Extended Data Fig. 7: Comparison of recombinase and CRISPR/Cas9 approaches for the presence of unintended deletion on the endogenous F8 locus in human cells.

Detection of inversion and excision (deletion) on the F8 locus in HEK293T cells assessed 48 h post mRNA transfection with D7, D7-ZF or Cas9 in combination with a gRNAs specific for the inverted repeat as published by Park et al.³². a Schematic representations of a fraction of the F8 gene, displaying the two possible orientations of the loxF8 locus (wild-type and inverted orientation) and two possible deletion outcomes (recombinase- or Cas9-induced) are shown (adapted from Lansing et al.⁸). Primers used for PCR to detect the orientation of the locus are displayed as green, blue, and red arrows. The position of the loxF8 sites, gRNA binding sites and the distance between them are indicated. b Agarose gel images of PCR products generated using the indicated primer combinations. The HEK293T cells treated with GFP mRNA only were used as a wild-type control, whereas iPSCs derived DNA from a patient carrying the int1h inversion were used as an inversion control. Primer combinations are indicated. M = Marker. The assay was performed three times (n = 3 biologically independent replicates). c Depiction of a potential precise Cas9-induced 142 060 bp deletion between the two gRNA binding sites. Sequences of the clones obtained from TA cloning of the deletion product induced by Cas9 are schematically shown below, indicating the size of the genomic fragments that were deleted in addition to the 142 060 bp.