Nature Methods: doi: /nmeth Supplementary Figure 1

Transcription

1 Supplementary Figure 1 ATP1A1 variants with in-frame deletions are enriched in ouabain-resistant cell populations. (a) Total editing efficacy along with spectrum and frequency of individual indels as determined by the TIDE assay in K562 cells treated with ATP1A1 G2 sgrna. Analysis was performed on genomic DNA samples shown in Figure 1b. (b) Same as (a) but for ATP1A1 G4 sgrna. (c) Same as (a) but for ATP1A1 G3 sgrna.

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3 Supplementary Figure 2 HDR-driven editing at ATP1A1 induces cellular resistance to ouabain. (a) Schematic representation of the intronic SpCas9 target site G3 and partial sequences of single-stranded oligodeoxynucleotides (ssodns) donors used to introduce the Q118D/R and N129D/R mutations. Annotated are novel restriction sites to monitor the insertion of ssodn-specified mutations. (b) K562 cells stably expressing SpCas9 were co-transfected with an ATP1A1 G3 sgrna expression vector (500ng) along with the indicated ssodns (10pmol). Genomic DNA was harvested 10 days post-transfection and a PvuI RFLP assay was used to determine the frequency of HDR at the cleavage site, indicated as the % HDR at the base of each lane. Where indicated, cells were treated with 0.5 M ouabain for 7 days starting 3 days post transfection. An expression vector encoding EGFP (-) was used as a negative control. (c) Same as in (b) but using BmgBI. (d) Same as in (b) but using ClaI. (e) Same as in (b) but using Hpy188I. (f) Same as in (e) but cells initially selected at 0.5 M ouabain were cultivated in the presence of increasing concentrations of the drug for a week. (g) Uncropped gel images for panels (e) and (f). A naturally occurring Hpy188I (TCNGA cleavage site) is present in the amplicon creating the strong RFLP signal indicated by the red arrows. The HDR-specific signal (black arrow; predicted at 77bp) is running below the 100bp marker and is best viewed when the gels are overexposed making it is impossible to quantify this signal using RFLP-based assays.

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5 Supplementary Figure 3 Ouabain-based selection is portable to various cell lines and compatible with CRISPR AsCpf1-driven editing. (a) U2OS cells were treated and analyzed as in Figure 1b. (b) htert-rpe-1 cells were treated and analyzed as in Figure 1b and Supplementary Figure 2d. (c) Schematic representation of AsCpf1 target sites surrounding DNA encoding the first extracellular loop of human ATP1A1. Annotated are the positions of residues Q118 and N129, exon/intron boundary, protospacer adjacent motifs (PAM) and five potential AsCpf1 target sequences (Targets 1-5). Also shown are partial sequences of single-stranded oligodeoxynucleotides (ssodns) donors used to introduce the Q118R and N129D mutations. Annotated are novel restriction sites to monitor the insertion of ssodn-specified mutations. (d) K562 cells stably expressing AsCpf1 were transfected with ATP1A1 crrna expression vectors (G1-G5) (500 ng) and the Surveyor assay was used to determine the frequency of AsCpf1-induced insertions and deletions, indicated as the % Indels at the base of each lane. Where indicated, cells were treated with 0.5 M ouabain for 7 days starting 3 days post transfection. An expression vector encoding EGFP (-) was used as a negative control. (e) K562 cells were transfected with a vector expressing espcas9(1.1) and the ATP1A1 G3 sgrna (500ng each) or with a vector expressing AsCpf1 and the ATP1A1 G5 crrna (500ng each) (see Supplementary Figure 12 for details on the vectors) along with the indicated ssodns. Cells were treated as in (d) and assayed using BmgBI and EcoRI RFLP assays.

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7 Supplementary Figure 4 Selection for CRISPR Cas9-driven targeted mutagenesis by coediting ATP1A1 via NHEJ. (a) Experimental strategy for the co-enrichment of CRISPR-driven editing at a second locus. GOI, gene of interest. (b) Typical selection process. Timing can vary according to initial modification rates at ATP1A1. (c) Schematic of the dual espcas9(1.1) and tandem U6- driven sgrnas expression vector (see also Supplementary Figure 12). (d) Surveyor assays to determine on-target and off-target activity for the EMX1 sgrna on samples reported in Table 1. (e) Same as (d) but for co-targeting AAVS1. (f) Surveyor assays on samples from K562 cells transiently co-transfected with WT SpCas9 and sgrna expression vectors targeting AAVS1 (0.5 g and 1 g of each vector).

