hfCas12Max Polypeptide Engineering for Genome Editing Specificity
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Previous Cas12i systems, such as Cas12i1 and Cas12i2, exhibit low editing efficiency, limiting their utility for therapeutic gene editing due to high off-target cleavage and low specificity.
Innovation Solution
Engineering of xCas12i with arginine substitutions at the PAM-interacting, REC, and RuvC domains to produce hfCas12Max, which enhances editing activity and minimizes off-target effects, and using xCas12i-based base editors to expand genome-editing capabilities via ribonucleoprotein and lipid nanoliposomes for ex vivo and in vivo applications.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If previous Cas12i systems (Cas12i1 and Cas12i2) are used for genome editing, then the system can perform DNA cleavage, but the editing efficiency is low due to high off-target cleavage and low specificity
Solution Approach 1:
The patent applies parameter changes by introducing arginine substitutions at specific positions (N243R, E336R, V880R, G883R, D892R, M923R) in the Cas12i protein sequence. These amino acid substitutions modify the protein's interaction parameters with DNA and guide RNA, thereby improving both specificity and editing efficiency simultaneously. The mutated positions correspond to key functional regions including PAM-interacting, REC, and RuvC domains.
Solution Approach 2:
The patent applies local quality by making targeted modifications at specific locations within the Cas12i protein structure. The arginine substitutions are localized to specific domains (PAM-interacting, REC, RuvC) rather than uniform changes throughout the protein. This localized modification strategy allows precise control over specific functional aspects while maintaining overall protein functionality.
2Productivity
If Cas12i systems are engineered with arginine substitutions to improve editing activity, then editing efficiency increases, but the complexity of the system increases
Solution Approach 1:
The patent modifies the Cas12i system by changing amino acid parameters at six specific positions through arginine substitutions. These parameter changes result in the hfCas12Max variant, which achieves significantly elevated editing activity. The modifications are focused and quantifiable, transforming the system from low-efficiency to high-efficiency without requiring fundamental redesign.
Solution Approach 2:
The patent segments the Cas12i protein into functional domains (PAM-interacting, REC, RuvC) and introduces mutations at specific positions within these segments. This segmentation approach allows independent optimization of specific functional regions while maintaining the overall modular structure of the CRISPR-Cas system, thereby improving editing activity without proportionally increasing overall system complexity.
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
hfCas12Max demonstrates significantly elevated editing activity with reduced off-target cleavage, making it a potent tool for therapeutic genome editing with improved specificity and efficiency.
Implementation Method 1
a guide RNA (also referred to as 'CRISPR RNA' or 'crRNA') or a polynucleotide encoding the guide RNA, the guide RNA comprising: (i) a direct repeat sequence capable of forming a complex with the Cas12i polypeptide or the fusion protein; and (ii) a spacer sequence capable of hybridizing to a target sequence on a target strand of a target dsDNA
Implementation Method 2
Cas12i mediates cleavage of dsDNA with a single RuvC domain, by preferentially nicking the non-target strand and then cutting the target strand
Data Source
AI summary
Provided are Cas12i polypeptides, fusion proteins comprising such Cas12i polypeptides, CRISPR-Cas12i systems comprising such Cas12i polypeptides or fusion proteins, and methods of using the same.