Synonymous mutations of the crRNA and the PAM do not affect self versus non-self discrimination.

<p><b>A</b>) Infectivity of a library of M13 phage containing PAM mutants adjacent to the g8 protospacer was tested against cells expressing Cascade, Cas3 and g8 CRISPR containing mutations at the −1, −2 and −3 nucleotides of the CRISPR repeats. PAM mutations are shown on the left, with the PAM sequences indicated in the 3′ to 5′ direction. CRISPR repeat mutations at positions −1, −2 and −3 are indicated on the top in the 5′ to 3′ direction. Underscored sequences have been tested for binding affinity by EMSA. Base pairing potential between the PAM positions and the repeat is indicated using numbers (0–7) that correspond to a base pairing pattern that is shown in the panel on the right. A 0 signifies no base pairing, a 1 signifies base pairing at the −3 position, a 2 signifies base pairing at the −2 position, etc. Black circles with white digits indicate resistance against phage infection (e.o.p.<10⁻⁴), grey circles indicate partial resistance (e.o.p.∼10⁻²) and red digits without circle indicate susceptibility to phage infection (e.o.p. = 1), as determined by phage spot assays. Letters B, C, D, E indicate combinations shown in detail in the corresponding panels. <b>B</b>) Combination of g8^C-3AC-2T CRISPR and M13 phage with CAT PAM, gives rise to full base pairing and a lack of resistance (red font). <b>C</b>) Combination of g8^C-3AC-2T CRISPR and M13 phage with CTC PAM, gives rise to only base pairing at the −1 position and yields a lack of resistance (red font). <b>D</b>) Combination of g8^C-3AC-2A CRISPR and M13 phage with CTT PAM gives rise to full base pairing and a lack of resistance (red font). <b>E</b>) Combination of g8^C-3AC-2A CRISPR and M13 phage with CTC PAM gives rise to only two potential base pairs at the −1 and −2 positions and yields a lack of resistance (red font). Note in (A–E) PAMs are oriented in 3′ to 5′direction to display base pairing potential with the last three nucleotides of the crRNA repeat.