tracrRNA

tracrRNA, short for trans-activating CRISPR RNA, is a small RNA component of the type II CRISPR-Cas adaptive immune system. It is required for the maturation of crRNA and for forming an active complex with the Cas9 nuclease that targets DNA. In bacteria such as Streptococcus pyogenes, tracrRNA is trans-encoded and contains an anti-repeat region that base-pairs with the repeat sequence of pre-crRNA. This RNA duplex is recognized by RNase III in the presence of Cas9, leading to processing of pre-crRNA into mature crRNA that remains associated with tracrRNA. The resulting Cas9–crRNA–tracrRNA complex then recognizes target DNA sequences adjacent to a PAM and cleaves the DNA.

Structurally, tracrRNA provides a scaffold and an anti-repeat element that enables base-pairing with crRNA. The interaction between tracrRNA, crRNA, and Cas9 is essential for guide formation and for the catalytic activity of Cas9 in many natural type II CRISPR systems.

In biotechnology, the natural two-RNA system was simplified into a single-guide RNA (sgRNA) by combining crRNA and tracrRNA into one molecule. The sgRNA retains the anti-repeat–crRNA pairing region and a Cas9-binding scaffold, enabling programmable DNA targeting with a single RNA molecule. This development greatly facilitated CRISPR-Cas9 genome editing and many other genome engineering applications.

Discovery and characterization of tracrRNA, along with the CRISPR-Cas9 mechanism, were key milestones in the CRISPR field, contributing to the rapid advancement of gene editing technologies in research and medicine.