Table 1.
Kinetic parameters for Cas9 enzymes.
| DNA binding and R-loop formation | HNH cleavage | RuvC alignment | RuvC cleavage | |||||
|---|---|---|---|---|---|---|---|---|
| Enzyme | 1/K11 (nM) | Kd,net2 (nM) | k2 (s−1) | k-2 (s−1) | k3 (s−1) | k4 (s−1) | k-4 (s−1) | k5 (s−1) |
| SpCas9 on-target | [3]3 | [1]3 | 2.5 ± 0.2 | 1.2 ± 0.4 | 5.6 ± 0.4 | 1.8 ± 0.3 | 2.3 ± 0.6 | 4.4 ± 0.4 |
| SpCas9 off-target | 24 | 8.1 | 1.1 ± 0.1 | 0.5 ± 0.1 | 0.17 ± 0.01 | 0.7 ± 0.1 | 0.30 ± 0.05 | 0.38 ± 0.02 |
| Hypa on-target | 4.3 | 1.6 | 0.7 ± 0.03 | 0.4 ± 0.02 | 0.042 ± 0.0007 | 1.0 ± 0.02 | 0.08 ± 0.006 | 0.054 ± 0.001 |
| Hypa off-target | 52 | 17 | 0.8 ± 0.1 | 0.4 ± 0.01 | 0.005 ± 0.001 | 0.04 ± 0.001 | 0.0004 ± 0.0003 | 0.008 ± 0.003 |
| Cas9HF1 on-target | 1.6 | 0.12 | 2.2 ± 0.2 | 0.2 ± 0.08 | 0.038 ± 0.002 | 0.71 ± 0.04 | 0.02 ± 0.01 | 0.032 ± 0.002 |
| Cas9HF1 off-target | 33 | 9.8 | 2.5 ± 2 | 0.7 ± 0.3 | 0.0002 ± 0.00002 | 1.1 ± 0.8 | 0.4 ± 0.2 | 0.0002 ± 0.00005 |
1We used a nominal value of k1 = 1 nM-1s-1 and fit data to derive k-1 to compute K1 = k-1k1.
2Kd,net = 1/(K1(1 + K2)), where K2 = k2/k-2
3Values for DNA dissociation rates are a function of k-1 and k-2, and for SpCas9 on-target DNA these values are not well defined by the data because of the low kinetic partitioning for DNA dissociation (Fig. 4a). For global data fitting, we locked rate constants k-1 = 3 s−1 at a nominal upper limit. Because this rate constant was not well-defined by the data, locking it at a reasonable upper limit had little effect on the values for other rate constants.