Figure 1.
Rationale for CTS and Proof of Feasibility
(A) Cells are edited simultaneously with gene-specific CRISPR/Cas9 and ssODN containing a variant of interest (red asterisk) as well as plasmids expressing AAVS1-specific TALENs and a puromycin resistant (PuroR) donor cassette.
(B) Components from (A) are transfected into iPSCs where HDR-receptive cells (green) are more likely to incorporate donor DNA than HDR-refractory cells (red).
(C) Precision-edited versus indel-containing alleles, detected via direct Sanger sequencing of relevant PCR products in hB53 hiPS6 iPSCs following CTS (n = 39) compared with no selection (n = 46) or transient exposure to puromycin (n = 48) (left panel), where n is the number of individual iPSC clones analyzed. Percent of clones bearing the CRYAB:c.325G>C variant (middle panel) and the number of heterozygous/homozygous clones (right panel).
(D) Representative chromatograms showing local CRYAB sequence of a wild-type (WT) clone (top) and that of a clone bearing a CRYAB:c.325G>C (homozygous) knockin allele (black arrow, c.325G>C variant; gray arrow, Cas9-blocking silent variant). See also Figures S1, S2, S4 and Table S1.