Engineered Cas Nuclease DNA Binding Cleft Mutations
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Solution Overview
Problem
Current CRISPR-Cas technologies often produce unpredictable indel mutations due to non-homologous end joining and related DNA repair pathways, limiting precise genome editing, especially in dividing and non-dividing cells.
Innovation Solution
Engineering Cas nucleases with specific mutations in the DNA binding cleft to inhibit indel-producing pathways and enhance homology-driven repair pathways, such as HDR and MMEJ, thereby reducing NHEJ activity and increasing precise editing efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If conventional CRISPR-Cas nucleases are used for genome editing, then DNA double-strand breaks are created at target loci, but indel mutations are produced through non-homologous end joining and other repair pathways, reducing editing precision
Solution Approach 1:
The patent applies parameter changes by introducing specific amino acid mutations (e.g., D54N, S55N, D54Q, S55Q) in the DNA binding cleft of the Cas nuclease. These parameter changes in the protein structure alter the interaction with DNA repair pathways, specifically suppressing non-homologous end joining while preserving homology-directed repair, thereby reducing indel mutations and improving editing precision
Solution Approach 2:
The engineered Cas nuclease acts as an intermediary that modulates the interaction between the DNA break and repair pathways. By modifying the Cas nuclease DNA binding cleft, it mediates a shift in repair pathway preference from error-prone NHEJ to high-fidelity HDR, reducing harmful indel mutations without requiring direct inhibition of repair enzymes
2Productivity
If non-homologous end joining pathways are active for DNA repair, then rapid repair occurs, but unpredictable indel mutations are generated, limiting precise editing outcomes
Solution Approach 1:
The patent changes the parameters of the Cas nuclease by introducing mutations in the DNA binding cleft that alter its interaction with DNA ends. This parameter change suppresses NHEJ pathway activation while maintaining DNA break creation capability, thereby improving editing accuracy without significantly compromising overall repair efficiency
Solution Approach 2:
Instead of trying to enhance HDR directly, the patent inverts the approach by suppressing NHEJ through Cas nuclease engineering. This indirect strategy allows HDR to become the dominant repair pathway by default, achieving precise editing through the repair pathway that naturally provides higher fidelity
3Manufacturing precision
If homology-directed repair pathways are enhanced for precise editing, then specific mutations can be introduced accurately, but the process requires competitive suppression of dominant NHEJ pathways
Solution Approach 1:
The patent simplifies the system by making a single parameter change in the Cas nuclease (mutations in the DNA binding cleft) that simultaneously achieves both goals: enhancing HDR efficiency and suppressing NHEJ. This eliminates the need for complex combinatorial approaches involving multiple proteins or small molecules, reducing device complexity while improving precise editing frequency
Data Source
AI summary
The present disclosure provides a method of editing a genome in a cell including exposing the cell to an engineered Cas nuclease comprising one or more mutations within the DNA binding cleft of the Cas nuclease, wherein exposure to the engineered Cas nuclease decreases, inhibits, or prevents non-homologous end joining (NHEJ) in the cell, and wherein exposure to the engineered Cas nuclease increases one or more homology-driven repair pathways within the cell. The mutant Cas nuclease is also disclosed herein.