Table 2.
Analysis of targeted addition of donor DNA with payload by HDR‐ or NHEJ‐mediated integration mechanisms
| Kanamycin resistance | Red fluorsc. | Junct. PCRs | NGS analysis | |||
|---|---|---|---|---|---|---|
| Calli | Positive | Analyzed calli | Single targeted insertion | Targeted insertion + X | ||
| Payloada (HDR) | ||||||
| 5 kb | 141 | 39 (28%) | 41 (29%) | 39 | 8 (21%) | 18 (46%) |
| 10 kb | 126 | 24 (19%) | 36 (29%) | 35 | 1 (3%) | 24 (69%) |
| 15 kb | 105 | 11 (11%) | 23 (22%) | 20 | 3 (15%) | 9 (45%) |
| 20 kb | 121 | 17 (14%) | 24 (20%) | 18 | 2 (11%) | 8 (44%) |
| 20 kb ZFN2+4 | 33 | 6 (18%) | 11 (33%) | 12 | 1 (8%) | 4 (33%) |
| Payloada (NHEJ) | ||||||
| 5 kb | 90 | 15 (27%) | 41 (46%) | 24 | 5 (21%) | 10 (42%) |
| 10 kb | 106 | 22 (21%) | 49 (46%) | 39 | 16 (41%) | 16 (41%) |
| 15 kb | 102 | 18 (18%) | 29 (28%) | 26 | 4 (15%) | 15 (58%) |
| 20 kb | 80 | 16 (20%) | 9 (11%) | 9 | 1 (11%) | 3 (33%) |
| 20 kb ZFN2+4 | 38 | 4 (11%) | 13 (34%) | 16 | 6 (38%) | 7 (44%) |
Junction PCRs were performed with primers npt5_F5 and nptII_3′ UTR_rev2 for nptII and primers dbbr_F2 and rfp3 or rfp3_R2 for DsRed (see Table S1 for sequences). Success rates are given for each payload/mechanism relative to all events tested for the phenotypical markers and the junction PCRs in number of calli and percentage. For NGS, numbers are given relative to all events analyzed by NGS in calli and percentage.
Abbreviations: fluorsc.: fluorescence; Junct., Junction; X, extra copies of donor DNA integrated either at the target site or at the target site and other non‐target genomic sites.
Payload is only the exchangeable part of the donor vector; integrated fragments are 2.2 kb larger (marker genes) for HDR and 5.2 kb larger (marker genes and donor vector backbone) for NHEJ.