Adaptive immune systems must accurately distinguish between self and non-self in order to defend against invading pathogens while avoiding autoimmunity. Type III CRISPR-Cas systems employ guide RNA to recognize complementary RNA targets, which triggers the degradation of both the invader’s transcripts and their template DNA. These systems can broadly eliminate foreign targets with multiple mutations but circumvent damage to the host genome. Biochemical and structural studies did not reveal the molecular underpinnings for these unique features, indicating that they are linked to the differential dynamics of the enzyme-target assemblies. In collaboration with the Marraffini Lab, we use single-molecule fluorescence resonance energy transfer (FRET) to study the interactions between a type III-A ribonucleoprotein complex and various RNA substrates. Our data show that Cas10—the DNase effector of the complex—undergoes rapid conformational fluctuations on foreign RNA targets but is locked in a static configuration on self RNA. Target mutations modulate Cas10’s dynamics and, accordingly, tune the CRISPR interference activity in vivo. These findings highlight that the internal dynamics of CRISPR-Cas complexes play a central role in differentiating self from non-self and specifying targets for immunity.