SYNTHETIC CAS12A FOR ENHANCED MULTIPLEX GENE CONTROL AND EDITING

Smart overview of the Invention

Engineered Cluster Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated (Cas) 12a proteins have been developed to enhance gene editing and modulation. These proteins are designed for applications in gene therapy, particularly targeting a broader range of diseases beyond inherited and monogenic conditions. By improving the efficacy of Cas12a, these engineered proteins aim to facilitate multiplex gene therapy, which can address complex and degenerative diseases.

Limitations of Current Gene Therapy Approaches

Current gene therapy techniques, particularly those utilizing CRISPR/Cas9, face limitations such as size constraints and immunogenicity. Cas9 is less effective for editing multiple genes simultaneously due to its larger size and lower efficiency in multiplexing. Although Cas12a offers advantages by processing multiple CRISPR RNAs from a single transcript, its lower activity compared to Cas9 restricts its application in vivo. Enhancing Cas12a's functionality is crucial for its therapeutic use.

Engineered Cas12a Variants

The engineered Cas12a proteins exhibit improved performance over wild-type variants. Specific mutations have been introduced to enhance their activation and repression capabilities, making them more effective for gene knockout, knockin, and mutagenesis. These modifications allow the engineered proteins to maintain activity under varying crRNA concentrations, facilitating better control over gene expression.

Applications and System Configuration

The engineered Cas12a system comprises the modified protein along with one or more CRISPR RNAs (crRNAs) that can target specific nucleic acids. These components can be packaged in expression vectors driven by various promoters, allowing for tailored gene modulation strategies. This system can simultaneously modulate multiple target nucleic acids, resulting in transcriptional activation or repression, epigenetic modifications, or precise editing of genetic material.

Potential for Therapeutic Development

The disclosure also includes methods for using the engineered Cas12a system in both in vitro and in vivo contexts. Additionally, pharmaceutical compositions containing these engineered proteins or systems are proposed, which could lead to significant advancements in treating a variety of diseases by enabling precise genetic modifications. This innovation represents a step forward in expanding the capabilities of gene therapy beyond current limitations.