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9 Supplementary Figure 5 Selection for CRISPR AsCpf1-driven targeted mutagenesis by coediting ATP1A1 via NHEJ. (a) Schematic of the dual AsCpf1 and U6-driven crrna array expression vector (see also Supplementary Figure 12). (b) K562 cells were transfected with 500ng of the vectors shown in (a), treated and assayed for indels as shown in Supplementary Figure 4d. (c) Same as in (a). (d) Same as in (b) but using 1 g of the vector. (e) HEK293 cells were treated and analyzed as in (b) but the TIDE assay was used to determine the frequency of indels. Value for ATP1A1 relates to the co-selection performed with the DNMT1 crrna. (f) Same as in (e) but using the Surveyor assay.

10 Supplementary Figure 6 Off-target analysis in K562 and HEK293 cells coselected with the AsCpf1 crrna array vector targeting DNMT1. (a) Schematic of the dual AsCpf1 and U6-driven crrna array expression vector. (b) Surveyor assays to determine on-target and offtarget activity performed on samples reported in Table 1 and Supplementary Table 1. To facilitate the interpretation of the results, the gels for on-target activity presented in Supplementary Figure 5b,f have been reproduced here. Off-target activity at DNMT1-OT1 was assayed using two different sets of primers since it is found within a repetitive element making its detection more challenging. (c) Ontarget activity at DNMT1 was also determined using the TIDE assay in both co-selected and transiently transfected cells. (d) Surveyor assays to determine on-target and off-target activity in K562 cells transiently transfected with 1 g and 2 g of the vector shown in (a).

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12 Supplementary Figure 7 Selection for CRISPR-driven targeted integration at LMNA by coediting ATP1A1 via HDR. (a) Targeting scheme for the integration of Clover and mruby2 to the N-terminus of Lamin A/C. (b) Fluorescence imaging of ouabaintreated cells expressing the Lamin A/C-Clover fusion. Scale bar, 25 m (c) A ClaI RFLP assay was used to determine the frequency of SpCas9-induced HDR at the ATP1A1 locus in samples shown in Figure 2. % HDR is indicated at the base of each lane. (d) FACSbased quantification of Clover targeting to the N-terminus of Lamin A/C at various doses of WT SpCas9 and espcas9(1.1) in presence or absence of co-selection with ouabain. (e) Same as in (d) but in cells transfected with both Clover and mruby2 donors. (f) Same as in (d) but targeting LMNA using an AsCpf1 crrna array. Transfection conditions are indicated in Supplementary Table 2.

13 Supplementary Figure 8 Sequencing of non-hdr alleles from LMNA coselection experiments. (a) Schematic of a PCR-based assay (out-out PCR) used to detect targeted integration of the Clover or mruby2 sequences at the N- terminus of Lamin A/C. Primers are located outside of the homology arms and are designed to yield a longer PCR product if the fluorescent protein is inserted. The upper band representing the targeted integration of either Clover or mruby2 was TOPO cloned and sequenced to confirm the accurate integration of the fluorescent proteins via HDR. (b) The lower band from (a) representing nontargeted LMNA alleles was TOPO cloned, sequenced and analyzed using the web tool CrispRVariantsLite. (c) ATP1A1 alleles from the same experiment were also sequenced and non-hdr alleles were analyzed as in (b). Samples are from the experiment shown in Figure 2. Ref: Lindsay, H. et al. CrispRVariants charts the mutation spectrum of genome engineering experiments. Nat Biotechnol. 34, (2016)

