CRISPR-CAS COMPONENT SYSTEMS, METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION

Smart overview of the Invention

The patent application describes innovative systems, methods, and compositions for manipulating genetic sequences using CRISPR-Cas technology. It focuses on vector systems that encode components of a CRISPR complex, facilitating precise genetic modifications. The invention aims to enhance genome editing capabilities in eukaryotic cells by directing CRISPR complex formation and introducing targeted mutations.

Technical Background

Advancements in genome sequencing and analysis have highlighted the need for precise genome-targeting technologies. Traditional genome-editing tools like zinc fingers and TALEs have limitations, prompting the development of more efficient and scalable methods. The CRISPR-Cas system offers a versatile alternative by using a single Cas enzyme programmed with RNA to target specific DNA sequences, thus simplifying genetic analysis and manipulation.

Key Features

The invention includes a vector system with several components:

• A regulatory element linked to a tracr mate sequence and guide sequences for directing CRISPR complex binding.
• An enzyme-coding sequence linked to another regulatory element, encoding the CRISPR enzyme with a nuclear localization sequence.

These components can be on the same or different vectors, enabling flexibility in design. The system supports multiple guide sequences for targeting different DNA sites within eukaryotic cells.

CRISPR Complex Optimization

The invention emphasizes optimizing CRISPR complexes to enhance their activity in eukaryotic cells. This includes using nuclear localization sequences to increase complex accumulation in cell nuclei. The CRISPR enzyme, often a Cas9 variant, is optimized for eukaryotic expression and can be designed to cleave DNA strands or act without cleavage activity, depending on the application.

Applications and Benefits

The described CRISPR-Cas system supports diverse applications in synthetic biology, biotechnology, and medicine by enabling precise gene editing. It simplifies the methodology for genetic research and accelerates the mapping of genetic factors related to biological functions and diseases. The flexibility in vector design and guide sequence targeting expands its utility across various genomic studies.