Table 1. Point mutation KI efficiencies in the MECP2 locus.
PM = point mutation. DSB = double-strand break.
| Point
mutation |
Selection,
plasmid amount |
Donor molecule
(ssODN is 10 μM) |
sgRNA:
ssODN |
PM ➔ DSB
distance |
Number of
upstream mutations |
Number of
KI clones |
KI efficiency | |
|---|---|---|---|---|---|---|---|---|
| 1 | R306C | Puro, 1.2 μg | 1.9 μg 2 kb plasmid | - | 31 bp | 1 | 0/191 | 0% |
| 2 | R306C | Puro, 2 μg | 4 μl 110 bp ssODN 1 | Not comp | 5 bp | 1 | 1/69 | 1.6% |
| 3 | R306C | GFP, 2.5 μg | 8 μl 110 bp ssODN 2 | Comp | 31 bp | 1 | 2/69 | 2.9% |
| 4 | R111G | GFP, 2 μg | 10 μl 100 bp ssODN 3 | Comp | 3 bp | 0 | 7/27 | 26% |
| 5 | R133C | GFP, 2 μg | 10 μl 100 bp ssODN 4 | Comp | 6 bp | 2 | 2/54 | 3.7% |
| 6 | T158M | GFP, 2 μg | 10 μl 100 bp ssODN 5 | Comp | 4 bp | 1 | 1/18 | 5.5% |
| 7 | T158M | GFP, 2 μg | 10 μl 100 bp ssODN 5 | Comp | 5 bp | 1 | 1/13 | 7.7% |