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15 Supplementary Figure 9 Selection for CRISPR-driven targeted integration at endogenous loci by coediting ATP1A1 via HDR. (a) Targeting scheme for the integration of mag1 to the C-terminus of H2BK. (b) K562 cells were transfected with a vector expressing espcas9(1.1) and tandem U6-driven sgrnas targeting ATP1A1 and HIST1H2BK along with ATP1A1 ssodn RD and a plasmid donor containing a mag1 cassette. In addition, cells were transfected with a wild-type SpCas9 expression vector and two independent sgrna vectors along with donors. Cells were treated or not with 0.5 M ouabain for 14 days starting 3 days post-transfection and flow cytometry was used to determine the % of mag1 positive cells in each population. (c) Fluorescence imaging of ouabain-treated cells expressing the H2BK-mAG1 fusion. Scale bar, 10 m (d) FACS-based quantification of targeted integration of a PGK1-EGFP-pA expression cassette at HPRT1 following co-selection using WT SpCas9 and various amounts of sgrnas. (e) FACS-based quantification of targeted integration of a SA-2A-EGFP-pA gene trap cassette at AAVS1 following co-selection using an all-in-one vector expressing ATP1A1 and AAVS1 sgrnas in addition to espcas9(1.1). (f) Same as in (e) but using a PGK1-TurboGFP-pA cassette. Transfection conditions are indicated in Supplementary Table 2.

16 Supplementary Figure 10 Endogenous tagging of the NuA4 TIP60 complex in coselected HEK293 cells. (a) Schematic representation of the experiment. (b) Gene tagging scheme. (c) Western blot analysis on whole cell extracts from HEK293 cells to detect the expression of EPC1-tag and EP400-tag proteins. The FLAG M2 antibody was used to detect tagged proteins and the tubulin antibody was used as a loading control.

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18 Supplementary Figure 11 Introduction of the sickle mutation in primary human cord blood (CB) CD34 + cells by coediting ATP1A1 via HDR. (a) Typical selection process. Timing can vary according to initial modification rates at ATP1A1. (b) Schematic representation of SpCas9 target site in ATP1A1 and predicted HDR outcome dictated by the ssodn donor used to introduce the Q118R and N129D mutations. Annotated are novel restriction sites to monitor the insertion of ssodn-specified mutations. (c) Cultured CD34 + cells were electroporated with ATP1A1 and HBB RNPs along with HBB ssodn #1 and ATP1A1 G4 RD ssodn and treated as shown in (a). Genomic DNA was harvested at indicated time points and a BmgBI RFLP assay was used to determine the frequency of SpCas9- induced HDR at ATP1A1, which is indicated as the % HDR at the base of each lane. Recombinant Cas9 was used as a negative control (-). (d) Same as in (c) but using the Surveyor assay to determine the total frequency of edited alleles at ATP1A1 (NHEJ + HDR). (e) Schematic representation of SpCas9 target site in HBB and predicted HDR outcome dictated by ssodns donor #2 used to introduce the E6V mutation. Annotated are the positions of E6 residue, 5 UTR, protospacer adjacent motifs (PAM) and novel restriction site to monitor the insertion of ssodn-specified mutations. (f) Same as in (c) but using HBB ssodn #2 and a PstI RFLP assay to determine the frequency of SpCas9-induced HDR at HBB. (g) Same as in (f) but using the Surveyor assay to determine the total frequency of HBB edited alleles (NHEJ + HDR). (h) Same as in (f) but using a BmgBI RFLP assay to determine the frequency of SpCas9-induced HDR at ATP1A1. (i) Same as in (g) but using the Surveyor assay to determine the total frequency of ATP1A1 edited alleles (NHEJ + HDR).

19 Supplementary Figure 12 espcas9(1.1) and AsCpf1 vectors for targeting ATP1A1, available from Addgene. Ref for py036: Engineered Cpf1 Enzymes with Altered PAM Specificities. Linyi Gao, David B.T. Cox, Winston X Yan, John Manteiga, Martin Schneider, Takashi Yamano, Hiroshi Nishimasu, Osamu Nureki, Feng Zhang. biorxiv ; doi:

