Table 1.

SMRT Sequencing confirms on- and off-target activity at sites ranked by PROGNOS

We interrogated 138 highly ranked genomic loci for the novel TALENs and ZFNs using SMRT, and observed off-target activity in 13 cases, nine of which were outside the globin gene family. The ‘match type’ indicates the orientation of the left (L) and right (R) nucleases at the site and the length of the spacer sequence. In sequences, lower-case red letters indicate mutations compared to the target site. Site sequences are listed as 5′—(+) half-site—spacer—(–) half-site—3′. Therefore, the (–) half-site for TALENs and the (+) half-site for ZFNs are listed in the reverse anti-sense orientation compared to the DNA sequence that the nuclease binds. Rankings by the initial PROGNOS algorithms Homology (column H), RVDs for NK (RK), RVDs for NN (RN) and Conserved G’s (C) are displayed as well as the rankings by the refined ‘TALEN v2.0’ algorithm for NK (TK) and for NN (TN) and the ‘ZFN v2.0’ algorithm (Z). 293T modification frequency is the percentage of observed sequences showing evidence of non-homologous end-joining repair. For the CCR5 ZFNs, 15 off-target sites ranked by PROGNOS that had not been previously investigated were interrogated using SMRT, validating a novel off-target site. Additionally, six known highly active off-target sites were sequenced as positive controls. The PROGNOS rankings for the site near KDM2A are listed as ‘N/A’ because the site was not found by PRGONOS due to the high number of mismatches. *P < 0.05 in cells expressing active nuclease compared to cells expressing empty vector. **P < 0.05 for the difference in activity between NK and NN at that site.