METHOD FOR SITE-SPECIFIC CONJUGATION OF NUCLEIC ACID TO CRISPR FAMILY PROTEIN, AND CONJUGATE THEREOF AND USE THEREOF

Smart overview of the Invention

A novel method enables the site-specific conjugation of nucleic acids to CRISPR family proteins by utilizing unnatural amino acids with orthogonal chemical reactivity. This approach begins with the targeted mutation of a CRISPR protein, allowing for precise attachment of nucleic acids. The resulting conjugate is designed to enhance gene editing efficiency, including gene cutting and base editing capabilities.

Technical Background

CRISPR technology, derived from bacterial immune systems, allows for precise DNA modifications by creating double-strand breaks, which can be repaired through various cellular mechanisms. Despite its success in genetic engineering and biomedicine, challenges remain, such as low homologous recombination efficiency and issues with protein delivery. Current methods for site-specific protein modification often compromise protein stability and functionality.

Addressing Existing Challenges

The method presented aims to tackle the limitations of existing CRISPR applications by covalently linking donor DNA to CRISPR proteins. This strategy enhances the proximity of donor DNA to target sites during cleavage, thereby facilitating more efficient homologous recombination repair. Additionally, improving the affinity of AsCas12a to its guide RNA is proposed to increase cleavage efficiency and overall editing effectiveness.

Utilization of Unnatural Amino Acids

The innovation involves incorporating unnatural amino acids into CRISPR proteins using advanced translation systems. These amino acids feature bioorthogonal reactive groups that allow for selective chemical reactions with modified nucleic acids. This technique not only enhances the specificity of protein-nucleic acid conjugation but also addresses previous issues related to random modifications that affect protein performance.

Conclusion and Implications

This method for site-specific nucleic acid conjugation to CRISPR proteins represents a significant advancement in genetic editing technologies. By improving the efficiency and accuracy of gene editing processes, it holds promise for applications in agriculture, animal husbandry, and medical therapies targeting genetic diseases. The strategic use of unnatural amino acids provides a pathway for developing more effective biopharmaceuticals with controlled properties.