Supplementary Information

Transcription

1 Supplementary Information Supplementary Figure 1: Failure of NgAgo to cleave episomal or chromosomal targets in human cells. Supplementary Figure 2: Each component of NgAgo system is efficiently delivered by transfection despite the lack of observed indels at endogenous targets. Supplementary Figure 3: NgAgo does not induced mutations at various endogenous target sites in human cells. Supplementary Methods 1 Supplementary Methods 2 Supplementary Methods 3 Supplementary References 1

2 Supplementary Figure 1: Failure of NgAgo to cleave episomal or chromosomal targets in human cells. (a) Failure to disrupt an episomal EGFP marker gene. HEK293T cells were transfected with pegfp-n3, expression plasmids for NgAgo and guide-dna (G3 and/or G4) to target EGFP and a mcherry expression plasmid to normalize for transfection efficiency. As controls, plasmids encoding SpCas9 and guide-rna to target EGFP or components to target CCR5 were transfected. The extent of EGFP knockdown was evaluated by flow cytometry and is indicated as fold reduction of the mean fluorescent intensity (MFI) normalized to mcherry expression. Three independent transfections were performed; error bars indicate SEM. Ago N1, NgAgo expression plasmid from Addgene; Ago N2, self-made NgAgo expression plasmid;, just guide-dna; UT, untransfected cells. (b) Failure to disrupt an integrated EGFP marker gene. U2OS cells, containing a single copy of an integrated EGFP expression cassette, were transfected

3 with expression plasmids for NgAgo and guide-dna (G3 and/or G4) to target EGFP. As controls, plasmids encoding SpCas9 and guide-rna to target EGFP or components to target CCR5 were transfected. The extent of EGFP knockout was evaluated by flow cytometry and is indicated as percentage of GFPnegative cells. Three independent transfections were performed; error bars indicate SEM. Student s two tailed t-test was performed on EGFP targeted samples (GFP, G3, G4) versus their relative CCR5 controls; *p<0,05, ****p<0,0001. (c, d) Failure to disrupt an endogenous locus. Genomic DNA was extracted from cells transfected as described in panels (a) and (b). The EGFP and CCR5 loci were subjected to T7E1 (c) and/or TIDE (d) analysis to evaluate the extent of nuclease induced indel formation. The expected sizes and the position of the digestion products are indicated. (e) NgAgo mrna is transcribed in human cells. After transfection of HEK293T cells with various NgAgo expression plasmids, the extent of RNA expression was determined by RT-PCR. GAPDH and controls without reverse transcriptase (-RT) are shown. (f) NgAgo is translated in human cells. Western Blot analysis for HA-tagged NgAgo proteins was performed in parallel to a β-actin control. N, native coding sequence of NgAgo; CO, codon-optimized sequence of NgAgo. See Supplementary Methods 1 for more details. 3

4 Supplementary Figure 2: Each component of NgAgo system is efficiently delivered by transfection despite the lack of observed indels at endogenous targets. (a) NgAgo and gdna is unable to induce indels. I. Reference human DYRK1A gene sequence with gdna sequences annotated. Arrowheads point 4

5 to 3 ends of the gdna. II. Representative chromatogram results from Sanger sequencing. The.ab1 files were further analyzed with TIDE web tool. (b) Delivery of gdna and plasmid DNA to cells. Cells were cotransfected with egfp expression plasmid and 3 -Alexa Fluor 594-tagged gdna harboring the same sequence as the G5 guide. egfp and Alexa 594 expression was assessed at 4, 12 and 24 hours posttransfection. Shown are the representative images from four independent experiments, taken with a 40x objective and a 4x zoom on Zeiss LSM 880. (c) NgAgo-GK plasmid is transfected efficiently to all three cell lines. Cells were transfected with G5 gdna + NgAgo-GK plasmid or GFP expression plasmid as indicated. 48 hours later, DNA was extracted and PCR was performed with indicated primers. Products were run on 2% agarose gel to assess the uptake of the plasmids. Shown is a representative image of three independent experiments. See Supplementary Methods 2 for more details. 5

