The diversity of activity among guide RNAs
The guide RNA activity distributions of negative control(selective1) and screening experiment(selective2) were shown (Fig.1c). As there is almost no selection pressure of DNA cleavage, the guide RNA activity in the SE1 nearly obeyed a normal distribution with mean approximately equal to 1 as expected, while in SE2 there was significant variability of guide RNA activities. Concretely, the majority of guide RNAs in the designed library exhibited high activities that the best of which resulted in 1000-fold slowdown in growth rate of E. coli cells. To validate such results from the screening experiment, we chose a series of guide RNAs consisting of 9 with high scores and 2 with low scores to retest their activities individually by measuring colony number after transformation, and the results were observed in a very good consistency with which in the screening experiment (Fig.1d). Thus, it suggested that our screening method to evaluate guide RNA activity was reliable and such activity dataset could be used for further analysis.