Clustered, regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) genes, a diverse family of prokaryotic adaptive immune systems, have emerged as a biotechnological tool and therapeutic. The discovery of protein inhibitors of CRISPR-Cas systems, called anti-CRISPR (Acr) proteins, enables the development of more controllable and precise CRISPR-Cas tools. Here we discuss applications of Acr proteins for post-translational control of CRISPR-Cas systems in prokaryotic and mammalian cells, organisms and ecosystems.