Development of a Robust Pipeline for Synthesizing Various Components of CRISPR/Cas

CRISPR/Cas systems have emerged as powerful gene editing tools. With CRISPR/Cas we have the power to correct genetic mutations and thereby cure many as of yet incurable diseases. However, it has been difficult to find a reliable and efficient way to deliver CRISPR/Cas complexes in mammalian cells, as the large ribonucleoprotein complexes make the delivery challenging. The current best method is to deliver Cas proteins, is by transfecting cells with a plasmid, however they are very large with uptake issues. However, by delivering an mRNA coding for CRISPR/Cas proteins some of the current problems can be alleviated such as the size issue, as mRNAs are much smaller than plasmids (1). Furthermore, the mRNA delivery approach with lipid nanoparticles has been shown to be safe and effective with vaccines during the COVID-19 pandemic (2). To investigate the efficacy of this method of expressing Cas proteins, a DNA fragment that encodes for enhanced green fluorescent protein (EGFP) was transcribed into an mRNA, then transfected into HEK 293T cells, and monitored for protein expression using fluorescence microscopy. We tested three different mRNA constructs: 1) created straight from the purchased DNA fragment, 2) synthesize from that purchased fragment that underwent polymerase chain reaction (PCR), and 3) stabilized with pseudo-uridine made straight from the purchased gene fragment. It was found that the EGFP mRNA construct that was stabilized with the pseudo-uridine produced the brightest fluorescence after just 17 hours with ~90% of the cells glowing green. We also found that by day 8 almost all the green fluorescence disappeared, and by day 11 it had completely vanished, indicating that the EGFP protein had fully degraded. In conclusion, we have demonstrated potential efficacy of transiently delivering a Cas protein as an mRNA in mammalian cells by showing success in expression and degradation of the EGFP transfect. We are currently applying this approach to various CRISPR/Cas proteins.

References:

(1) Stadelmann C. Molecular Therapy – Nucleic Acids. 2022; 28; 47-57

(2) Chaudhary N. Nat Rev Drug Discov. 2021; 20(11): 817-838