Supplementary Materials for

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1 advances.sciencemag.org/cgi/content/full/3/4/e /dc1 Supplementary Materials for CRISPR-Cpf1 correction of muscular dystrophy mutations in human cardiomyocytes and mice Yu Zhang, Chengzu Long, Hui Li, John R. McAnally, Kedryn K. Baskin, John M. Shelton, Rhonda Bassel-Duby, Eric N. Olson This PDF file includes: Published 12 April 2017, Sci. Adv. 3, e (2017) DOI: /sciadv fig. S1. Genome editing of DMD exon 51 and Dmd exon 23 by LbCpf1 or AsCpf1. fig. S2. Genomic PCR and T7E1 assay of off-target sites. fig. S3. Capillary electrophoresis and fragment analysis of DMD g1 off-target sites. fig. S4. Capillary electrophoresis and fragment analysis of DMD g2 off-target sites. fig. S5. Histological analysis of muscles from WT, mdx, and LbCpf1-edited mice (mdx-c). fig. S6. Immunohistochemistry of skeletal muscles, heart, and brain from WT, mdx, and LbCpf1-edited mice (mdx-c). fig. S7. H&E staining of skeletal muscles, heart, and brain from WT, mdx, and LbCpf1-edited mice (mdx-c). fig. S8. Western blot analysis of skeletal muscles, heart, and brain from WT, mdx, and LbCpf1-edited mice (mdx-c). fig. S9. T7E1 and Tse I RFLP analysis of germ cells from LbCpf1-edited mice (mdx-c) and uncorrected mdx mice. table S1. Sequence of the on-target site and 10 potential off-target sites. table S2. Primers used in the off-target site analysis. table S3. Comparison of CRISPR-Cas9 and CRISPR-Cpf1 mediated HDR correction in mdx mice. table S4. Sequence of primers.

2 Supplementary Materials Supplementary Figures

3 fig. S1. Genome editing of DMD exon 51 and Dmd exon 23 by LbCpf1 or AsCpf1. (A) DNA sequencing of DMD exon 51 from a mixture of DMD-patient skin fibroblast-derived ipscs (RIKEN 51) edited by LbCpf1 or AsCpf1 using g1. Sequences of individual edited DMD allele are shown beneath the uncorrected DMD allele. Δ denotes nucleotide deletion. (B) DNA sequencing of DMD exon 51 from a single clone of DMD-patient skin fibroblastderived ipscs (RIKEN 51) edited by LbCpf1 or AsCpf1 using g1. (C) DNA sequencing of Dmd exon 23 from 10T1/2 cells following LbCpf1-editing with g2 or g3. WT sequence is on top and INDEL sequences are on the bottom.

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5 fig. S2. Genomic PCR and T7E1 assay of off-target sites. (A) Off-target analysis of LbCpf1-editing with g1 targeting DMD exon 51. Upper panel is undigested PCR products and lower panel is T7E1 digestion from T7E1 assay on DMD on-target site and genome-wide top ten off-target (OT) sites. (B) Off-target analysis of LbCpf1-editing with g2 targeting DMD intron 50. Upper panel is undigested PCR products and lower panel is T7E1 digestion from T7E1 assay on DMD on-target site and genome-wide top ten off-target (OT) sites. (C) Offtarget analysis of LbCpf1-editing with g2 targeting Dmd exon 23. Upper panel is undigested PCR products and lower panel is T7E1 digestion from T7E1 assay on DMD on-target site and genome-wide top ten off-target (OT) sites. Red arrowheads point to cleavage products. and + denote the genomic DNA from unedited and LbCpf1-edited samples. M, marker. Offtarget sites location information can be found in table S1 and primers used for off-target sites analysis can be found in table S2.

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7 fig. S3 Capillary electrophoresis and fragment analysis of DMD g1 off-target sites. (A) Reconstructed gel images from the Fragment Analyzer electropherogram showing DMD g1 on-target site and predicted top-ten off-target sites. (B) Automatically calculated concentration of each peak and percentage cleavage value for DMD g1 on-target site and predicted top-ten off-target sites after T7E1 cleavage. and + denote the genomic DNA from unedited and LbCpf1-edited samples.

