Cas Endonuclease Base Editing System for Precise Nucleic Acid Modification
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Solution Overview
Problem
Current nucleic acid editing systems, particularly those using CRISPR/Cas systems, face limitations in specificity and efficiency due to the need for double-strand breaks, which can lead to off-target effects and random mutations, and there is a lack of functional characterization of CRISPR/Cas enzymes from uncultivated microbial species.
Innovation Solution
An engineered nucleic acid editing system comprising a class 2, type II Cas endonuclease with a RuvC domain lacking nuclease activity, coupled with a base editor and a guide RNA structure, is developed. This system is derived from uncultivated microorganisms and is configured to bind to specific protospacer adjacent motifs (PAMs), allowing for precise base editing without inducing double-strand breaks.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If CRISPR/Cas systems induce double-strand breaks for nucleic acid editing, then editing capability is achieved, but off-target effects and random mutations increase
Solution Approach 1:
The invention divides the Cas9 endonuclease into two separate functional components: a nickase that creates single-strand breaks and a base editor that performs the actual base conversion. This segmentation allows the system to achieve editing capability without requiring double-strand breaks, thereby reducing off-target effects and improving editing specificity.
Solution Approach 2:
The invention introduces a base editor as an intermediary component that mediates the editing process. Instead of directly using Cas9 to create double-strand breaks, the base editor is delivered to the target site by the nickase-guide RNA complex and performs the base conversion. This intermediary approach enables precise editing while avoiding the harmful effects of double-strand breaks.
2Productivity
If CRISPR/Cas systems use double-strand breaks for editing, then editing efficiency is achieved, but random mutations occur
Solution Approach 1:
The invention converts the harmful double-strand break mechanism into a beneficial single-strand nicking mechanism. By using a nickase instead of wild-type Cas9, the system creates controlled single-strand breaks that guide the base editor to the target site without causing random mutations. The single-strand nicks are repaired by the cell's natural base excision repair pathway, which is more accurate and less prone to errors compared to double-strand break repair pathways.
3Ease of manufacture
If endonuclease from cultivated microorganisms is used, then system development is facilitated, but functional diversity is limited
Solution Approach 1:
The invention performs preliminary bioinformatic screening and identification of Cas enzymes from uncultivated microorganisms using metagenomic data. This preliminary action allows the team to identify and characterize functional properties of Cas enzymes from uncultivated sources without requiring actual cultivation, thereby expanding functional diversity while avoiding the limitations of traditional cultivation-based approaches.
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
The system achieves precise and efficient nucleic acid editing with reduced off-target effects by enabling base conversion without the need for double-strand breaks, leveraging unique functionalities from uncultivated microbial sources to enhance editing specificity and accuracy.
Implementation Method 1
a guide ribonucleic acid sequence configured to hybridize to a target deoxyribonucleic acid sequence
Implementation Method 2
an endonuclease configured to bind to a protospacer adjacent motif (PAM) sequence
Data Source
AI summary
The present disclosure provides for endonuclease enzymes having distinguishing domain features, as well as methods of using such enzymes or variants thereof.