...allow for two sets of microhomologies (5’ and 3’) to anneal, knocking the GFP-Puro cassette into the locus...protocol for MMEJ-mediated knock-in of a GFP-Puro cassette into a given locus, just upstream of a stop codon...microhomology to the insertion locus flanking the GFP-Puro cassette. Three double stranded breaks are necessary for...for knock-in: one on either side of the GFP-Puro cassette and one in between the 5’ and 3’ microhomologies...of PITCh. The PITCh plasmid contains a GFP-Puro cassette flanked by 5' and 3' microhomology and PITCh-gRNA...creates two DSBs in the PITCh plasmid to release the cassette, and an insertion-locus-specific gRNA cleaves ...and 3’ microhomologies are added to the GFP-Puro cassette via PCR, and this construct is inserted into the...