Modified CRISPR-Cas9 system for enhanced gene editing

This invention enhances the CRISPR-Cas9 gene-editing system by modifying the Cas9 protein’s Rec3 clamp region, improving its fidelity, specificity, and speed, reducing off-target effects, and increasing precision for therapeutic and research applications.

Background

Genome engineering is a rapidly advancing field that involves the precise modification of an organism’s genetic material. This technology holds immense potential for applications in medicine, agriculture, and biotechnology. One of the most prominent tools in genome engineering is the CRISPR-Cas9 system, which allows for targeted editing of DNA sequences. The need for such technology arises from its ability to address genetic disorders, enhance crop resilience, and facilitate research into gene functions.

However, the precision and efficiency of gene editing are critical, as off-target effects can lead to unintended genetic alterations, which may have harmful consequences. Despite the promise of CRISPR-Cas9, current approaches face significant challenges. The primary issue is the occurrence of off-target DNA cleavage, where the Cas9 protein inadvertently cuts DNA sequences that are similar but not identical to the intended target. This lack of specificity can result in unintended mutations, potentially leading to negative outcomes in therapeutic applications.

Additionally, while some Cas9 variants have been engineered to improve specificity, they often suffer from reduced on-target activity, meaning they are less efficient at editing the desired DNA sequence. The underlying mechanisms of Cas9’s interaction with DNA mismatches are not fully understood, further complicating efforts to enhance its precision and reliability. Therefore, there is a pressing need for improved Cas9 variants that can offer higher fidelity and efficiency in gene editing.

Technology description

This technology centers around an enhanced version of the Cas9 protein, a pivotal element of the CRISPR-Cas9 gene-editing system. The modified Cas9 targets the Rec3 clamp region, where specific mutations have been introduced to improve the protein’s fidelity, specificity, and speed of processivity. These enhancements are vital for minimizing off-target effects and increasing the precision of gene editing. The technology also encompasses methods and compositions for gene editing using this modified Cas9, including kits that incorporate the engineered protein. This advancement holds promise for more effective therapeutic applications and research in genome engineering.

What differentiates this technology is its focus on the Rec3 clamp region of the Cas9 protein, which plays a crucial role in the protein’s interaction with DNA. By engineering mutations in this specific region, the modified Cas9 achieves a higher level of precision and efficiency in gene editing compared to its unmodified counterpart. This targeted approach addresses a significant limitation of the traditional CRISPR-Cas9 system, which is prone to off-target effects. The improved specificity and fidelity make this technology particularly valuable for therapeutic applications, where precision is paramount to avoid unintended genetic alterations.

Benefits

Enhanced fidelity, specificity, and speed of processivity in gene editing
Reduced off-target effects for increased precision in gene editing
Improved on-target activity and fidelity of Cas9 variants
Potential for more effective therapeutic applications in genome engineering
Methods and compositions for performing gene editing with modified Cas9
Applications in treating genetic disorders and modifying organisms for desired traits