(27y) Characterizing Cas12i Proteins As Novel Enzymes for Diagnostics and Gene Editing

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas (CRISPR-Associated protein) systems are powerful biotechnology tools used for genome editing and diagnostics. Within the vast array of the CRISPR effectors, Type V systems are characterized by RNA-guided Cas12 proteins which are capable of nonspecific cleavage of single-stranded DNA. Several Cas12 proteins have not been as extensively explored as Cas12a and Cas12b, thus here we are characterizing an understudied Type V Cas enzyme called Cas12i. First identified in 2018, Cas12i belongs to type V-I family of CRISPR enzymes. Similar to Cas12a, Cas12i has also been shown to pre-process its own crRNA to perform CRISPR-mediated interference and therefore required a significantly shorter guide RNAs as compared to other Cas12 enzymes such as for e.g., Cas12b. In this work, we explore the nuclease activity of this novel enzyme by characterizing its cis-cleavage and trans-cleavage capabilities and study its potential to be used for diagnostics and gene editing purposes. Notably, Cas12i trans-cleavage activity can be substantially amplified by using engineering crRNAs, opening the door for designing an enhanced version of the enzyme. Additionally, we experimented into combining Cas12a and Cas12i properties by interchanging their canonical cRNA. This type of phenomenon has not been explored between two different classes of Cas12 proteins, which promotes designing universal crRNAs that can work for multiple different enzymes simultaneously. In conclusion, we seek to widely explore and enhance Cas12i’s activity in order to extend the use of this protein and eventually apply it as another tool for improved diagnostics and gene editing within the CRISPR-related systems.