CRISPR-Mediated Targeted Gene Integration

The pCas-9-GFP, pCas9D10A-GFP, and gRNA cloning vectors were obtained from Addgene. The traditional MEF2C FlagBio target vector containing the FlagBio ORF and Frt-Neo-Frt cassette flanked by 1 kb and 4 kb homolog arms was made by recombineering [7] (link). The dsDNA donor with 50 bp homolog arms on each side was made by PCR, using primers harboring 50 bp homology sequences. The Oct4-GFP donor with 200 bp homolog arms was made using the Gibson assembly technique [8] (link) using a kit from New England Biolabs, with pCR-Blunt II-TOPO vector (Life Technologies) as backbone.
Oligonucleotides were synthesized by Integrated DNA Technologies. Primer sequences are indicated in Table 1. gRNAs were designed using the CRISPR design tool (crispr.mit.edu) [9] (link).