Figure 2.

Gene Editing in the PKLR Locus

(A) Diagram showing where therapeutic matrix is introduced by HR in the PKLR locus. The strategy to identify the integrated matrix by PCR (horizontal arrows) and Southern blot (vertical arrows) indicating the expected DNA fragment sizes is shown, and the line over the Puro^R/thymidine kinase fusion cassette indicates probe location. Small squares at the beginning and end of the partial codon-optimized (cDNA)RPK indicate splicing acceptor and FLAG tag sequences present in the cassette, respectively; light gray squares represent endogenous (mRNA)RPK exons; dark gray squares represent the first LPK exon and 3′ UTRs at the beginning and at the end of the PKLR gene, respectively; and black squares represent homology arms.

(B) DNA electrophoresis of gDNA from Puro^R-PKD2iPSC clones, amplified by PCR to identify specific matrix integration.

(C) Southern blot of gDNA from edited PKD2iPSC clones, digested by ScaI or SpeI to confirm the precise integration of the matrix in the PKLR locus.

See also Figures S3 and S4 and Table S2.