Engineered CRISPR-Cas12i Systems for High-Fidelity Multiplex Editing
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Existing CRISPR-Cas systems, particularly Cas9 and Cas12a, are limited by size and efficiency in genome editing, with Cas12i showing promise but needing enhancements for improved multiplex high-fidelity editing and reduced off-target effects.
Innovation Solution
A Cas12i polypeptide with amino acid substitutions at specific positions (E336, V880, G883, D892, and/or M923) is developed, combined with a guide nucleic acid and spacer sequence to form a complex for targeted DNA modification, using a system that includes ribonucleoproteins and lipid nanoparticles for enhanced specificity and efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If Cas12i is used for genome editing, then multiplex high-fidelity editing is achieved, but off-target effects increase
Solution Approach 1:
The patent applies parameter changes by introducing specific amino acid substitutions (E336R, V880R, G883R, D892R, M923R) at defined positions in the Cas12i protein sequence. These parameter modifications in the protein structure enable enhanced discrimination between target and off-target sequences, thereby reducing off-target effects while maintaining high-fidelity editing capability.
Solution Approach 2:
The patent replaces the conventional Cas9 system with a engineered Cas12i system that utilizes a different molecular mechanism - the Cas12i protein's intrinsic ability to process precursor crRNA and its preferential nicking of the non-target strand followed by cutting of the target strand. This substitution of the editing mechanism enables multiplex high-fidelity editing with reduced off-target effects.
2Productivity
If Cas9 and Cas12a are used for genome editing, then editing capability is achieved, but system size increases
Solution Approach 1:
The patent extracts and utilizes only the essential functional components needed for genome editing - specifically the engineered Cas12i polypeptide with amino acid substitutions and a guide nucleic acid. By taking out and optimizing these critical elements, the system achieves high editing capability while minimizing unnecessary components that would increase system size.
Solution Approach 2:
The patent applies parameter changes by optimizing the Cas12i protein structure through specific amino acid substitutions that enhance editing capability. These parameter modifications enable the system to achieve high productivity in genome editing while maintaining a compact size, as the engineered Cas12i is smaller than Cas9 and Cas12a.
3Productivity
If conventional CRISPR systems are used, then genome editing is achieved, but efficiency is limited
Solution Approach 1:
The patent replaces conventional CRISPR systems (Cas9, Cas12a) with an engineered Cas12i system that has a different molecular mechanism. The Cas12i protein preferentially nicks the non-target strand and then cuts the target strand, which enables multiplex high-fidelity editing with improved efficiency and fidelity compared to conventional systems.
Solution Approach 2:
The patent applies parameter changes by introducing specific amino acid substitutions (E336R, V880R, G883R, D892R, M923R) in the Cas12i protein that enhance both editing efficiency and fidelity. These parameter modifications optimize the protein's ability to process guide nucleic acids and cleave target DNA while reducing off-target effects.
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 modified Cas12i system achieves improved multiplex high-fidelity genome editing with reduced off-target effects, enabling precise DNA modification and potential applications in disease diagnosis, treatment, and detection.
Implementation Method 1
a spacer sequence capable of hybridizing to a target sequence of a target DNA, thereby guiding the complex to the target DNA
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
The disclosure provides Cas12i polypeptides, fusion proteins comprising such Cas12i polypeptides, CRISPR-Cas12i systems comprising such Cas12i polypeptides or fusion proteins, and methods of using the same.