Fig. 1

Inserting PAC into a single Evx1 locus using CRISPR/Cas9-mediated homology-directed repair. A Schematic showing electroporation of mESCs to incorporate 3 plasmids: SpCas9, Evx1 guide RNA (gRNA), and the pWS-TK3-Evx1-PAC donor vector. The donor vector contains homology arms (5′ and 3′ HAs) against Evx1 genomic sequences flanking the cut site, PAC, floxed PGK-Neo for positive selection, and the negative selection marker TK3. B Schematic showing SpCas9 being guided to the Evx1 genomic locus by Evx1 gRNA. The Cas9 cut site is flanked by homologous sequences to the HAs of the donor vector, which is inserted into the double-stranded break (DSB) through homology-directed repair (HDR). Cre-mediated recombination removes the Neo cassette so that only PAC remains in the Evx1 locus. C jPCR images showing the wildtype RW4 mESCs as a control, with PAC insertion in both Evx1-PAC and Neo-excised (n.e.) Evx1-PAC, as well as presence of Neo in Evx1-PAC and absence in Evx1-PAC n.e. mESCs. D Copy number assay using the previously established Hb9-puro mESC line as a control for one copy of PAC and RW4 mESCs as a control for two copies of GAPDH as a reference. Evx1-PAC and Evx1-PAC n.e. mESCs show one copy of PAC. Error bars show the range of possible copies, not S.E.M