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12 Supplementary Figure 3: NgAgo does not induced mutations at various endogenous target sites in human cells. (a) Schematics of the endogenous loci in mammalian cells targeted by NgAgo or SpCas9. The target/guide DNA sequences and single-guide RNA sequences are indicated by red and black lines, respectively. 5 phosphorylation of each guide DNA is indicated by 5 -P. PAM sequence for each singleguide RNA is indicated by blue color. (b) Targeting of endogenous loci after NgAgo plasmid lipofection was analyzed by targeted deep sequencing. Two kinds of vectors for NgAgo expression in mammalian cells were delivered to HEK293T cells by lipofection. The concentration of the co-transfected guide DNA 12

13 is indicated in each panel. The SpCas9 expression vector was transfected as a positive control. The experiment was performed three times for each sample for data analysis. Error bars indicate SEM (n=3) (c) The two NgAgo expression vectors were transfected into HeLa cells by lipofection using the same conditions. (d) The T7E1 assay after lipofection delivery to HEK293T cells. Cleaved fragments are indicated by red arrows. (e) The T7E1 assay after electroporation delivery to HEK293T cells. (f) Targeting of endogenous loci after NgAgo plasmid electroporation was analyzed by targeted deep sequencing. Two kinds of vectors for NgAgo expression in mammalian cells were delivered to HEK293T cells by electroporation. The experiment was performed three times for each sample for data analysis. Error bars indicate SEM (n=3) (g) The same electroporation conditions were used to deliver the NgAgo vectors into HeLa cells. The SpCas9 expression vector was transfected as a positive control. The experiment was performed three times for each sample for data analysis. (h) Dose escalation of the transfected NgAgo plasmid. Fivefold the original amount (1ug) of the NgAgo expression plasmid with guide DNA was delivered to target the DYRK1A locus in HEK293T cells. (i) The T7E1 assay for mutation analysis after electroporation delivery to HEK293T cells. (j) The T7E1 assay for mutation analysis after electroporation delivery to HeLa cells. Cleaved fragments are indicated by red arrows. (k) Test of the dependency of potential NgAgo mutation-inducing activity on the Mg++ concentration. The NgAgo expression plasmid was delivered to target the DYRK1A locus in HEK293 cells with various concentrations of Mg++ in the medium. (l) Representative mutation patterns at various gene loci induced by co-delivery of SpCas9 and sgrna expression plasmids to mammalian cells. Target sites for sgrnas are indicated in blue text and PAM sequences are shown underlined in red. (m) Quality assessment of the 5 phosphorylation of DNA guide oligonucleotides by mass spectrometry. Results are shown for guide DNAs (G5, G6, G7) for DYRK1A gene targeting, guide DNAs (G27, G28) for EMX1 gene targeting, guide DNAs (G40, G41) for GATA4 gene targeting, and guide DNAs (G43, G44) for GRIN2B gene targeting. (n) Western blotting assay of NgAgo and SpCas9 expression in HEK293T cells. Protein levels after delivery of the NgAgo or SpCas9 expression plasmid were compared. The same amount of plasmid was transfected with Lipofectamine 2000 for both NgAgo and SpCas9. See Supplementary Methods 3 for more details