20 Supplementary Note 1 Several approaches have been implemented to capture and isolate cells expressing high amounts of nucleases. First, co-transfection of fluorescent proteins combined with fluorescence-activated cell sorting (FACS) can be used to isolate nuclease-expressing cells 1. Second, direct coupling of expression of fluorescent proteins and nucleases via 2A peptide sequences allows for efficient isolation of cell populations with increasingly higher nuclease expression levels, which translates into increasingly higher genome editing rates 2. Fluorescence-based surrogate target gene reporters have also been successfully used to enrich for cells with high nuclease activity 3, 4. A limitation of these methods is that single-cell FACS enrichment is not suitable for more sensitive cell lines. In addition, multi-dsbs arising from cleavage of the episomal reporter plasmids are likely to induce a gene-independent antiproliferative response as recently described for CRISPR-Cas9-induced DNA breaks 5, 6. SUPPLEMENTARY REFERENCES 1. Whyte, J.J. et al. Gene targeting with zinc finger nucleases to produce cloned egfp knockout pigs. Mol Reprod Dev 78, 2 (2011). 2. Duda, K. et al. High-efficiency genome editing via 2A-coupled co-expression of fluorescent proteins and zinc finger nucleases or CRISPR/Cas9 nickase pairs. Nucleic Acids Res 42, e84 (2014). 3. Certo, M.T. et al. Tracking genome engineering outcome at individual DNA breakpoints. Nat Methods 8, (2011). 4. Kim, H. et al. Surrogate reporters for enrichment of cells with nuclease-induced mutations. Nat Methods 8, (2011). 5. Aguirre, A.J. et al. Genomic Copy Number Dictates a Gene-Independent Cell Response to CRISPR/Cas9 Targeting. Cancer Discov 6, (2016). 6. Munoz, D.M. et al. CRISPR Screens Provide a Comprehensive Assessment of Cancer Vulnerabilities but Generate False-Positive Hits for Highly Amplified Genomic Regions. Cancer Discov 6, (2016). Nature Methods doi: /nmeth.4265

21 Supplementary Data 1 Total editing efficacy along with spectrum and frequency of individual indels as determined by the TIDE assay for Table 1 and Supplementary Table 1. espcas9(1.1) tandem U6-driven sgrnas EMX1 + ATP1A1 (K562-50ng) EMX1 untreated EMX1 ouabain ATP1A1 untreated ATP1A1 ouabain espcas9 (1.1) tandem U6-driven sgrnas EMX1 + ATP1A1 (K ng) EMX1 untreated EMX1 ouabain ATP1A1 untreated ATP1A1 ouabain Nature Methods doi: /nmeth.4265

22 espcas9 (1.1) tandem U6-driven sgrnas AAVS1 + ATP1A1 (K562 50ng) AAVS1 untreated AAVS1 ouabain ATP1A1 untreated ATP1A1 ouabain espcas9 (1.1) tandem U6-driven sgrnas AAVS1 + ATP1A1 (K ng) AAVS1 untreated AAVS1 ouabain ATP1A1 untreated ATP1A1 ouabain Nature Methods doi: /nmeth.4265

23 AsCpf1 crrna array DMNT1 + ATP1A1 (K ng) DNMT1 untreated DNMT1 ouabain ATP1A1 untreated ATP1A1 ouabain AsCpf1 crrna array EMX1 + ATP1A1 (K ng) EMX1 untreated EMX1 ouabain ATP1A1 untreated ATP1A1 ouabain Nature Methods doi: /nmeth.4265

24 AsCpf1 crrna array VEGFA + ATP1A1 (K ng) VEGFA untreated VEGFA ouabain ATP1A1 untreated ATP1A1 ouabain AsCpf1 crrna array GRIN2b + ATP1A1 (K ng) GRIN2B untreated GRIN2B ouabain ATP1A1 untreated ATP1A1 ouabain Nature Methods doi: /nmeth.4265

25 AsCpf1 crrna array DNMT1 + EMX1 + VEGFA + GRIN2b + ATP1A1 (K562-1µg) DNMT1 untreated DNMT1 ouabain EMX1 untreated EMX1 ouabain VEGFA untreated VEGFA ouabain GRIN2B untreated GRIN2B ouabain ATP1A1 untreated ATP1A1 ouabain Nature Methods doi: /nmeth.4265

26 AsCpf1 crrna array DNMT1 + ATP1A1 (HEK293LTV - 500ng) DNMT1 untreated DNMT1 ouabain ATP1A1 untreated ATP1A1 ouabain AsCpf1 crrna array EMX1 + ATP1A1 (HEK293LTV - 500ng) EMX1 untreated EMX1 ouabain ATP1A1 untreated ATP1A1 ouabain Nature Methods doi: /nmeth.4265

