Figure 3.

Illustration of a C–terminal P2A-eGFP–floxed puromycin expression cassette knock-in for gene of interest (GOI) by CRISPR/Cas9 editing. (A) A gRNA was designed to overlap with the stop codon of a GOI, and the donor vector contained homology arms of 800–1000 bp. After HDR, a P2A-eGFP–floxed puromycin expression cassette will be inserted in frame just after the coding region and before the stop codon. Screening primer 1 was designed outside of the left homology arm to prevent detection of the donor plasmid. The insert–specific primer 2 was designed on eGFP. (B) Results of pooled genomic PCR from mouse embryonic stem cells that detects the insertion of the desired sequence into the genome. (C) Sequence results from a single clone indicate precise integration of the desired sequence into the genome. Only the results of the 5′ sequence are shown. UTR, untranslated region.