14 Supplementary Methods 1 These experiments were conducted by the Cathomen group. Plasmids used in Supplementary Figure 1 Plasmid nls-ngago-gk (Addgene #78253) was obtained from Addgene. The coding sequences of SpCas9 and NgAgo (both native and codon-optimized) were synthesized and cloned by Gibson assembly in a pvax1-derived expression plasmid (Thermo Fisher Scientific) under control of the CMV promoter. The grnas were cloned into plasmid MLM3636 (Addgene #43860) plasmid. Correct assembly of plasmids was verified by Sanger sequencing. Episomal gene disruption assay HEK293T and HeLa cells were purchased from ATCC, maintained in Dulbecco s Modified Eagle s Medium (DMEM) with stable glutamine, 3.7 g/l NaHCO3 and 4.5 g/l D-glucose (GIBCO), supplemented with 10% fetal bovine serum (PAA) and 100 U/ml penicillin, and checked for mycoplasma contamination on a regular basis. 100,000 cells per well were seeded in 24-well plates one day before transfection. Lipofection was performed following the manufacturer s instruction: add DNA to 50 µl of OptiMEM (GIBCO) and mix with 1,25µl of Lipofectamine 2000 (Thermo Fisher Scientific) diluted in 50 µl of OptiMEM. After 20 Min. of incubation the cocktail was added to cells. DNA mix included 50 ng of pegfp- N3 reporter plasmid, 300 ng of NgAgo expression plasmid with or without 200 ng of 5 -phosphorylated guide DNA, and 50 ng of mcherry expression plasmid to normalize for transfection efficiency. As positive controls, 300 ng of Cas9 expression plasmid was used in combination with 200 ng of grna expression plasmid. Cells were analyzed by flow cytometry (BD Accuri C6; BD Biosciences) 48 hours posttransfection. The fold reduction of GFP expression was calculated as the ratio of the mean fluorescence intensity (MFI) of mcherry divided by the MFI of GFP, using untransfected (UT) cells as a reference for gating. Chromosomal GFP knockout assay U2OS-EGFP cells 1 were maintained in DMEM with 3.7 g/l NaHCO3 and 1 g/l D-glucose (Biochrome), supplemented with 10% fetal bovine serum (PAA), 2 mm L-glutamine (Biochrome) and 100 U/ml penicillin, and checked for mycoplasma contamination on a regular basis cells per sample were transfected with a 4D-Nucleofector (Lonza) according to the manufacture s instruction using program DN100 and nucleofection kit SE, and then seeded into 24-well plates. DNA mix contained 600 ng of 14

15 NgAgo or Cas9 expression plasmid and 200 ng of 5 -phosphorylated gdnas or grna expression plasmid, respectively. 100 ng of an mcherry expression plasmid was used in all samples to normalize for transfection efficiency. The percentage of GFP-negative cells was determined in the mcherry-positive fraction by flow cytometry (BD Accuri C6; BD Biosciences) 48 hours post-transfection, using untransfected (UT) cells as a reference for gating. T7E1 assay T7 endonuclease I (T7E1; New England Biolabs) assay was performed as previously described 2. Briefly, for each T7E1 reaction 200 ng of PCR product (see below) was denatured, reannealed and digested with 10 U of T7E1 for 20 Min at 37 C. The reaction was analyzed on a 2% TAE agarose gel. TIDE analysis A purified PCR amplicon (see below) was mixed with a sequencing primer (see below) and sequenced. The TIDE analysis 3 considered an indel spectrum of 40 bp around the cleavage site, a cutoff value for decomposition at p<0.01. NgAgo mrna expression RNA was extracted 48 hours after treatment from HEK293T cells lipofected with NgAgo expression plasmids with RNeasy MiniKit (Qiagen). QuantiTect Reverse Transcription kit (Qiagen) was utilized together with random primers according to the manufacturer s instruction to obtain complementary DNA. Specific primers for GAPDH and NgAgo (native or codon-optimized) were used (see below) to amplify the DNA by a 25-cycle PCR followed by analysis of the PCR amplicons on a 2% TAE agarose gel. NgAgo protein expression HEK293T cells transfected with HA-tagged NgAgo expression plasmids were harvested in RIPA buffer 4 48 hours after treatment. 20 µg of protein lysate was separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The membrane was blocked overnight at 4 C with 5% milk, incubated with both anti-ha (1:1000 dilution, Novus Biologicals NB ) and antiß-actin (1:1000 dilution, Cell Signaling Technology #4967) antibodies, and specific proteins visualized after incubation with an HRP-linked secondary antibody (1:5000 dilution, Cell Signaling Technology #7074) with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific). To detect DsRed- 15