27 AsCpf1 crrna array VEGFA + ATP1A1 (HEK293LTV - 500ng) VEGFA untreated VEGFA ouabain ATP1A1 untreated ATP1A1 ouabain AsCpf1 crrna array GRIN2b + ATP1A1 (HEK293LTV - 500ng) GRIN2B untreated GRIN2B ouabain ATP1A1 untreated ATP1A1 ouabain Nature Methods doi: /nmeth.4265

28 Supplementary Table 1 Co-selection for CRISPR-induced indels in HEK293 cells. TIDE Assay (% indels 1 ) Surveyor Assay (% indels 1 ) Target Nuclease Dose (ng) Untreated Ouabain Untreated Ouabain DNMT1 AsCpf EMX1 " VEGFA " GRIN2b " ATP1A1 3 " Rounded to the nearest one. Undetectable set to 0. 2 U6-driven crrna array (2 guides) coupled to AsCpf1 expression from a single vector. 3 Value taken from DNMT1 co-selection experiment. Supplementary Table 2 Transfection mixes for HDR-based co-selections Figure 2 SpCas9_LMNA_G2 (px330) 100 ng Clover_LMNA_Plasmid_donor 250 ng mruby2_lmna_plasmid_donor 250 ng ATP1A1_G3_pUC19_sgRNA 100 ng ssodn_atp1a1_rd 5 pmol Supplementary Figure 7d espcas9(1.1)_no_flag_atp1a1_g3_dual_lmna_g2 50 ng 100 ng 500 ng Clover_LMNA_Plasmid_donor 250 ng 250 ng 250 ng ssodn_atp1a1_rd 5 pmol 5 pmol 5 pmol SpCas9_LMNA_G2 (px330) Clover_LMNA_Plasmid_donor ATP1A1_G3_pUC19_sgRNA ssodn_atp1a1_rd 100 ng 250 ng 100 ng 5 pmol Supplementary Figure 7e espcas9(1.1)_no_flag_atp1a1_g3_dual_lmna_g2 100 ng Clover_LMNA_Plasmid_donor 250 ng mruby2_lmna_plasmid_donor 250 ng ssodn_atp1a1_rd 5 pmol SpCas9_LMNA_G2 (px330) Clover_LMNA_Plasmid_donor mruby2_lmna_plasmid_donor ATP1A1_G3_pUC19_sgRNA ssodn_atp1a1_rd 100 ng 250 ng 250 ng 100 ng 5 pmol Nature Methods doi: /nmeth.4265

29 Supplementary Figure 7f py036_atp1a1_g5_array_lmna Clover_LMNA_Plasmid_donor ssodn_atp1a1_rd2 Supplementary Figure 9b espcas9(1.1)_no_flag_atp1a1_g3_dual_h2bk H2BK_mAG1_Plasmid_donor ssodn_atp1a1_rd 1000 ng 250 ng 5 pmol 100 ng 250 ng 5 pmol Transfection for WT SpCas9 was done with 1E6 cells in an amaxa cuvette hcas9 (WT) 500 ng H2BK_mAG1_Plasmid_donor 1250 ng ATP1A1_G3_pUC19_sgRNA 500 ng HIST1H2BK_pUC19_sgRNA 500 ng ssodn_atp1a1_rd 10 pmol Supplementary Figure 9d Transfection was done with 1E6 cells in an amaxa cuvette hcas9 (WT) 500 ng 500 ng hpgk1_egfp_plasmid_donor 1000 ng 1000 ng ATP1A1_G3_pUC19_sgRNA 50 ng 500 ng HPRT1_pUC19_sgRNA 50 ng 50 ng ssodn_atp1a1_rd 10 pmol 10 pmol Supplementary Figure 9e espcas9(1.1)_no_flag_aavs1 AAVS1_SA_2A_EGFP_Plasmid_donor ATP1A1_G3_pUC19_sgRNA ssodn_atp1a1_rd 100 ng 250 ng 100 ng 5 pmol Supplementary Figure 9f espcas9(1.1)_no_flag_atp1a1_g3_dual_aavs1 50 ng AAVS1_PGK_TurboGFP_Plasmid_donor 500 ng ATP1A1_RD_Plasmid_donor 5 pmol Figure 3 and Supplementary Figure 10 espcas9(1.1)_no_flag_atp1a1_g3 P400_FS_Plasmid_donor P400_sgRNA_pUC19_sgRNA ssodn_atp1a1_g3_rd 100 ng 250 ng 100 ng 5 pmol Nature Methods doi: /nmeth.4265

