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. 2019 Apr 30;10:365. doi: 10.3389/fgene.2019.00365

FIGURE 4.

FIGURE 4

Targeting of the beta-2 microglobulin gene (A) The beta-2 microglobulin locus (B2M) was targeted in HEK cells by HDR mediated insertion of a T2A-mCherry coding region placed upstream of the B2M Stop codon and 3′untranslated region (3′-UTR) using a donor vector with 600 bp homology regions (0.6). (B) Four days after the transfection of HEK cells with expression vectors for Cas9 or Cas9 fusion protein, sgRNA against the B2M target region and the donor vector, the frequency of Cherry positive cells was determined by flow cytometry (FACS). (C) Bar graph representation of Cherry positive cells, indicating HDR at the B2M locus upon transfection with an expression vector for Cas9, or Cas9 in C-terminal fusion with MRE11A, Rad52, CtIP wildtype (WT), or the CtIP mutant T847E (mut). Bars show mean values of four independent samples with standard deviation. These values were used to calculate p-values (T-test) to determine the significance in levels of cherry+ cells between samples 1 and 2–5.