16 tagged NgAgo, HEK293T cells were assessed by flow cytometry (BD Accuri C6; BD Biosciences) 48 hours after lipofection of cells with the DsRed-tagged NgAgo expression plasmid. Oligonucleotides used in Supplementary Figure 1 Guide DNA oligonucleotides were synthesized by Integrated DNA Technologies (IDT) and PAGE purified. The presence of a 5 -phosphate was verified by mass spectrometry. gdna Sequence (5 to 3 ) Gene G3 5 P-AAGGGCGAGGAGCTGTTCACCGGG GFP G4 5 P-GTGGTCGGGGTAGCGGCTGAAGCA GFP CCR5 5 P-CCAATCTATGACATCAATTATTAT CCR5 sgrna Sequence (5 to 3 ) Gene GFP GUGGUCACGAGGGUGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAG GFP UCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU CCR5 GUGAGUAGAGCGGAGGCAGGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAG UCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU CCR5 Primers Sequence (5 to 3 ) Application GFP_For CACTTGGCAGTACATCAAGTGTATCATATGC T7E1 GFP_Rev CTCGGCGCGGGTCTTGTAG T7E1 CCR5_For GTGGACAGGGAAGCTAGC T7E1 CCR5_Rev CAAAGTCCCACTGGGCG T7E1 AgoNat_F GACGGGCTAATGCTTCTCAC RT-PCR AgoNat_R CGCGAACGAGGTAGTCATC RT-PCR AgoCo-opt_F CAGCCTGTGGGATGATTACC RT-PCR AgoCo-opt_R GTGGCCGACTTCAACACTG RT-PCR GAPDH_F AGCCACATCGCTCAGACAC RT-PCR GAPDH_R GCCAATACGACCAAATCC RT-PCR 16

17 Supplementary Methods 2 These experiments were conducted by the Ekker group. Plasmids used in Supplementary Figure 2 NLS-NgAgo-GK (plasmid # 78253, provided by Chunyu Han) was obtained from Addgene and verified by Sanger Sequencing. pegfp N1 was obtained from Clontech. Cell culture and transfection HEK293, HeLa and K562 cell lines were purchased from ATCC and the identities of all cell lines were confirmed by the Medical Genome facility at Mayo Clinic Center (Rochester, MN) using short tandem repeat profiling upon receipt. HEK293 and HeLa were maintained in DMEM (Invitrogen) while K562 was maintained RPMI-1640 (Invitrogen). DNA stain (DAPI) was used to rule out mycoplasma contamination in all the cell lines. Media were supplemented with 10% FBS (Sigma). Cells were seeded into 24 well plates 16 hours before transfection at a density of 20,000 cells per well. Cells were transfected using Lipofectamine 2000 (Invitrogen) using manufacturer s guidelines. Briefly, all reagents were diluted to 100ng/μL in sterile 0.5X Tris EDTA (TE, (5 mm Tris-HCl, 0.5 mm EDTA ph 8.0). 200ng of NgAgo-expressing plasmid and 200ng of ssdna guides were used for transfection in each well. 500ng of pegfp N1 was used per well as control for transfection efficiency. DNA and Lipofectamine 2000 were mixed separately in 50μL of OPTI-MEM (Invitrogen) and incubated for 5 minutes at room temperature. The mixtures were combined and incubated for an additional 20 minutes at room temperature. Media was aspirated from the well and 100μL of the mixture was added to each well of 293 and HeLa cells. 400μL of DMEM with 10% FBS was added to each well after 4 hours. Since the K562 are non-adherent cells, 100μL of the transfection mix was directly added to the well (total volume 500μL) without change of media. Target site sequencing and PCR HeLa and HEK293 cells were collected 48 hours following transfection by trypsin digestion. K562 cells were collected by spinning down the media at 1000 rpm for 5 minutes. DNA was extracted with 100mM NaOH. The solution was incubated at 95 C for 15 minutes and neutralized with 1M Tris- HCl (ph 8.0). Phenol chloroform extraction was performed using MaxTract tubes (Qiagen) and DNA was precipitated with isopropanol. The following PCR targets were used: DYRK1A, pegfp-n1 and NgAgo-GK. 35 cycles were run with 10 to 25ng of DNA and an annealing temperature of 57 C. The product was gel purified and the amplicon was sequenced using Sanger sequencing using the forward primer. Reference DNA 17

