The CRISPR-Cas system is celebrated for its transformative impacts in areas like basic research, diagnostics, and therapeutics, primarily because of its unmatched precision in targeting DNA or RNA. However, there are hurdles when adapting it for therapeutic uses. The delivery of the system in vivo via adeno-associated viruses (AAVs) presents a significant challenge, mainly due to protein size limitations. In response to this, smaller Cas enzymes, such as CasMINI and Cas12f, have emerged, offering potential solutions given their ability to work around these size constraints.
The focus of many in the field has been on the miniaturization of Cas proteins. Two dominant strategies exist: one explores metagenomic datasets to find naturally compact Cas enzymes, although these often lack the advantages seen in optimized Cas proteins. The other strategy involves tweaking the functions of Cas enzymes, a move that tends to diminish their DNA/RNA cleavage activity but preserves their binding proficiency.
The study in question sheds light on the intricacies of the CRISPR-Cas13 system, which is tailored for RNA targeting. The system is further divided into subtypes, namely Cas13a-d. A unique attribute of all Cas13 effectors is their dual-center composition, consisting of a pre-crRNA processing catalytic center and a target RNA cleavage center. The article furnishes a thorough breakdown of the distinctions and attributes of the Cas13 subtypes.
At the heart of this research is the Interaction, Dynamics, and Conservation (IDC) strategy aimed at protein miniaturization. Rooted in protein structure, the IDC methodology factors in the interactions at the functional sites of Cas enzymes, the dynamics concerning their conformational alterations, and the structural conservation seen in the Cas13d protein family. The IDC strategy came to fruition through the marriage of meticulous structural analysis and the computational prowess of AlphaFold2.
Implementing this strategy, the team successfully produced five compact variants of Cas13, which, notably, matched the RNA binding and cleavage activity of their full-sized analogues. Furthermore, by using the AAV produced at PackGene Biotech to target certain RNA transcripts, with Pcsk9 as a case in point, the study emphasized the potential of the compact RNA base editor, termed mini-Vx, revealing its commendable efficiency in editing.
In essence, the fusion of the IDC strategy and AlphaFold2’s capabilities marks a leap in the miniaturization of Cas proteins. This approach ensures the retention of the enzyme’s effectiveness while maneuvering around size-related barriers crucial for therapeutic implementations. This breakthrough signals a promising trajectory for more potent and streamlined gene therapies in the not-so-distant future.
The remarkable work from the miniaturization of CRISPR-Cas system serves as a compelling example of how adeno-associated viruses (AAVs) play a pivotal role in turning discoveries into tangible advances. At PackGene Biotech, we are dedicated to enhancing AAV production technology to ensure that high-quality AAVs are readily accessible from research stages to clinical applications. Our state-of-the-art π-Alpha high-yield AAV platform has powered groundbreaking studies like the one highlighted in the work above.
1. A strategy for Cas13 miniaturization based on the structure and AlphaFold. F Zhao, T Zhang, X Sun, X Zhang, L Chen, H Wang, J Li, P Fan, L Lai, T Sui, Z Li. Nat Commun. 2023 Sep 8;14(1):5545. doi: 10.1038/s41467-023-41320-8.
Tauopathy’s Role in Aβ Oligomer Formation: New Insights from Transgenic Primate Models on Alzheimer’s Pathogenesis
Tauopathy, characterized by protein tau and Aβ oligomer accumulation, is a significant aspect of Alzheimer's disease. The causal relationship between these accumulations and neurodegeneration remains unclear despite their links to various cognitive and movement...
Huntington's disease (HD) is a devastating neurodegenerative condition caused by a mutation in the Huntingtin gene (HTT). This leads to the production of a mutant form of the huntingtin protein (mHTT) with an extended polyglutamine (polyQ) repeat, which has a...
Collaboration provides end-to-end coverage of mRNA drug and vaccine development and manufacturing workflow HOUSTON, TX and NEEDHAM, MA – PackGene Biotech (PackGene) and Kudo Biotechnology (Kudo Bio) have partnered to provide high-quality, customized messenger RNA...
Zolgensma’s Success in SMA Treatment Advocates for AAV-based Gene Therapy and Promises a Bright Future for Genetic Disorders
At the 2023 Muscular Dystrophy Association Clinical and Scientific Conference, Novartis presented new data highlighting the continued efficacy and durability of its gene therapy treatment, Zolgensma, in treating spinal muscular atrophy (SMA). Zolgensma has been shown...
Plasmid CDMO Services
We provide high quality plasmids that are ideal for any phase of the drug development cycle.
AAV cGMP Manufacturing
AAV Capsid Engineering
Proven technology paving your path to effective therapies for cancer or genetic disorder