Orthogonal Cas9 Fusion Editing for Precise Genomic Deletions
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Solution Overview
Problem
Existing CRISPR/Cas9 genome editing methods struggle to generate precise and reproducible deletions in the genome, often resulting in variable deletion sizes due to error-prone non-homologous end-joining, which complicates genetic engineering for clinical applications.
Innovation Solution
Employing an orthogonal Cas9-Cas9 fusion system comprising a Streptococcus pyogenes (Spy)Cas9 cleavase with a R1333K mutation and a Neisseria meningitidis (Nme)Cas9, Campylobacter jejuni (Cje) Cas9, or Simonsiella muelleri (Smu) Cas9 cleavase, guided by specific RNA molecules to target distinct genomic loci, minimizing off-target cleavage and locus inversions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional CRISPR/Cas9 genome editing is used to generate deletions, then genome editing efficiency is improved, but deletion precision and reproducibility deteriorate due to error-prone non-homologous end-joining
Solution Approach 1:
The invention divides the single Cas9 cleavage event into two separate orthogonal Cas9 cleavage events. The first Cas9 (e.g., SpyCas9) makes a cut at the first genomic locus, and the second Cas9 (e.g., NmeCas9) makes a cut at the second genomic locus. These two independent cutting events are coordinated to achieve precise deletions, circumventing the imprecision of single Cas9-mediated NHEJ repair.
Solution Approach 2:
The invention introduces an intermediary mechanism - the orthogonal Cas9 system with different PAM requirements - that mediates between the need for efficient cutting and precise deletion. By using two different Cas9 enzymes with distinct PAM specificities (e.g., SpyCas9 requires 5'-NGG-3' while NmeCas9 requires 5'-NRN-1'), the system ensures that each cut is made at the intended location with high fidelity, enabling precise deletions while maintaining editing efficiency.
2Device complexity
If single Cas9 system is used for genome editing, then system complexity is reduced, but off-target cleavage and locus inversions increase
Solution Approach 1:
The invention segments the cleavage function into two independent Cas9 components with different PAM specificities. This segmentation ensures that off-target effects are minimized because each Cas9 enzyme only recognizes its specific PAM sequence, reducing the likelihood of unintended cleavage at wrong genomic locations.
Solution Approach 2:
The invention changes the PAM recognition parameter by using two different Cas9 enzymes with distinct PAM requirements. SpyCas9 recognizes 5'-NGG-3' PAMs while NmeCas9 recognizes 5'-NRN-1' PAMs. This parameter change in PAM specificity allows the system to distinguish between different genomic loci more accurately, thereby improving cleavage specificity and reducing off-target effects.
3Device complexity
If traditional CRISPR/Cas9 deletion method is used, then editing process is simplified, but variability in deletion size increases
Solution Approach 1:
The invention segments the deletion process into two controlled cutting events rather than relying on a single cut followed by error-prone repair. The first Cas9 cuts at a defined location and the second Cas9 cuts at another defined location, ensuring that the deletion size is precisely determined by the distance between these two cutting sites, thereby eliminating variability in deletion size.
Data Source
AI summary
Methods and compositions for genetically modifying a cell are provided.