Extended Data Fig. 3: Data rejecting alternative explanations for the impact of mutating the leader region associated with SpyCas9.

a, Assessing targeting by the mutated ecrRNA guide by plasmid clearance in E. coli. The native and mutated ecrRNAs were encoded as single-spacer arrays with the native leader. There was no significant difference in plasmid clearance with (Student’s two-tailed t-test with unequal variance, P = 0.36, n = 3) or without (Student’s two-tailed t-test with unequal variance, P = 0.80, n = 3) outgrowth. b, Western blotting analysis of SpyCas9-3xFLAG levels with the native or mutated leader. Results are representative of two independent experiments. c, Plasmid clearance with SpyCas9-3xFLAG in E. coli. The SpyCas9-3xFLAG fusions were tested using an sgRNA with a guide derived from spacer 1 (S1) in the native array. The transformations were conducted without non-selective outgrowth. The results showed that the fusion did not compromise clearance activity by SpyCas9, and introducing the mutations into the CRISPR leader did not significantly affect SpyCas9 activity (Student’s two-tailed t-test with unequal variance, P = 0.168, n = 3). d, Assessing transcription of the CRISPR array with the mutated leader. The native or mutated leader through the first spacer was cloned upstream of gfp in the pUA66 plasmid. E. coli cells harboring either plasmid were then subjected to flow cytometry analysis. There was no significant difference (Student’s two-tailed t-test with unequal variance, P = 0.103, n = 3) in the background-subtracted GFP fluorescence between the constructs. Values represent the mean and standard deviation of triplicate independent measurements starting from separate colonies. Values in a, c and d represent the geometric mean and standard deviation from independent experiments starting from three separate colonies. n.s.: not significant. n.s.: P > 0.05. Statistical tests were performed using a two-tailed Student’s t-test with unequal variance, n = 3.

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