30 espcas9(1.1)_no_flag_atp1a1_g3 EPC1_FS_Plasmid_donor EPC1_sgRNA_pUC19_sgRNA ssodn_atp1a1_g3_rd 100 ng 250 ng 100 ng 5 pmol Supplementary Table 3 Sequences for SpCas9 guides Target Sequence ATP1A1 G1 GAACTCACATTATCGTTTTG ATP1A1 G2 GATCCAAGCTGCTACAGAAG ATP1A1 G3 GAGTTCTGTAATTCAGCATA ATP1A1 G4 GTTCCTCTTCTGTAGCAGCT AAVS1 GGGGCCACTAGGGACAGGAT LMNA G2 GCCATGGAGACCCCGTCCCAG EMX1 GAGTCCGAGCAGAAGAAGAA EPC1 GTGACGTAGCTTCCTCCGAG EP400 GGCCCTGACTACTGGCACGG HPRT1 GATGTGATGAAGGAGATGGG HIST1H2BK GGGGCTTTAAGACGCTTACT Supplementary Table 4 Sequences for AsCpf1 guides Target Sequence ATP1A1 G1 TTTCTTGGCTTATAGCATCC ATP1A1 G2 TTGGCTTATAGCATCCAAGC ATP1A1 G3 AGGTTCCTCTTCTGTAGCAG ATP1A1 G4 GAGGTTCCTCTTCTGTAGCA ATP1A1 G5 TAGTACACATCAGATATCTT ATP1A1 G3 (23bp for crrna arrays) AGGTTCCTCTTCTGTAGCAGCTT ATP1A1 G5 (23bp for crrna arrays) TAGTACACATCAGATATCTTCTC DNMT1 CTGATGGTCCATGTCTGTTACTC EMX1 TGGTTGCCCACCCTAGTCATTGG GRIN2b GTGCTCAATGAAAGGAGATAAGG VEGFA CTAGGAATATTGAAGGGGGCAGG LMNA CGGGACCCCTGCCCCGCGGGCAG Nature Methods doi: /nmeth.4265