18 sequence: Reference mrna sequence: TIDE sequence analysis Whole PCR amplicons were subjected to Sanger sequencing with F primer for DYRK1A by Mayo s sequencing core. The resultant.ab1 files were analyzed by TIDE web software ( with a cut off value of p<0.01. Subcloning Randomly selected DYRK1A amplicons (G6 and G10 from HeLa cells as well as corresponding GFP control) were subcloned into Strataclone vector (Agilent Technologies) per manufacture s protocol. Once the vector was transformed into Strataclone competent cells, these were plated onto Carbenicillin plates with Blue-White screening at 37 degrees, overnight. 24 random white colonies were subjected to colony PCR with M13F and M13R primers for 45 cycles with annealing temperature of 55 C. These amplicons were subjected to agarose gel electrophoresis to ensure the presence of a DNA insert. 15 amplicons per condition were then sent in for Sanger sequencing with M13F primer by Mayo Clinic s sequencing core. The resulting.ab1 files were analyzed by Snapgene application (GSL Biotech) Confocal microscopy to verify co-delivery of plasmid and gdna 5 -phosphorylated, 3 -Alexa Fluor 594 tagged G5 gdna was synthesized (IDT). Cells were seeded on coverslips and transfected with the guide DNA as described above. At 4, 12, and 24 hours posttransfection, cells were fixed with 3% paraformaldehyde for 15 minutes at room temperature and the coverslips were mounted on slides. These slides were then examined for GFP expression and Alexa Fluor signals using a 40x objective with 4x zoom on a fluorescent confocal microscope (Zeiss LSM 880). Oligonucleotides used in Supplementary Figure 2 Guide Sequence Gene G5 5'P-CCTACCAGAATCGCCCAGTGGCTG-3' DYRK1A G6 5'P-CAGCCACTGGGCGATTCTGGTAGG-3' DYRK1A G10 5'P-CCAAAGTCCAAGGTATTAGCAGCC-3' DYRK1A G12 5'P-CATTCTGTCCAAAGTCCAAGGTAT-3' DYRK1A G13 5'P-GCTCCATTCTGTCCAAAGTCCAAG-3' DYRK1A 18

19 Primers DYRK1A F DYRK1A R pegfp N1 F pegfp N1 R NgAgo-GK F NgAgo-GK R Sequence 5 -GTTCTTTCAGGTGCGTCA-3' 5 -GGGACTCTTCTCTATCAGCC-3' 5 -CCACCATGGTGAGCAAGGG-3' 5 -CTTTACTTGTACAGCTCGTCCATGC-3' 5 -CCAGGTCCATAACTCGACTGC-3 5 -GCCGATTTCGGCCTATTGG-3 19