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9 fig. S4. Capillary electrophoresis and fragment analysis of DMD g2 off-target sites. (A) Reconstructed gel images from the Fragment Analyzer electropherogram showing DMD g2 on-target site and predicted top-ten off-target sites. (B) Automatically calculated concentration of each peak and percentage cleavage value for DMD g2 on-target site and predicted top-ten off-target sites after T7E1 cleavage. and + denote the genomic DNA from unedited and LbCpf1-edited samples.

10 fig. S5. Histological analysis of muscles from WT, mdx, and LbCpf1-edited mice (mdx- C). (A) Immunohistochemistry and H&E staining of whole tibialis anterior (TA) muscle. Dystrophin staining is red. (B) Immunohistochemistry and H&E staining of whole gastrocnemius/plantaris (G/P) muscles. Dystrophin staining is red.

11 fig. S6. Immunohistochemistry of skeletal muscles, heart, and brain from WT, mdx, and LbCpf1-edited mice (mdx-c). Immunohistochemistry of quadriceps, soleus, diaphragm, heart and brain from WT, mdx, and LbCpf1-edited mdx-c mice (HDR-8%, HDR-25%, HDR- 50% corrected allele) using antibody to dystrophin (red) shows restored dystrophin expression after LbCpf1-mediated correction. Scale bar = 100 microns.

12 fig. S7. H&E staining of skeletal muscles, heart, and brain from WT, mdx, and LbCpf1- edited mice (mdx-c). H&E staining of quadriceps, soleus, diaphragm, heart and brain from WT, mdx, and LbCpf1-edited mdx-c mice (HDR-8%, HDR-25%, HDR-50% corrected allele) shows reduced fibrosis and inflammatory infiltration after LbCpf1-mediated correction. Scale bar = 100 microns.

13 fig. S8. Western blot analysis of skeletal muscles, heart, and brain from WT, mdx, and LbCpf1-edited mice (mdx-c). Western blot analysis of limb muscles, diaphragm, heart and brain from WT, mdx, and LbCpf1-edited mdx-c mice (HDR-8%, HDR-25%, HDR-50% corrected allele) shows restored dystrophin expression after LbCpf1-mediated correction. Vinculin (VCL) is loading control.

14 fig. S9. T7E1 and TseI RFLP analysis of germ cells from LbCpf1-edited mice (mdx-c) and uncorrected mdx mice. (A) T7E1 assay of germ cells from LbCpf1-edited mice (mdx-c) and uncorrected mdx mice. Blue arrowhead denotes uncleaved DNA and red arrowhead shows T7E1 cleaved DNA. (B) TseI RFLP assay of germ cells from LbCpf1-edited mice (mdx-c) and uncorrected mdx mice. Blue arrowhead denotes uncorrected DNA. Red arrowhead points to TseI cleavage indicating HDR correction in germline. mdx-c6-c9 denotes LbCpf1-edited mdx mice.

15 Supplementary Tables table S1. Sequence of the on-target site and 10 potential off-target sites.

16 table S2. Primers used in the off-target site analysis.

17 table S3. Comparison of CRISPR-Cas9 and CRISPR-Cpf1 mediated HDR correction in mdx mice. Strain Dose of Cas9/sgRNA/ssODN (ng/ul) Dose of Cpf1/sgRNA/ssODN (ng/ul) Injection Method No. of Transferred Zygotes No. of Pups/zygotes (%) No. of Mutant Founders/Pups (%) No. of HDR/Pups (%) mdx 10/10/10 - Nuc (28%) 4 (14%) 1 (3.4%) - Nuc+cyt (39%) 7 (12%) 4 ( 6.9%) mdx 50/20/10 - Nuc (47%) 2 (6.7%) 0 - Nuc+cyt (19%) 3 (39%) 2 (8.9%) mdx - 50/20/10 Nuc+cyt (44%) 28 (23.7) 11 (9.3%)

18 table S4. Sequence of primers.