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. Author manuscript; available in PMC: 2015 Aug 7.
Published in final edited form as: Cell Stem Cell. 2014 Jun 12;15(2):215–226. doi: 10.1016/j.stem.2014.05.018

Figure 3. Single and multiplexed gene targeting.

Figure 3

(A) Strategy of gene targeting. (B and C) Allelic sequence distribution in HUES8 clonal lines generated with single gRNAs targeting NGN3 (Cr5, 6), GATA4 (Cr2), GATA6 (Cr1, 8), TET1 (Cr2), TET2 (Cr4), TET3 (Cr4) (B) or multiplexed gRNAs targeting GATA4 and 6 (Cr2, Cr1 respectively) or TET1, 2 and 3 (Cr2, Cr4, Cr4, respectively) (C). (D) Representative sequences of various knockout (KO) mutant clones with PAM sequences labeled in red. pm: point mutation. (E) Analysis of 5hmC levels in DNA isolated from TET1, 2, 3 triple-targeted hESC clones by dot blot assay using an anti-5hmC antibody. Wild-type HUES8 iCas9 cells and human foreskin fibroblasts (HFF) are used as controls. See also Figure S3, Table S2, S3.