Conditionally Active CRISPR-Cas9 System
CRISPR-Cas9 is an endonuclease protein that was originally discovered in S. pyogenes. The most commonly-used Cas9 protein is derived from S. pyogenes and uses a single guide RNA (sgRNA) to guide its activity. The Cas9 sgRNA recognizes a 20 bp target DNA sequence, which it binds to and cleaves to alter the DNA sequence. However, target sequence binding can tolerate mismatches up to several base pairs, meaning there are often thousands of possible binding sites. If these complexes do not bind at the target, they will create off-target double-strand breaks and cause unintended, potentially harmful genetic modifications.
Thus, the 2021 Yale iGEM team set out to design and validate a conditionally activated CRISPR-Cas9 system to precisely knock out disease-causing genes in cell culture, with the ultimate, long-term goal of implementing genetic logic inside living cells.
There were two main objectives to this project: (1) validation in vitro of gene knockout, and (2) validation in vitro of conditional gene knockout (blocking and reactivation of Cas9).
We started by testing for gene knockout in vitro We conducted in vitro cleavage (IVC) experiments using sgRNAs that we designed and concluded that our sgRNAs successfully guided the Cas9 protein to the correct target DNA location. In other words, we confirmed that the fundamental gene knockout mechanism would function properly using our synthetic guide.
The objective of our second phase was to demonstrate inactivation and reactivation of Cas9. The idea was to turn OFF Cas9’s gene-knockout activity, then turn it back ON in the presence of a specific “trigger” molecule. This phase proved significantly more challenging due to the addition of two non-natural components to the Cas9 system, but we achieved successful inhibition and reactivation of Cas9 in vitro. In the future, we aim to achieve successful inhibition and reactivation in vivo.