 nt for each of the twoTAL effector-binding sites preceded by a T and either a fixed-length spacer of 15 nt or a more flexible spacer of 14 or 16 nt. Thebinding sites identified are stored as genomic coordinates ina MySQL database (5.0.26). For display of the TAL effector-binding sites, which allows user-specific searches, we installeda local instance of the genome browser GBrowse Version 2.0 (2).Turning on and off genome browser tracks allows the user todisplay the transcripts of each of the genomes and their exon–intron structure and the identified TAL effector-binding sites,respectively (Fig. S1).

Nuclease Expression Vectors. The Rab38-specific zinc-finger nucle-ase (ZFN) expression vectors pCAG-Rab-ZFN-L and -R weredescribed previously (3). For the expression of TALENs inmammalian cells, we designed the generic expression vectorpCAG-TALEN, which contains a CAG hybrid promoter regionand a transcriptional unit comprising a sequence coding for theN-terminal amino acids 1–176 of TAL nucleases, located up-stream of a pair of BsmBI restriction sites. This N-terminal regionsincludes an ATG start codon, a nuclear localization sequence,a FLAG Tag sequence, a glycine-rich linker sequence, a segmentcoding for 110 aa of the TAL protein AvrBs3, and the invariableN-terminal TAL repeat of the Hax3 TAL effector. Downstream ofthe central BsmBI sites, the transcriptional unit contains 78 codons,including an invariable C-terminal TAL repeat and 44 residuesderived from the TAL protein AvrBs3, followed by the codingsequence of the FokI nuclease domain and a polyadenylation signalsequence. DNA segments coding for arrays of TAL repeats can beinserted into the BsmBI sites of pCAG-TALEN in frame with the

up- and downstream coding regions to enable the expression ofpredesigned TAL-Fok nuclease proteins. To derive TAL elementDNA-binding domains, we used the TAL effectormotif (repeat) 11of the Xanthomonas Hax3 protein (LTPEQVVAIASNIGGKQA-LETVQRLLPVLCQAHG) to recognize A, the TAL effectormotif 5 (LTPQQVVAIASHDGGKQALETVQRLLPVLCQAHG)derived from the Hax3 protein to recognize C, and the TALeffector motif 4 (LTPQQVVAIASNGGGKQALETVQRLLP-VLCQAHG) from the Xanthomonas Hax4 protein to recognizeT (bold letters indicate repeat variable diresidues). To recognizea target G nucleotide, we used the TAL effector motif 4 from theHax4 protein with replacement of the amino acids 12 into N and 13intoN (LTPQQVVAIASNNGGKQALETVQRLLPVLCQAHG).These elements were obtained by gene synthesis (Genscript) andfurther amplified by PCR using primers that include BsaI sitesoutside of the coding region. For a 15-bp TALEN target sequence,seven elements each are pooled in a pair of reactions together withBsaI and T4DNA ligase to create and ligate unique overhangs. Full-length ligation products were recovered by gel extraction and in-serted by seamless cloning (Gibson assembly; NewEngland Biolabs)into pCAG-TALEN opened with BsmBI. The integrity of allTALENexpression vectors was confirmed byDNA sequencing. Thecomplete sequence of the vector encoded TALEN proteins andrepeat variable diresidue (RVD) arrays is shown below [blue letters,nuclear localization sequence; red letters, FLAG tag; green letters,AvrBs3 N- and C-terminal sequences; black letters, TAL repeats(RVDs are underlined); orange letters, FokI nuclease domain; grayletters, linker sequences]:

TALENRab38 (1). MGPKKKRKVAAADYKDDDDKPGGGGSG-GGGVPASPAAQVDLRTLGYSQQQQEKIKPKVRSTVAQ-HHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIA-ALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPP-LQSGLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPL-NLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTP-QQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQV-VAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIA-SNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIG-GKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQ-ALETVQRLLPVLCQAHGLTPQQVVAIASHDGGKQALE-TVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQ-RLLPVLCQAHGLTPQQVVAIASHDGGKQALETVQRLL-PVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVL-CQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAH-GLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLT-PQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQ-VVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAI-ASNGGGRPALESIVAQLSRPDPALARSALTNDHLVALA-CLGGRPALDAVKKGLPHAPALIKRTNRRIPEGSDRLN-QLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQD-RILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVG-SPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQ-TRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKA-QLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEV-RRKFNNGEINF

