Split Cas9 Protein Delivery via Intein-Mediated Splicing
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Solution Overview
Problem
The size of the Cas9 protein (>4 kb) poses a limitation for efficient delivery via recombinant adeno-associated virus (rAAV) in genome-editing applications, hindering precise genome targeting in gene therapy.
Innovation Solution
The Cas9 protein is split into N-terminal and C-terminal portions, each fused with an intein, allowing for delivery on separate rAAV vectors and subsequent intein-mediated protein splicing to form a complete and functional Cas9 protein within cells, along with the use of bipartite nuclear localization signals and regulatory elements for high expression levels.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If Cas9 protein is delivered via rAAV vector, then genome-editing capability is achieved, but delivery efficiency deteriorates due to large size (>4 kb)
Solution Approach 1:
The Cas9 protein is divided into two separate portions (N-terminal and C-terminal), each encoded by a separate nucleotide sequence on individual rAAV vectors. This segmentation reduces the size of each vector below the 4.7 kb packaging limit, enabling efficient delivery while maintaining the ability to reconstruct functional Cas9 through intein-mediated protein splicing in target cells.
2Productivity
If Cas9 protein is split into portions and delivered on separate vectors, then delivery efficiency is improved, but system complexity increases
Solution Approach 1:
An intein-mediated protein splicing system is introduced as an intermediary mechanism to join the N-terminal and C-terminal portions of Cas9 after delivery. The intein sequences are fused to the respective Cas9 portions, enabling automatic in vivo splicing when the two portions are expressed, thus simplifying the overall system by eliminating the need for complex in vitro assembly procedures.
3Productivity
If nuclear localization signals are added to split Cas9 portions, then nuclear import efficiency is improved, but protein sequence complexity increases
Solution Approach 1:
Bipartite nuclear localization signals (NLS) are merged into the N-terminal and C-terminal portions of the split Cas9 protein. This combination ensures that both portions are efficiently imported into the nucleus where genome editing occurs, while the NLS sequences are strategically positioned to work synergistically with the intein splicing mechanism.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach enables efficient delivery and expression of functional Cas9 proteins, overcoming the size limitations and enhancing genome-editing capabilities in various cell types, including human cells, for therapeutic applications.
Implementation Method 1
The resulting fusion proteins, when delivered on separate vectors (e.g., separate rAAV vectors) into one cell and co-expressed, may be joined to form a complete and functional Cas9 protein or nucleobase editor (e.g., via intein-mediated protein splicing).
Implementation Method 2
the first nucleotide sequence or second nucleotide sequence is operably linked to a nucleotide sequence encoding at least one bipartite nuclear localization signal
Data Source
AI summary
Provided herein are methods of delivering “split” Cas9 protein or nucleobase editors into a cell, e.g., via a recombinant adeno-associated virus (rAAV), to form a complete and functional Cas9 protein or nucleobase editor. The Cas9 protein or the nucleobase editor is split into two sections, each fused with one part of an intein system (e.g., intein-N and intein-C encoded by dnaEn and dnaEc, respectively). Upon co-expression, the two sections of the Cas9 protein or nucleobase editor are ligated together via intein-mediated protein splicing. Recombinant AAV vectors and particles for the delivery of the split Cas9 protein or nucleobase editor, and methods of using such AAV vectors and particles are also provided.