Cas9 Targeting Complex Cell Cycle Synchronization for DNA Modification
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Solution Overview
Problem
Current methods for site-specific DNA modification in eukaryotic organisms, such as those using Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), are limited by the need for specific protein engineering and suffer from off-target activity, while CRISPR/Cas systems require optimization for increased efficiency and effectiveness in eukaryotic cells.
Innovation Solution
The use of a Cas9 targeting complex, comprising a Cas9 protein and a guide RNA with a targeting sequence that hybridizes to a specific DNA site, is enhanced by cell cycle enrichment and blocking, allowing for site-specific modification of DNA or associated proteins in eukaryotic cells, with the Cas9 protein optionally having a heterologous polypeptide sequence for additional activities.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If ZFNs or TALENs are used for site-specific DNA modification, then DNA cleavage activity is achieved, but protein engineering complexity increases and off-target activity occurs
Solution Approach 1:
The patent uses guide RNA sequences as programmable elements that can be easily designed and synthesized to target specific DNA sequences, replacing the need for complex protein engineering. The guide RNA acts as a simple copy or representation of the target sequence, enabling precise targeting without engineering custom proteins for each target site.
Solution Approach 2:
The Cas9 nuclease serves as a universal tool that can target any DNA sequence by simply changing the guide RNA sequence. This single Cas9 protein can perform site-specific DNA modification at multiple different locations in the genome, eliminating the need for multiple different engineered proteins required by ZFNs and TALENs.
2Manufacturing precision
If ZFNs or TALENs are used for site-specific DNA modification, then DNA cleavage activity is achieved, but off-target activity increases
Solution Approach 1:
The patent replaces the protein-DNA recognition mechanism of ZFNs and TALENs with an RNA-DNA hybridization mechanism. The guide RNA base-pairs with the target DNA sequence through complementary base pairing, providing high specificity and reducing off-target effects compared to protein-DNA interactions.
3Productivity
If Cas9 targeting complex is used without cell cycle enrichment, then DNA modification can be performed, but modification efficiency is low
Solution Approach 1:
The patent applies cell cycle enrichment or blocking procedures before introducing the Cas9 targeting complex to pre-synchronize cells in phases with optimal DNA repair activity. This preliminary action ensures that when the Cas9 complex is introduced, the cells are in the most receptive state for efficient DNA modification and repair, maximizing productivity.
Solution Approach 2:
The patent optimizes the timing of Cas9 complex introduction relative to cell cycle phase by using enrichment or blocking methods. This changes the temporal parameter of when the modification occurs, aligning it with cell cycle phases that have highest HDR or NHEJ activity, thereby improving overall efficiency.
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 significantly increases the efficiency of homology-directed repair (HDR) and non-homologous end joining (NHEJ) by up to 10-fold, compared to methods without cell cycle blocking or enrichment, thereby improving the precision and effectiveness of DNA modification in eukaryotic cells.
Implementation Method 1
a guide RNA comprising: a targeting sequence that hybridizes to a target sequence of the target DNA
Data Source
Figure 1A~1B
Figure 1C
Figure 1D
AI summary
The present disclosure provides compositions and methods of site-specific modification of a target DNA, or a protein associated with a target DNA, in a eukaryotic cell. The present disclosure provides methods of binding a target DNA in a eukaryotic cell.