Split Cas9 Protein Delivery via Segmented Viral Vectors
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
The large size of the Cas9 protein poses a challenge for efficient packaging and intracellular delivery using viral vectors, limiting its application in gene regulation due to low virus production and delivery efficiency.
Innovation Solution
The Cas9 protein is divided into two domains, each expressed from separate recombinant vectors, which are then fused within non-human cells to form the full-length Cas9 protein, enabling effective gene regulation and overcoming packaging limitations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If the Cas9 protein is inserted into a viral vector for intracellular delivery, then gene regulation can be achieved, but the efficiency of virus production and intracellular delivery is low due to the limited packaging capacity of the viral vector
Solution Approach 1:
The Cas9 protein is divided into two separate domains (first domain and second domain), each encoded by a separate recombinant vector. This segmentation allows each vector to be packaged efficiently into viral particles, overcoming the packaging capacity limitation while still enabling complete Cas9 function when both domains are expressed in the target cell
2Reliability
If the Cas9 protein is inserted into a viral vector for intracellular delivery, then gene regulation can be achieved, but the efficiency of intracellular delivery is low due to the large size of the Cas9 protein
Solution Approach 1:
The large Cas9 protein is segmented into two smaller domains that can each be accommodated within the packaging capacity of viral vectors. This segmentation directly addresses the delivery efficiency problem by reducing the size of individual genetic payloads while maintaining the functional capability of the complete Cas9 protein upon co-expression in target cells
Data Source
Figure 1
Figure 2
Figure 3
AI summary
The present invention relates to a method for regulating gene expression, comprising introducing into a cell each of a recombinant vector which expresses a first domain comprising N-terminus of a Cas9 protein, and a recombinant vector which expresses a second domain comprising C-terminus of a Cas9 protein, a composition comprising the recombinant vectors, a kit for regulating gene expression, and a method for intracellular production of Cas9 protein. Moreover, the present invention relates to a transformed cell introduced with a viral vector which packages the first domain, and a viral vector which packages the second domain, and to a composition comprising a virus produced therefrom.