Design of man-made miniature CRISPR-Cas systems using computational technologies

Purpose: An RNA-guided targeted genome engineering platform, CRISPR/Cas system is one of the breakthroughs of the twenty-first century. Despite the wealth of its advancement, there are some associated limitations that need to be overcome for the betterment of this revolutionized technology. Among them, the larger size of the available Cas proteins that are essential for the functioning of these tools limits their in vivo administration due to the low delivery efficiency. To address this issue, we have used computational chemistry tools to design smaller versions or compact size Cas proteins that can be used as an alternative. Methods: The available crystal structures of CRISPR-Cas systems were utilized and the reduction was done preserving the regions that are essential for the DNA binding and cleavage functions using Chimera, Yasara, and the Swiss Model software. Molecular Dynamics (MD) simulations were performed to obtain stable conformations of the reduced structures. The minimized sequences were used to generate their structures by the Swiss Model. Results/Conclusions: Four stable man-made miniature Cas proteins were generated that are less than half the size of the currently used CRISPR systems such as Cas9 or Cas12a. The sequence-based modeling studies using the Swiss model have shown the similar folding of these reduced proteins compared to their original counterparts. Further experimental validation of their ds-DNA cleavage activities remains to be determined at this point of the study.