Enzymatic Assembly for CRISPR Split-Cas9 System: The Emergence of a Sortase-based Split-Cas9 Technology

CRISPR-Cas9 has been widely used in scientific research and medical investigations as a pioneering technology. However, challenges such as the large size of the Cas9 sequence and potential off-target effects have impeded its widespread adoption. In response, various alternatives, such as split-Cas9 technology, have emerged. Split-Cas9 systems allow the large Cas9 sequence to be divided into two segments to aid in the delivery of the enzyme. Nevertheless, challenges persist in achieving precise control over the timing and location of Cas9 reassembly and activity to minimize off-target effects. This study presents an enzymatic-based split-Cas9 system, introducing a new approach utilizing the Sortase enzyme for the reconstitution of the full Cas9 protein. The developed method eliminates the need for chemical or physical induction and allows for precise genome editing in specific cells through the utilization of various specific promoters or targeted drug delivery. Experimental validation of the enzymatic system was conducted in E. coli , HEK cells, and Jurkat cells, demonstrating successful assembly and activity of the assembled Cas9 enzyme. In addition, this study explored the incorporation of nuclear localization signals, the evaluation of inducible promoters, and the delivery of the system’s components in mRNA or protein form. Furthermore, we investigated the potential of S/MAR minicircle technology instead of viral vectors within the system. Overall, we highlighted the feasibility and utility of the Sortase-based split-Cas9 system to enhance control and efficiency compared to traditional CRISPR-Cas9 approaches. Additionally, this study revealed the potential of using the Sortase enzyme for posttranslational modifications and protein assembly in human cells.