Compositions for Inducing Modifications of Target Endogenous Nucleic Acid Sequences in Nucleuses of Eukaryotic Cells

Smart overview of the Invention

The patent application focuses on targeted genome editing techniques in eukaryotic cells, utilizing compositions and methods to induce modifications in endogenous nucleic acid sequences. The process involves the use of CRISPR/Cas9 technology, where a Cas9 protein and a single-guide RNA (sgRNA) are prepared and combined in a cell-free buffer to form a transfection mixture. This mixture is introduced into eukaryotic cells, enabling precise modifications of DNA sequences within cell nuclei.

Technical Context

CRISPR/Cas systems serve as a prokaryotic immune mechanism, offering acquired immunity by incorporating short DNA segments from foreign invaders into the host genome. These segments, known as spacers, help recognize and neutralize external genetic elements. The Cas9 protein, when paired with guide RNAs, forms an endonuclease capable of cleaving specific DNA sequences. This system provides advantages over traditional methods like zinc finger proteins due to its RNA-based targeting, which is simpler to design.

Challenges and Innovations

Despite the potential of CRISPR/Cas systems, a comprehensive method for genome editing using RNA-guided endonucleases (RGENs) had not been fully developed prior to this invention. The inventors have addressed this gap by creating a programmable RGEN for targeted DNA cleavage in eukaryotic organisms. Additionally, they have innovated methods to use RGENs in genotyping recurrent mutations, enhancing the accuracy and efficiency of mutation detection compared to traditional methods like RFLP.

Objectives and Solutions

The invention aims to provide compositions for cleaving target DNA in eukaryotic cells using guide RNAs and Cas proteins. It also offers kits for targeted mutagenesis and genotyping mutations, facilitating the creation of genome-modified organisms. Methods include co-transfecting or serial-transfecting cells with the necessary components, enabling precise genetic modifications. These solutions enhance the capability to edit genomes in various organisms, including plants and animals.

Applications

• Preparation of engineered eukaryotic cells through targeted DNA modification.
• Development of kits for inducing mutagenesis and genotyping mutations in biological samples.
• Creation of genome-modified organisms, such as plants and animals, using CRISPR/Cas technology.
• Improvement in genotyping methods for detecting both naturally occurring and engineered mutations.