20 Supplementary Methods 3 These experiments were conducted by the Kim group. Plasmids used in Supplementary Figure 3 Two kinds of plasmids were used: The NLS-NgAgo-GK (plasmid # 78253) was obtained from Addgene and its full sequence was verified by Sanger sequencing. The other plasmid was generated by cloning in our laboratory. The HA-NLS-NgAgo sequence (Invitrogen) was synthesized and cloned into a mammalian expression vector (pcdna3.1). For SpCas9-based gene targeting, U6 promoter-driven sgrna expression plasmids were prepared by cloning appropriate target sequences (gx20). All plasmids used in Suppl. Fig. 3 were prepared using a Midiprep kit (NucleoBond Xtra Midi / Maxi EF) for endotoxin-free plasmid preparation. Guide DNA oligonucleotides Guide DNA oligonucleotides were synthesized by Bio Basic Canada Inc. The quality of the 5 phosphorylation of each oligonucleotide was assessed by mass spectrometry. Cell culture and transfection HEK293T (ATCC, CRL-11268) and HeLa (ATCC, CCL-2) cells were maintained in Dulbecco s modified Eagle s medium (Cat. LM , WELGENE), supplemented with 10% FBS and a 1% penicillin and streptomycin mixture, and with growth conditions of 37 C and 5% CO 2. Both lipofection and electroporation were used to deliver the NgAgo or SpCas9 expression plasmids to cells. For lipofection, HEK293 or HeLa cells were seeded into 24-well plates one day (24 hours) before transfection, allowing them to reach 80% confluency. Adherent HEK293T or HeLa cells were then transfected with a pre-incubated mixture, which was composed of 1.5ul Lipofectamine 2000 (Invitrogen), 50ul of (1 ) Opti- MEM (Invitrogen), and either the NgAgo plasmid (0.2ug-1ug) and guide DNA (0.1ug-1ug) or the SpCas9 (1ug) and sgrna (1ug) expression plasmids. Cells were incubated for 48hours after transfection before preparation of genomic DNAs. For electroporation, the NgAgo expression plasmid (0.2ug-1ug) was incubated with guide DNA (0.1-1ug) in 20ul of nucleofection solution. The mixture was then cotransfected into HEK293T or HeLa cells using the Amaxa SF Cell Line 4D-Nucleofector kit (Lonza). In the case of the Mg++ supplementation experiment, 0 to 10mM of Mg++ was added to the medium. 1 ug of SpCas9-encoding plasmid and 1 ug of sgrna-expressing plasmid in 20ul nucleofection solution was cotranfected as a positive control. Cells were harvested 48hours after tranfection for analysis. 20

21 T7E1 assay For the T7E1 assay, genomic DNA from HEK293 or HeLa cells was amplified (95 C for melting (30sec), 58 C for annealing (30sec), 72 C for extension (30sec), 30cycles) with PCR primers specific for the target genes (see below). PCR products were denatured by heating and annealed slowly with a thermocycler. Annealed products were incubated with T7 endonuclease I (ToolGen) for 20 min at 37 C and fragments were separated using 2% agarose gel electrophoresis. Targeted deep sequencing and data analysis For deep sequencing analysis, genomic DNA samples from HEK293T or HeLa cells were amplified (95 C for melting (30sec), 58 C for annealing (30sec), 72 C for extension (30sec), 30cycles) with PCR primers specific for the target genes (see below). Nested PCR was then performed (95 C for melting (30sec), 62 C for annealing (30sec), 72 C for extension (12sec), 28cycles) to label each sample by conjugating an adaptor sequence. The resulting PCR amplicons were subjected to paired-end sequencing with MiSeq (Illumina). After Miseq, paired-end reads were joined by the ea-utils (Fastq-join) program using default values. Paired end reads were then analyzed by comparing wild-type and mutant sequences. Every experiment was performed three times for each sample for data analysis. Western blotting One day before transfection, HEK293T cells were seeded into 12-well plates ug of the cloned NgAgo expression vector, or the same amount of SpCas9 expression plasmid, were mixed with 2ul Lipofectamin 2000 (Invitrogen) in 100ul (1 ) Opti-MEM (Invitrogen) solution and then added to each well for tranfection. 24hours after transfection, cells were collected and suspended in lysis buffer (25mM Tris HCl ph 7.6, 150mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS). After centrifuge at 16,000xg in a tube for 10min, only supernatant solution was prepped and each protein amount was measured by microplate absorbance reader (BIO-RAD). Equal amount of protein sample was loaded and size separated by 10% SDS-PAGE. Then loaded sample was transferred to the membrane and blotted with western device (ibind, Life technologies). Anti-HA tag (Abfrontier, LF-MA0048) and anti-gapdh (Santa Cruz Biotechnology, sc ) antibody were used for blotting. HRP-conjugated secondary antibody (Santa Cruz Biotechnology, sc-2031) blotted membrane was treated with detection reagents (Amersham ECL prime, GE Healthcare) and protein band was detected by imaging device (Amersham Imager 600, GE healthcare). 21

