Fig. 2

Imperfect microhomology simultaneously creates iPSCs with patient mutations and their isogenic controls. a Schematic overview of the MMEJ method for editing HPRT_Munich and control alleles. Left and right homology arms overlap, generating a 13 bp tandem µH (blue) flanking the selection cassette (red). The patient mutation (c.312C > A, red) is present in one µH (unilateral) or both (bilateral). A silent bilateral point mutation (c.306G > T, blue) generates an AflII site. Complementary ps1 protospacers (black) are nested divergently between the µH and cassette, with sequences and cut site positions indicated in green above. Gene targeting used AvrHPRT1_B TALENs (yellow bolt). Upon transfection of targeted clones with CRISPR-Cas9 (pX-ps1), DSBs are generated flanking the cassette, proximal to the engineered µH (green bolts). Repair by MMEJ scarlessly excises the cassette, resulting in two possible editing outcomes. b Detailed schematic of HPRT1 gene targeting and MMEJ resolution. Exons (gray), overlapping homology arms (HA-L/R, white), µH (blue), ps1 CRISPR-Cas9 target sites (green), and engineered bases are indicated. 2A-puroΔTK is inserted in-frame with HPRT1 exon3. Black bars indicate Southern blot probes for the indicated restriction fragments. Genotyping primers are shown in red. c FACS scheme used to enrich mCh^neg cassette-excised iPSCs. Sorted populations were plated with or without HAT selection for clonal analysis to determine the frequency and fidelity of repair. d Southern blot analysis of select excised clones revealing restoration of the HPRT1 locus (HPRT-B probe, top) and removal of the cassette (mCherry probe, bottom). Parental 1383D6 and intermediate 033-U-45 and 033-B-43 targeted iPSCs are included as controls. Genotypes (S, Silent only; M, Munich and Silent) are indicated above. “x” indicates one clone with aberrant banding. e Sequencing examples of iPSC clones with Munich and/or Silent mutations derived from clone 033-U-45 (unilateral). Both types of clones are isolated from the same excision experiment