An Approach to Proximity Ligation by T4 RNA Ligase to Screen sRNA That Regulate CRISPR-Cas Systems

CRISPR-Cas systems of prokaryotes are immune defense mechanisms against foreign nucleic acids via RNA-guided endonuclease activities. Small regulatory RNAs (sRNA) are essential for controlling various aspects in gene expression and function in bacteria. Here, we describe a protocol that utilizes T4 RNA ligase 1 (single-stranded RNA ligase) to link two base-paired RNA molecules to investigate potential interaction between sRNA and CRISPR-Cas system. Our goal is to provide readers a detailed method of identifying candidate sRNAs that may regulate CRISPR-Cas adaptation and/or other functions through unbiased screening and validation.