Figure 5.

Analysis of HDR Efficiency

(A) PCR analysis of genomic DNA from CRISPR/Cas9-treated EBS hKc using a forward primer binding to exon 6 and a reverse primer binding a sequence downstream of KRT14. To minimize the size of the PCR product and to facilitate subcloning, we performed a nested PCR using a forward primer binding to exon 6 and a reverse primer binding to intron 7, resulting in a specific product of 507 bp. An EcoRI restriction digest confirmed the presence of recombined KRT14 alleles in treated EBS cells. Upon bacterial transformation, single colonies were picked for colony PCR and amplified product digested with EcoRI to identify recombined alleles. (B) Sequence analysis of one representative single clone, containing a modified wild-type allele (green arrow: wild-type sequence at mutation site [position 178] within exon 6), is shown. The allele additionally contains the introduced restriction sites NheI/EcoRI and the silent mutations (blue stars). Sequence alignment was performed with the software “Multiple sequence alignment with hierarchical clustering.”²¹