TALENRab38 (2). MGPKKKRKVAAADYKDDDDKPGGGGSG-GGGVPASPAAQVDLRTLGYSQQQQEKIKPKVRSTVAQ-HHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIA-ALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPP-LQSGLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPL-NLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLT-

Wefers et al. www.pnas.org/cgi/content/short/1218721110 1 of 6

http://www.talen-design.dehttp://www.ensembl.orgwww.pnas.org/cgi/content/short/1218721110

PQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQ-VVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVA-IASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASHD-GGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGK-QALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQAL-ETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETV-QRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLL-PVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLC-QAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAH-GLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTP-QQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQV-VAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIA-SNGGGRPALESIVAQLSRPDPALARSALTNDHLVALA-CLGGRPALDAVKKGLPHAPALIKRTNRRIPEGSDRLN-QLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDR-ILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGS-PIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQT-RNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQ-LTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVR-RKFNNGEINF

Reporter Vectors. The nuclease reporter plasmid pTAL-Rep con-tains BstBI and NruI sites for cloning of TALEN target sites(underlined) as oligodeoxynucleotides (ODNs) (Metabion); Rab38target sense oligonucleotide: 5′-CGGCCACCATGGTCGTATCA-AGCGCTATGTGCACCAAAACTTCTCCTCGCACTACCG-GGCCACCATTGGT-3′; Rab38 target anti-sense oligonucleotide:5′-ACCAATGGTGGCCCGGTAGTGCGAGGAGAAGTTTTG-GTGCACATAGCGCTTGATACGACCATGGTGGC-3′.The integrity of all reporter plasmids was confirmed by DNA

sequencing.

Single-Stranded Oligodesoxynucleotide and Targeting Vector Design.The single-stranded oligodesoxynucleotide ODNIDG-Cht, targetingthe first exon of the mouse Rab38 gene, was synthesized by Met-abion and had a length of 144 nt (5′-CACCTCACAAGGAGC-ACCTGTACAAGCTGCTGGTGATCGGCGACCTGGTAG-TGGGCAAGACCAGCATTATTAAACGGTACGTGCATCA-AAATTTCTCCTCTCATTATCGAGCCACCATTGGTGTG-GACTTCGCGCTGAAGGTGC-3′). The targeting vectorpRab38IDG-WT comprised two homology regions encompassing918 and 2789 bp of genomic sequence flanking exon 1 of themouse Rab38 gene.

Nuclease Assay in HEK293 Cells. HEK293 cells were grown inDMEM supplied with 10% FCS (vol/vol) under standard con-ditions. A total of 1 × 106 cells were transfected by coelec-troporation with 5 μg of TALEN reporter, 5 μg of each TALENexpression plasmid, and 5 μg of the luciferase expression plasmidpCMV-FLuc. Control transfections were done with 5 μg ofTALEN reporter, 10 μg of pBluescript plasmid DNA, and 5 μg ofluciferase expression plasmid pCMV-FLuc. After transfection,cells were split into three wells and treated as technical repli-

cates. Forty-eight hours after transfection, cells were lysed, andwhole-protein samples were extracted. β-Galactosidase activitywas determined using the β-galactosidase reporter gene assay(Roche Diagnostics) following the manufacturer’s instructions,and signals were detected using a Centro LB 960 luminometer(Berthold Technologies). In parallel, luciferase activity wasmeasured as transfection control. Luciferase substrate [25 mMglycylglycin, 15 mM MgSO4, 4 mM EGTA, 2 mM ATP, 1 mMDTT, 100 μM coenzyme A, 75 μM luciferin, 15 mM K2HPO4/KH2PO4 (pH 8.0)] was added to the samples, and chemilumi-nescent signals were measured in the luminometer. The triplicateβ-galactosidase values of each transfected DNA mixture werenormalized in relation to the levels of luciferase activity of thesame samples, and the mean value and SEM of β-galactosidaseactivity were calculated. The values derived from cotransfectionof TALEN expression and reporter plasmids were comparedwith the transfection of reporter plasmid without TALEN plas-mids and expressed as relative increase.

