Orthogonal Cas9 Proteins for Multiplex Gene Regulation and Editing
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Solution Overview
Problem
Existing CRISPR-Cas systems primarily focus on targeted DNA degradation and editing, lacking efficient methods for simultaneous and independent regulation of gene expression or editing using orthogonal RNA-guided DNA binding proteins.
Innovation Solution
Utilizing orthogonal RNA-guided DNA binding proteins, such as nuclease-null Cas9 proteins, to form complexes with guide RNAs that co-localize with DNA, enabling simultaneous and independent regulation or editing of multiple genes by incorporating transcriptional activators or repressors.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If CRISPR-Cas systems are used for targeted DNA degradation and editing, then DNA cleavage and editing capability is improved, but the ability to simultaneously regulate multiple genes independently is limited
Solution Approach 1:
The invention divides the CRISPR-Cas system into multiple orthogonal variants (e.g., Cas9 from different species with different PAM requirements), allowing each variant to independently target specific genes without interfering with others. This segmentation enables simultaneous independent regulation of multiple genes while maintaining precise targeted cleavage for each gene.
Solution Approach 2:
The patent creates a universal platform where multiple orthogonal Cas9 proteins can function together in the same cell system. Each Cas9 variant maintains its DNA cleavage function while adding the capability to regulate different genes independently, achieving multi-functionality that combines precise editing with simultaneous gene regulation.
2Adaptability or versatility
If multiple guide RNAs and Cas9 proteins are used for simultaneous gene regulation, then adaptability and versatility are improved, but system complexity increases
Solution Approach 1:
The invention uses orthogonality as an intermediary mechanism where Cas9 proteins from different species recognize different PAM sequences and bind to different guide RNAs. This intermediary property allows multiple components to coexist and function simultaneously without cross-interference, enabling multiplexed regulation while managing system complexity through natural biological orthogonality.
Data Source
AI summary
Methods of modulating expression of a target nucleic acid in a cell are provided including use of multiple orthogonal Cas9 proteins to simultaneously and independently regulate corresponding genes or simultaneously and independently edit corresponding genes.