Fig. 5

ZFN-mediated gene editing is highly specific and characterized by high frequency of on-target small indels. (a) Experimental strategy to confirm homozygote clones and identified large deletions. Left: With the original 140 bp sequencing assay, clones with only 1 indel pattern may represent homozygote clones or clones with an uncaptured deletion spanning a primer site. Center: 12 homozygous clones could be confirmed by a sequencing assay that captures an informative SNP in 2 healthy donors. Right: For samples with no informative SNPs or uncaptured events, a 12 kb region around the target site was sequenced. Example shows a heterozygous sample with a large deletion that was not captured by the small 140 bp amplicon and therefore ruled out as a homozygote by the subsequent analysis. (b) Frequency of identified allele lengths in clones (N = 1175) derived from healthy donors (n = 4). A 12 kb region was sequenced around the BCL11A enhancer in clones with alleles potentially missed by short amplicon sequencing. Indels > 1 kb represent less than 1% of events. Unknown events include 3 insertions for which the length could not be determined. (c) Depiction of the largest deletion observed (5.9 kb). The deletion is contained within the BCL11A erythroid super-enhancer, itself located within the 77 kb intron 2 of BCL11A. (d) Total fraction of indels that disrupt the core GATAA site. (e) Enrichment of MMEJ indels with large deletions. The figure shows the frequency of small indels (MMEJ or NHEJ) as the second allele of clones carrying a large deletion (> 50 bp), a large insertion (> 50 bp), a complex indel, or a NHEJ indel. MMEJ indels are over-represented as the second allele of clones with deletions > 50 bp (Fisher exact test P = 0.008). Complex indels and insertions show over- and under-representation of large deletions (P = 0.02 and 0.03, respectively). Data from healthy donor cells (n = 4). MMEJ patterns represent those identified by the RGEN Microhomology-Predictor tool (methods). bp, base pairs; HD, healthy donor; MMEJ, microhomology-mediated end joining; NHEJ, non-homologous end joining; SNP, single nucleotide polymorphism.