ENGINEERING OF SYSTEMS, METHODS AND OPTIMIZED GUIDE COMPOSITIONS WITH NEW ARCHITECTURES FOR SEQUENCE MANIPULATION

Smart overview of the Invention

The patent application outlines innovative systems, methods, and compositions designed to modify the expression of target gene sequences using the CRISPR-Cas system. It provides structural insights into the Cas protein, which are utilized to generate modified components of the CRISPR complex. The application discusses vectors and vector systems that encode these components, as well as methods for designing and using such vectors. A particular focus is on engineered guide architectures for optimized CRISPR-Cas enzyme systems, which are critical for precise genome editing in eukaryotic cells.

Background

Advancements in genome sequencing and analysis have accelerated the understanding of genetic factors related to various biological functions and diseases. However, there is a need for precise genome targeting technologies that are affordable, scalable, and capable of targeting multiple positions within the eukaryotic genome. Existing techniques like designer zinc fingers and TALEs are available but have limitations. The CRISPR-Cas system offers a promising alternative, utilizing a single Cas enzyme programmed by a short RNA molecule to target specific DNA sequences, simplifying genome editing processes.

Key Innovations

The invention introduces non-naturally occurring or engineered CRISPR-Cas system guide RNAs, including chimeric single guide RNA molecules (sgRNA), capable of manipulating target nucleic acids. These guide RNAs feature modified architectures to enhance their functionality. Modifications include replacing specific nucleotide tracts and sequences, such as poly U with poly C, and incorporating GC-rich regions. Additionally, distinct RNA sequences that bind to adaptor proteins can be inserted, expanding the system's versatility by associating with functional domains like transcriptional activators or repressors.

Applications and Embodiments

Several embodiments of the invention involve the use of modified sgRNAs or guide RNAs in conjunction with CRISPR enzymes like Cas9. These systems can be implemented in various cell types, including mammalian and human cells, for gene expression alteration. The invention also encompasses polynucleotides encoding the guide RNAs, vectors containing these polynucleotides, and vector systems for delivering CRISPR components. Transformant organisms can be created to express the guide RNA and CRISPR enzyme, facilitating genomic modifications.

Potential Impact

The described CRISPR-Cas systems offer a robust and flexible platform for genetic engineering, with significant implications for biotechnology and medicine. By enabling precise and efficient genome editing, the invention supports the advancement of synthetic biology and the study of genetic diseases. The engineered guide architectures and associated methods enhance the CRISPR-Cas system's capability to target and modify specific genomic loci, paving the way for innovative therapeutic and research applications.