Table 3. Deep sequencing analysis of targeted gene editing versus off-target effects in human CD34+ haematopoietic cells following ex vivo treatment with SCF and γtcPNA4/donor DNA NPs.
| Gene locus | Sequences of partial homology (5′–3′) | Size of region sequenced | Alleles sequenced | Number modified | Frequency (%) |
|---|---|---|---|---|---|
| β-globin | TGCCCTGAAAGAAAGAGA | 128 | 12,489,910 | 606,230 | 5.02 |
| Serine Theronine Kinase | ATTCCTGAAAGAAAGCAC | 189 | 3,330,879 | 0 | 0 |
| Anoctamin-3 | AATTCTGAAAGAAAGACC | 150 | 5,805,518 | 1 | 0.000017 |
| 39s Ribosomal protein L17 | AGCCCTGAAAGAATACCA | 169 | 4,211,251 | 0 | 0 |
| Neuroblast differentiation associated | TCCCTGAAAGAAAAAAGA | 198 | 3,579,389 | 2 | 0.000055 |
| Transcription enhance factor TEF1 | TCTCCCTGAAAGAAAAAA | 244 | 80,548 | 0 | 0 |
| Rho GTPase activating protein | CAACATGAAAGAAAGAGA | 154 | 8,256,220 | 0 | 0 |
| Total off-target | 25,263,805 | 3 | 0.000012 |
The top six gene loci in the human genome with partial homology to the 18 bp γtcPNA4 target site in β-globin intron 2 were identified, with the sequences as indicated. Human CD34+ haematopoietic cells were treated ex vivo with SCF and with NPs containing γtcPNA4/donor DNA, and 2 days later genomic DNA from the cells was subject to deep sequencing analysis at these loci. The size of the region sequenced around each site is listed, along with the number of alleles sequenced and the number of alleles with modified sequences.