TABLE 1.
Kinetic parameters of AsCas12a pre-crRNA processing
| kon (M−1 sec−1) | koff (sec−1)b | Kd (pM) | kc (sec−1) | |
|---|---|---|---|---|
| pre-crRNA(−34:+24) | 2.9 (±0.2) × 106 | 1.6 (±0.5) × 10−6 | 0.56 (±0.18) | 2.3 (±0.3) × 10−3 |
| pre-crRNA(−23:+24) | 2.6 (±0.2) × 106 | 3.6 (±0.3) × 10−6 | 1.4 (±0.2) | 2.3 (±0.2) × 10−3 |
| pre-crRNA(−34:0) | 6.7 (±0.3) × 105 | 5.3 (±0.7) × 10−6 | 7.2 (±1.4) | 1.6 (±0.5) × 10−3 |
| pre-crRNA(−23:0) | 6.2 (±1.2) × 105 | 6.7 (±0.5) × 10−6 | 5.0 (±1.3) | 1.8 (±0.15) × 10−3 |
| *crRNA(−18:+24) | 5.9 (±0.7) × 106 | 4.5 (±0.3) × 10−6 | 0.76 (±0.09) | NA |
| crRNA(−18:+24)*a | 1.4 (±0.3) × 107 | 7.4 (±0.2) × 10−6 | 0.53 (±0.12) | NA |
| crRNA(−18:+24) | ND | 2.2 (±0.3) × 10−5c | ND | NA |
| crRNA(−18:0)a | 1.7 (±0.2) × 105 | 6.7 (±0.2) × 10−5 | 390 (±50) | NA |
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aFor crRNA variants, the asterisk indicates whether the radiolabel is at the 5′ end (*crRNA) or the 3′ end (crRNA*).
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bFor the determination of the pre-cRNA dissociation rate constants, we used a pre-crRNA substrate containing dU at position −19 to prevent Cas12a-mediated cleavage.
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cMeasured using excess pre-crRNA(−34: + 24) in multiple turnover reactions.
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(NA) Not applicable, (ND) not determined.
