FIGURE 2.

Genotyping analysis of HEK293 cells targeted with miR-21-directed TALENs and HR donor. (A) Combination of genome-specific and donor-cassette-specific primers amplified expected PCR products of 830 bp (5′ arm) and 1281 bp (3′ arm) in cells cotransfected with TALENs and the HR donor vector. Transfection with only the HR donor vector did not result in any detectable PCR product. (B) Subsequent sequencing of the amplified PCR products confirmed seamless integration of both HR donor vector arms. (C) Selection reveals RFP-positive puromycin-resistant single cell-derived colonies. (D) A triple primer PCR strategy was used to determine whether mono- or bi-allelic HR events had occurred in single cell-derived clones. A donor-specific and a wild type-specific forward primer were combined with a wild type-specific reverse primer. Alleles with donor cassette integration were recognized by a 1281-bp amplicon, while WT or NHEJ events result in a 1067-bp amplicon. (E) Examples of seed region modification by NHEJ, compared to the wild type.