Compact SMART Nucleases for Targeted Gene Editing Delivery
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
The large size of Class 2 Cas effectors poses a challenge for therapeutic applications, making delivery difficult.
Innovation Solution
Development of SMART (SMall ARchaeal-associaTed) nuclease systems with engineered endonucleases derived from uncultivated microorganisms, featuring a molecular weight of 96 kDa or less, comprising RuvC and HNH domains, and configured with guide ribonucleic acids for targeted DNA cleavage.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If Class 2 Cas effectors are used for genome engineering, then programmable endonuclease cleavage ability is achieved, but delivery for therapeutic applications becomes challenging due to large size
Solution Approach 1:
The patent changes the molecular weight parameter of the Cas effector by selecting and engineering small archaeal endonucleases (96 kDa or less) instead of using conventional large Class 2 Cas effectors. This parameter change enables therapeutic delivery while maintaining endonuclease cleavage functionality through the RuvC and HNH catalytic domains.
Solution Approach 2:
The patent segments the Cas effector into functional domains (RuvC domain for DNA cleavage, HNH domain for guide RNA binding and activation) within a compact protein structure. This segmentation allows the maintenance of essential functions while reducing overall protein size for improved delivery.
2Weight of moving object
If small endonucleases are used to improve delivery, then molecular weight is reduced to 96 kDa or less, but the complexity of engineering and characterizing novel enzymes increases
Solution Approach 1:
The patent identifies universal functional features across small archaeal endonucleases, including conserved RuvC and HNH domains, that can be leveraged for multiple applications. This universality simplifies engineering by allowing the use of standardized functional modules rather than designing enzymes from scratch for each application.
Solution Approach 2:
The patent uses computational methods to model and copy functional features from known endonucleases onto newly discovered small archaeal endonucleases from uncultivated microorganisms. This copying approach reduces the experimental complexity of characterizing novel enzymes by predicting their functions based on sequence homology and structural modeling.
3Adaptability or versatility
If endonucleases from uncultivated microorganisms are used, then novel biochemical mechanisms and small size are achieved, but the difficulty of obtaining and characterizing the enzymes increases
Solution Approach 1:
The patent replaces traditional wet-lab methods of enzyme acquisition and characterization with computational approaches, including AI-based sequence analysis, homology modeling, and in silico characterization. This substitution allows the study of enzymes from uncultivated microorganisms without requiring physical isolation or traditional biochemical analysis, significantly easing the manufacturing and characterization process.
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 SMART nuclease systems enable efficient and targeted DNA manipulation and gene editing with improved delivery and efficacy.
Implementation Method 1
a guide ribonucleic acid sequence configured to hybridize to a target deoxyribonucleic acid sequence
Implementation Method 2
These endonuclease effectors are defined by their small size (about 400 aa to about 1050 aa), the presence of RuvC and HNH catalytic domains
Data Source
AI summary
The present disclosure provides for endonuclease enzymes as well as methods of using such enzymes or variants thereof.