Microinjection of One-Cell Embryos. The injection of TALENmRNA and targeting molecules (vector plasmid or ODN, re-

spectively) was performed as described previously for ZFNs (3, 4).Briefly, TALEN mRNA was prepared by in vitro transcriptionfrom linearized plasmid DNA using the mMessage mMachine T7Ultra kit and the MEGAclear kit (Life Technologies). EachTALEN mRNA was then diluted in injection buffer [10 mM Tris,0.1 mM EDTA (pH7.2)] to a working concentration of 10 ng/μL.mRNAs were mixed and stored together with the supercoiledtargeting vector or oligodesoxynucleotide at −80 °C. The targetingvectors were precipitated and dissolved in injection buffer toa working concentration of 15 ng/μL. The targeting ODNs weredissolved in water and diluted with injection buffer to a workingconcentration of 15 ng/μL. For the targeting of wild-type alleles,zygotes were obtained by mating of C57BL/6N males with super-ovulated FVB females (Charles River) as described (3, 4). Fortargeting of the Rab38cht allele, zygotes from matings of homo-zygous Rab38cht males (5) (kindly provided byM. Seabra, ImperialCollege London, London, UK) with FVB females were used.Zygotes were injected with the mixture of the targeting constructand the TALENmRNAs in a two-step procedure, as described (3,4). Briefly, a first aliquot of the DNA/RNA mixture was injectedinto, whenever possible, the larger (male) pronucleus to deliverthe DNA vector, as used for the production of transgenic mice.Upon the withdrawal of the injection needle from the pronucleus,a second aliquot of the DNA/RNA mixture was injected into thecytoplasm to deliver the TALEN mRNAs directly to the trans-lation machinery. Injections were performed using a Leica mi-cromanipulator and microscope and an Eppendorf FemtoJetinjection device. Injected zygotes were transferred into pseudo-pregnant CD1 female mice, and viable adult mice were obtained.All mice showed normal development and appeared healthy.Micewere handled according to institutional guidelines approved bythe animal welfare and use committee of the Regierung von

TALEN RVD arrays

TALENTAL repeat diresidues (without last

half repeat)

TALENRab38 no. 1 NI NG HD NI NI NN HD NN HD NG NI NGNN NG↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓

Target sequence (5′–3′) A- T- C- A- A- G- C- G- C- T- A- T- G- T-TALENRab38 no. 2 NN NN HD HD HD NN NN NG NI NN NGNNHD NN

↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓Target sequence (5′–3′) G - G - C - C - C - G - G - T - A - G - T - G - C - G -

Wefers et al. www.pnas.org/cgi/content/short/1218721110 2 of 6

www.pnas.org/cgi/content/short/1218721110

Oberbayern and housed in standard cages in a specific pathogen-free facility on a 12 h light/dark cycle with ad libitum access to foodand water.

Southern Blot Analysis. Southern blot analysis was performed aspreviously described (4) using the Rab38 5′ probe. Hybridization

probes were obtained by PCR amplification (P-Rab1: 5′-CTGG-AAACTAAAATTCAAGGTGTTATAC-3′; P-Rab2: 5′-TATT-CATTCACTTAACCATTTGTTC-3′) from C57BL/6N genomicDNA, purified with the Qiaquick PCR Purification Kit (Qiagen),cloned with the Strataclone PCR cloning kit (Agilent Technolo-gies), and confirmed by sequencing.

1. Stajich JE, et al. (2002) The Bioperl toolkit: Perl modules for the life sciences. GenomeRes 12(10):1611–1618.

2. Stein LD, et al. (2002) The generic genome browser: A building block for a modelorganism system database. Genome Res 12(10):1599–1610.

3. Meyer M, Ortiz O, Hrabé de Angelis M, Wurst W, Kühn R (2012) Modeling diseasemutations by gene targeting in one-cell mouse embryos. Proc Natl Acad Sci USA109(24):9354–9359.

