Figure 1.

Generation of CRISPR-Cas9-mediated knockin H7 HK1-EGFP reporter

(A) Schematic illustration of the generation of the knockin reporter into the HK1 locus. HK1 sgRNA mediated a double-strand break within the HK1 locus replacing the stop codon with a donor cassette V5-2A-EGFP sequence in frame.

(B) Clonal expansion of H7 HK1-GFP knockin reporter cell. hESCs (H7) were electroporated with HK1 sgRNA and donor vector. The first round of fluorescent-activated cell sorting for GFP-positive cells showed 3.26% efficiency. The second round of fluorescent-activated cell sorting performed to enhance the clonal efficiency showed 65.8% efficiency.

(C) Representative images of H7 HK1-GFP clones. Green fluorescent colonies reflect the expression of HK1 gene in these clones. Scale bar, 100 μm.

(D) Western blot showing nine H7 HK1-GFP clones harboring the V5-2A-EGFP knockin sequence, which was absent in wild-type H7 ESCs. Cell lysates were probed with antibody against the V5 tag.

(E) PCR genotyping results indicated that all nine clones possess the V5-2A-EGFP knockin sequence.