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We acknowledge the efforts and expertise of the New York Genome Center. S. Wu assisted with data analysis. K.A.S. is supported by NIH grant F31EY026789. V.B.M. and A.G.B. are supported by NIH grants (R01EY026682, R01EY024665, R01EY025225, R01EY024698, R01NS098590 and R21AG050437) and Research to Prevent Blindness (RPB), New York, New York. The Bernard & Shirlee Brown Glaucoma Laboratory is supported by NIH grants (5P30EY019007, R01EY018213). The National Cancer Institute Core is supported by an NIH grant (5P30CA013696), the RPB Physician-Scientist Award, and unrestricted funds from RPB, New York, New York, USA. S.H.T. is a member of the RD-CURE Consortium and is supported by the Tistou and Charlotte Kerstan Foundation, the Schneeweiss Stem Cell Fund, New York State (grant C029572), the Crowley Family Fund, and the Gebroe Family Foundation.
The authors declare no competing financial interests.
Integrated supplementary information
Heat maps represent the percentage of specific point mutation nucleotide changes detected in CRISPR-treated animals. For both animals, most mutations were G to A and C to T mutations.
a. Example of a coding SNV confirmed to be heterozygous in F03. F05 is wildtype as is reported in Supplementary Tables 1 and 2. b. Example of an intronic SNV confirmed in both mice as reported in Supplementary Table 5. c. Example of an SNV in a snoRNA confirmed in both mice as reported in Supplementary Table 5.
Supplementary Figure 3 Sequence alignment of guide RNA to actual off-target regions does not show substantial homology.
a. The top-10 predicted, off-target regions aligned to the gRNA. Sequences are 80 to 95% homologous to the gRNA. b. Regions surrounding 10 selected, experimentally-observed SNVs in coding regions aligned to the gRNA. All regions were observed in both CRISPR-treated mice. Sequences are 15 to 45% homologous to the gRNA. c. Regions surrounding 10 selected, experimentallyobservedSNVs in non-coding regions aligned to the gRNA. All regions were observed in both CRISPR-treated mice. Sequences are 5-65% homologous to the gRNA. d. Regions surrounding 10 selected, experimentally-observed indels in both coding and non-coding regions aligned to the gRNA. All regions were observed in both CRISPR-treated mice. Sequences are 25 to 65% homologous to the gRNA.
Pie charts show the SNVs and indels detected in WGS of CRISPR-treated animals based on assigned biotype. Biotypes were assigned by SNPEff software (Cingolani P, et al. Fly, 2012.). Intragenic regions were not assigned a biotype.
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Schaefer, K., Wu, W., Colgan, D. et al. Unexpected mutations after CRISPR–Cas9 editing in vivo. Nat Methods 14, 547–548 (2017). https://doi.org/10.1038/nmeth.4293
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