The current methods to control RNA functions in living conditions are limited. The new RNA-controlling strategy presented in this study involves utilizing 5-formylcytidine (f5C)-directed base manipulation. This study shows that malononitrile and pyridine boranes can effectively manipulate the folding, small molecule binding, and enzyme recognition of f5C-bearing RNAs. We further demonstrate the efficiency of f5C-directed reactions in controlling two different clustered regularly interspaced short palindromic repeat (CRISPR) systems. Although further studies are needed to optimize the efficiency of these reactions in vivo, this small molecule-based approach presents exciting new opportunities for regulating CRISPR-based gene expression and other applications.