CRISPR/CAS9 GENE EDITING SYSTEM AND APPLICATION THEREOF

Smart overview of the Invention

A novel CRISPR/Cas9 gene editing system has been developed that combines a specific Cas9 protein with a single guide RNA (sgRNA). This system is designed to accurately locate and cleave target DNA sequences, resulting in double-strand breaks. The specific Cas9 proteins used are relatively small, comprising around 1000 amino acids, which simplifies the identification of the protospacer adjacent motif (PAM) sequence necessary for targeting DNA.

Technical Background

The CRISPR/Cas9 system, originally part of bacterial immune systems, has evolved into a powerful tool for gene editing. The mechanism involves the formation of a complex between crRNA, tracrRNA, and the Cas9 protein, which identifies and cleaves DNA at specific sites. Two primary repair pathways in cells—non-homologous end joining (NHEJ) and homologous recombination (HR)—are leveraged for gene modifications, allowing for gene knockouts or precise insertions when templates are provided.

Innovative Features of the New System

The newly developed CRISPR/Cas9 system addresses limitations found in existing technologies by utilizing smaller Cas9 proteins that exhibit high editing activity and specificity while recognizing simpler PAM sequences. This advancement enhances the potential for more extensive clinical applications, including gene therapy, as smaller proteins can be more effectively packaged into delivery vectors like AAV viruses.

Methodology for Gene Editing

The method for using this CRISPR/Cas9 system involves synthesizing a Cas9 gene sequence and cloning it into an expression vector. Following this, sgRNA corresponding to the target DNA sequence is synthesized and ligated into the vector. Finally, the vector is delivered into cells to facilitate targeted gene editing. The steps include synthesizing oligos for sgRNA and using various modifications to optimize performance.

Applications and Advantages

This CRISPR/Cas9 system can be utilized for diverse applications such as gene knockout, site-directed base changes, and regulation of transcription levels. Compared to previous systems, it offers a smaller Cas9 protein, enabling effective packaging and higher targeting efficiency due to its ability to recognize simpler PAM sequences. This positions the new system as a significant advancement in genetic research and therapeutic applications.