Regularly spaced short palindromic repeats (CRISPR) and their associated protein, CRISPR-associated protein 9 (Cas9), made international headlines a few years ago as a breakthrough genome editing system. Comprised of Cas9 and a strand of genetic material known as single-guide RNA (sgRNA), the system can target specific regions of DNA and work like “molecular scissors” to make precise changes. The direct delivery of Cas9-sgRNA complexes, i.e. Cas9 ribonucleoproteins (RNPs) into the cell nucleus is considered to be the safest and most efficient way to achieve genome editing. However, the Cas9 RNP has low cellular permeability and therefore requires a carrier molecule to transport it past the first hurdle of the cell membrane before it can reach the cell nucleus. These transporters must bind to Cas9 RNP, transport it into the cell, prevent its degradation by intracellular organelles called “endosomes” and finally release it without causing changes in its structure.
In a recent article published in June 2022 in volume 27 of Materials applied today, a research team from Kumamoto University has developed a transformable polyrotaxane (PRX) carrier that can facilitate genome editing using Cas9RNP with high efficiency and user-friendliness. “Although there have been PRX-based drug transporters for nucleic acids and proteins reported before, this is the first report of the PRX-based Cas9 RNP transporter. Additionally, our findings describe how Precisely controlling intracellular dynamics through multiple steps will prove invaluable for future research in this direction,” says Professor Keiichi Motoyama, corresponding author of the paper.
For their new medium, the research team focused on PRX with amine groups, i.e. amino-PRX, and went through several rounds of development and optimization before obtaining its end product. For example, the first generation (1G) of their carrier molecules exploited the autonomous processing properties of amino-PRX to efficiently complex it with Cas9 RNP and enable its delivery beyond the cell membrane. The second generation (2G) worked towards endosome escape. This was achieved via transformation of amino groups in amino-PRX into highly cationic (positively charged) particles in the endosome, which resulted in endosome rupture and leakage of Cas9 RNP-amino-PRX. Later generations addressed issues related to the release of Cas9 once the complex had escaped the endosome. Finally, they developed the fifth-generation (5G) multi-step transformable amino-PRX carrier that could accurately and efficiently deliver Cas9 RNP into the cell nucleus. The research team further performed in vitro and in vivo experiments to confirm the cytotoxicity of the system, as well as its genome editing efficiency. “Our delivery system has low cytotoxicity and its genome-editing activity is equal to the most efficient system currently on the market,” reveals Associate Professor Taishi Higashi of Kumamoto University, who is the other corresponding author. of the study. “In addition, our multiple attempts to optimize the delivery system across generations offer important insights into the types and positions of various biodegradable groups and amino groups that can be used in such a system to further customize and tailor their properties.”
The autonomous action, multi-step transformable properties and low cytotoxicity of the 5G amino-PRX transporter make it an extremely promising candidate for the safe and efficient delivery of Cas9 RNP. These findings could further be applied for the delivery of a wide range of molecules, such as enzymes, antibodies, and small interfering RNAs (siRNAs), making this novel carrier a significant achievement in the field of drug development. medicines and vaccines.
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Material provided by Kumamoto University. Note: Content may be edited for style and length.