Computational Design of Compact CRISPR-Cas Enzymes of Lachnospiraceae bacterium Cas12a Utilizing Bioinformatic Tools

Purpose: Nature has provided us with a popular genome editing tool known as the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas system, which has shown promise in both plants and animals. The CRISPR-Cas system utilizes a guide RNA (gRNA) and specific proteins known as Cas proteins to facilitate its function. A major limitation of the CRISPR/Cas system and any gene therapy is how it’s delivered within the organism. The most common "vehicle” for delivering gene therapies is adeno-associated viral vectors (AAVs), which have a maximum effective capacity of approximately 4.7 kb. The main issue with most Cas enzymes and other CRISPR components needed is that they are much bigger than this required maximum capacity. The most widely characterized CRISPR-Cas system is Cas9. However, the unique feature of Cas12a’s ability to process its own crRNA arrays without the requirement for tracrRNA makes it a promising candidate as well. In other CRISPR-Cas systems, the RNA CRISPR components need to be synthesized and packaged into an AAV, whereas in the Cas12a family, some of these components are not needed. Lachnospiraceae bacterium Cas12a (LbCas12a) has increased activity when compared to other species of Cas12a enzymes. To address the aforementioned size issue, we have used various bioinformatic tools to computationally design compact-size proteins of LbCas12a with similar functionality and comparable efficiency.

Methods: The best available crystal structure of LbCas12a was chosen from the Protein Data Bank (PDB). A structure reduction process was carried out using Yasara and UCSF ChimeraX. The intermediate steps of this process were verified using the homology-based modeling tool SWISS-MODEL and AI-based modeling tool Alphafold2 to ensure that the protein was still folding similarly to the original structure. Furthermore, the global and local structural features were analyzed, and the best candidate was subjected to molecular dynamics (MD) simulations along with gRNA and substrate DNA to determine its functional efficiency under realistic dynamic conditions and compared it with the original structure.

Results/Conclusions: A compact-size variant of LbCas12a was generated, which is 292 residues smaller than the original crystal structure. This man-made miniature protein contains all the regions that are needed for DNA cleavage activity. MD simulations confirm its stability in the presence of DNA and gRNA. Further validation of the designed protein and experimental testing is under investigation at this point of the study.