31 Supplementary Table 5 Sequences for ssodns ssodn Sequence ATP1A1 DR CAATGTTACTGTGGATTGGAGCGATTCTTTGTTTCTTGGCTT ATAGCATCGATGCTGCTACAGAAGAGGAACCTCAAAACGAT CGTGTGAGTTCTGTAATTCAGCATATCGATTTGTAGTACACA TCAGATATCTT ATP1A1 RD CAATGTTACTGTGGATTGGAGCGATTCTTTGTTTCTTGGCTT ATAGCATCAGAGCTGCTACAGAAGAGGAACCTCAAAACGAT GACGTGAGTTCTGTAATTCAGCATATCGATTTGTAGTACACA TCAGATATCTT ATP1A1 RD2 TCTTTGTTTCTTGGCTTATAGCATCAGAGCTGCTACAGAAGA GGAACCTCAAAACGATGACGTGAGTTCTGTAATTCAGCATA TGAATTCGTAGTACACATCAGATATCTTCTCCGTCTTTGTCT CCCACTTCTTCTCAATTACCACTCATTACTTAATGGTTAT ATP1A1 G4 RD AAGATATCTGATGTGTACTACAAATCCATATGCTGAATTAC AGAACTCACGTCATCGTTTTGAGGTTCCTCTTCTGTAGCAGC TCTGATGCTATAAGCCAAGAAACAAAGAATCGCTCCAATCC ACAGTAACATTG HBB #1 TCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACC ATGGTGCATCTGACTCCTGTCGAGAAGTCTGCAGTTACTGCC CTGTGGGGCAAGGTGAACGTGGATGAAGTTGGTGGTGAGGC CCTGGGCAG HBB #2 TCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACC ATGGTGCATCTGACTCCTGTGGAGAAGTCTGCAGTTACTGCC CTGTGGGGCAAGGTGAACGTGGATGAAGTTGGTGGTGAGGC CCTGGGCAG Supplementary Table 6 PCR primers used for Surveyor assays and expected size of cleavage products. Target Primer Size (bp) Surveyor Signal (bp) ATP1A1 SpCas9 G2 Forward GGATTAACATCTGCTCGTGCAGC ATP1A1 SpCas9 G2 Reverse CACTTGTAAGAGCATCTACAACG /238 ATP1A1 SpCas9 G3 Forward GGATTAACATCTGCTCGTGCAGC ATP1A1 SpCas9 G3 Reverse CACTTGTAAGAGCATCTACAACG /196 ATP1A1 SpCas9 G4 Forward GGATTAACATCTGCTCGTGCAGC ATP1A1 SpCas9 G4 Reverse CACTTGTAAGAGCATCTACAACG /246 ATP1A1 AsCpf1 G2 Forward GGATTAACATCTGCTCGTGCAGC ATP1A1 AsCpf1 G2 Reverse CACTTGTAAGAGCATCTACAACG /247 ATP1A1 AsCpf1 G3 Forward GGATTAACATCTGCTCGTGCAGC ATP1A1 AsCpf1 G3 Reverse CACTTGTAAGAGCATCTACAACG /246 ATP1A1 AsCpf1 G5 Forward GGATTAACATCTGCTCGTGCAGC ATP1A1 AsCpf1 G5 Reverse CACTTGTAAGAGCATCTACAACG /166 AAVS1 Forward TTCGGGTCACCTCTCACTCC AAVS1 Reverse GGCTCCATCGTAAGCAAACC /181 Nature Methods doi: /nmeth.4265

32 DNMT1 site 3 Forward DNMT1 site 3 Reverse EMX1 Forward EMX1 Reverse VEGFA Forward VEGFA Reverse GRIN2b-7 Forward GRIN2b-7 Reverse HBB Forward HBB Reverse DNMT1 site 3 OT1 Forward 1 DNMT1 site 3 OT1 Reverse 1 DNMT1 site 3 OT1 Forward 2 DNMT1 site 3 OT1 Reverse 2 AAVS1 OT1 Forward AAVS1 OT1 Reverse EMX1 OT1 Forward EMX1 OT1 Reverse EMX1 OT2 Forward EMX1 OT2 Reverse CTGGGACTCAGGCGGGTCAC CCTCACACAACAGCTTCATGTCAGC CCATCCCCTTCTGTGAATGT GGAGATTGGAGACACGGAGA GAGAAGGCCAGGGGTCACTCCAG AGCCCGCCGCAATGAAGG GCATACTCGCATGGCTACCT CTCCCTGCAGCCCCTTTTTA GACAGGTACGGCTGTCATCA CAGCCTAAGGGTGGGAAAAT GCCCCATTAGCCTGTGTTTT GTGTGCAAATAGGAAGGTGTCT CGGAAGTATTTCTGAGAGCATGG GAAGGTGTCTAACTCTCCAATCG GGGATGAGTCATGTCCACCACAG TTACATCCCCTCTGGGCATGAGC ATCTGCACATGTATGTACAGGAGTC TCTCTCCTTCAACTCATGACCAGC CACTGCCTACCTTCCTCTACTTC TCTTCATCCTCCTCATCTGAGGACTC / / / / / / / / / /200 Supplementary Table 7 Primers used for out-out PCR assays Target Primer Size (bp) LMNA OO Forward TGAGTCACACTGATGGGCACC LMNA OO Reverse TTCTAACAGGACCCTTTCTGCC 1404 P400 OO Forward CTCTCACCCTTTTCCCAAGA P400 OO Reverse AAGACCACGAGGCATTTTTC 988 EPC1 OO Forward AAGCCTGACACAAATCTCAGT EPC1 OO Reverse CCAAGGAGTCCACAGCTACC 943 Nature Methods doi: /nmeth.4265