22 Oligonucleotides used in Supplementary Figure 3. gdna Sequence (5 to 3 ) gene G5 5 P-CCTACCAGAATCGCCCAGTGGCTG DYRK1A G6 5 P-CAGCCACTGGGCGATTCTGGTAGG DYRK1A G7 5 P-ATCGCCCAGTGGCTGCTAATACCT DYRK1A G27 5 P-CCCACGAGGGCAGAGTGCTGCTTG EMX1 G28 5 P-CAAGCAGCACTCTGCCCTCGTGGG EMX1 G40 5 P-GGCGCCCGCGCCGTGCATGAAGGC GATA4 G41 5 P-AGCTCCGGTGGGGCCGCGTCTGGT GATA4 G43 5 P-GATAAGGTCCTTGAATTGCAGTAT GRIN2B G44 5 P-TTGCAGGGAGTCGACGAGTTGAAG GRIN2B sgrna Sequence (5 to 3 ) gene 1 GGGCCUACCAGAAUCGCCCAGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAG DYRK1A GCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU 2 GCCAAGGUAUUAGCAGCCACUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAG DYRK1A GCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU 3 GGGCAGAGUGCUGCUUGCUGCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAA EMX1 GGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU 4 GAAGCAGCACUCUGCCCUCGUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAG EMX1 GCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU 5 GCCGCGUCUGGUGCGGGGCCCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAG GATA4 GCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU 6 GAGCUCCGGUGGGGCCGCGUCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAG GATA4 GCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU 7 GAGGCUAGAUACUGCAAUUCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAG GRIN2B GCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU 8 GGUGCUCAAUGAAAGGAGAUAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAA GGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU GRIN2B The target sequence (gx20) in each sgrna sequence is shown in red. Primers Sequence (5 to 3 ) DYRK1A PCR-F GTTCTTTCAGGTGCGTCA DYRK1A PCR-R GGGACTCTTCTCTATCAGCC EMX1 PCR-F CCATCCCCTTCTGTGAATGT EMX1 PCR-R GGAGATTGGAGACACGGAGA GATA4 PCR-F CCCCTTTGATTTTTGATCTTCG GATA4 PCR-R TGTGCAGGACCGGGCTGT GATA4 PCR-F2 CAGTTCCTCCCACGCATATT GATA4 PCR-R2 GGTAGGGGCTGGAGTAGGAG GRIN2B PCR-F CAGGAGGGCCAGGAGATTTG GRIN2B PCR-R TGAAATCGAGGATCTGGGCG DYRK1A PCR-F3 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTGTCTTCCTCAACGACTTCTTCCTCGAC 22

23 DYRK1A PCR-R3 GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTTAGCAGAAAATTGGTATGTTGGATGTCC EMX1 PCR-F3 ACACTCTTTCCCTACACGACGCTCTTCCGATCTACGAAGCAGGCCAATGGGGAGGACATC EMX1 PCR-R3 GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCTGTCAATGGCGGCCCCGGGCTTCAA GATA4 PCR-F3 ACACTCTTTCCCTACACGACGCTCTTCCGATCTTGCCCACACCGCGGGTGCCCTCCTCCGT GATA4 PCR-R3 GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTAAGCGCGGCGACACCGGCGGCGGGGTG GRIN2B PCR-F3 ACACTCTTTCCCTACACGACGCTCTTCCGATCTTTTCATTTTCTCTCATTCTGCAGAGCA GRIN2B PCR-R3 GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTGGGCTTCATCTTCAACTCGTCGACTCC Nested PCR primers are described in F2/R2. The adaptor sequence in each primer (F3/R3) is shown in green. Accession code. SRA: SRX

24 Supplementary References 1. Händel, E.M. et al. Versatile and efficient genome editing in human cells by combining zincfinger nucleases with adeno-associated viral vectors. Hum Gene Ther 23, (2012). 2. Mussolino, C. et al. A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity. Nucleic Acids Res 39, (2011). 3. Brinkman, E.K., Chen, T., Amendola, M. & van Steensel, B. Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic Acids Res 42, e168 (2014). 4. Mussolino, C. et al. TALENs facilitate targeted genome editing in human cells with high specificity and low cytotoxicity. Nucleic Acids Res 42, (2014)