4. Meyer M, de Angelis MH, Wurst W, Kühn R (2010) Gene targeting by homologousrecombination in mouse zygotes mediated by zinc-finger nucleases. Proc Natl Acad SciUSA 107(34):15022–15026.

5. Loftus SK, et al. (2002) Mutation of melanosome protein RAB38 in chocolate mice. ProcNatl Acad Sci USA 99(7):4471–4476.

Wefers et al. www.pnas.org/cgi/content/short/1218721110 3 of 6

www.pnas.org/cgi/content/short/1218721110

Fig. S1. Screenshot of the TALEN-Designer browser (www.talen-design.de). (A) Region overview showing the different transcripts of mouse PR domain zincfinger protein 13 gene (Prdm13) (Ensembl genome database, www.ensembl.org. Accession no. ENSMUSG00000040478). The potential TAL effector-bindingsites identified with search patterns of high and medium stringency are displayed as separated genome browser tracks, respectively. (B) Detailed view of exon 2of Prdm13 with a selection of TAL effector sites showing the binding domain as a green box and the spacer as a thin black line. The genomic coordinates andthe sequence of pattern tal2434667 as identified on Prdm13 exon 2 sequence (ENSMUSE00000731842) is displayed upon activating a hyperlink. In the FASTA-formatted sequence panel, the two DNA-binding domains are highlighted by a gray background.

Wefers et al. www.pnas.org/cgi/content/short/1218721110 4 of 6

http://www.talen-design.dewww.ensembl.orgwww.pnas.org/cgi/content/short/1218721110

Fig. S2. Screenshot of the TALEN-Designer browser. Distribution of TALEN target sites in the first exon (202 bp) of the Rab38 gene (red bar), searching forbipartite 15-bp target sequences (red boxes), separated by a spacer regions of 14–16 bp (thin line).

Fig. S3. Comparison of TALEN activities. TALENs directed against sequences within 20 different genes were constructed and tested by the HEK293 reporterassay for specific nuclease activity against their target sequences cloned into pTALEN-Rep. The activities obtained from transfection of the reporter in thepresence or absence of TALEN expression vectors were compared and expressed as an index of nuclease activity. These values (filled columns) are comparedwith the value obtained from transfection of the Rab38-specific ZFN (ZFNRab38) (red column) (Fig. 1D).

Wefers et al. www.pnas.org/cgi/content/short/1218721110 5 of 6

www.pnas.org/cgi/content/short/1218721110

Fig. S4. Coat color of homozygous Rab38IDG-Cht and Rab38wt littermates. (A) Pup Rc6-4 carrying the Rab38IDG-Cht allele (Fig. 2D) was mated with C57BL/6 wild-type mice, and the derived heterozygous offspring were intercrossed. Pups derived from these matings were genotyped by ApaLI digestion of PCR productsamplified from Rab38, identifying homozygous Rab38wt and heterozygous and homozygous Rab38IDG-Cht mice. Compared with the black fur of a Rab38wt/wt

littermate (pup 159), homozygous Rab38IDG-Cht/IDG-Cht mutants (pup 160) exhibit the dark brown coat color characteristic for chocolate mutants (5). (B) TheRab38 genotype of pups 159 and 150 was confirmed by sequence analysis of PCR products.

Table S1. Overview of founders and Rab38 alleles obtained from embryo microinjections

Microinjection experiment No. of pupsFounder

IDNo. of Rab38NHEJ alleles

No. of Rab38HR alleles

No. of founders withNHEJ allele(s)

No. of founders withHR allele(s)

TALENRab38 and ODNIDG-Cht 117 Rc6 1 2 1 1Rc7 2 1 1 1Rc1 1 0 1 0Rc2 1 0 1 0Rc3 1 0 1 0Rc4 1 0 1 0Rc5 1 0 1 0

TALENRab38 and pRab38IDG-WT 50 Rw2 0 1 0 1Rw1 2 0 1 0

∑ 167 9 founders 10 4 8 (4.8%) 3 (1.8%)

NHEJ, nonhomologous end-joining.

Wefers et al. www.pnas.org/cgi/content/short/1218721110 6 of 6

www.pnas.org/cgi/content/short/1218721110