CRISPR-Cas Delta Endonuclease for Compact Gene Editing
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Solution Overview
Problem
Current CRISPR/Cas systems face limitations such as requiring multiple RNAs, complex PAM sequences, and varying sizes, which affect their efficiency and specificity in gene editing.
Innovation Solution
Development of a new CRISPR/Cas system with a novel RNA-guided endonuclease protein having specific amino acid sequences (SEQ ID NOs: 1, 2, and 3) and truncated variants, along with fusion proteins and nucleic acid molecules, enabling precise gene editing with improved efficiency and reduced off-target effects.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If CRISPR/Cas9 system is used for gene editing, then site-directed gene editing capability is achieved, but the system requires two guide RNAs and has complex PAM sequence requirements
Solution Approach 1:
The patent extracts and utilizes only the essential catalytic domain of Cas proteins (Casδ-1, Casδ-2, and their variants) while removing unnecessary components. This truncated Casδ system maintains gene editing functionality with simplified structure, reducing the need for multiple guide RNAs and complex PAM requirements while preserving the core nucleic acid editing capability
Solution Approach 2:
The patent modifies key parameters of the CRISPR system by using Casδ proteins with altered PAM recognition specificities and different nuclease activities compared to conventional Cas9. These parameter changes enable the system to function with simplified RNA guidance requirements and reduced off-target effects
2Manufacturing precision
If C2c1 is used for gene editing, then target site prediction is easier and off-target effects are reduced, but the system recognizes only rigorous 5'-TTN PAM motifs limiting target availability
Solution Approach 1:
The patent creates a universal Casδ system that can recognize multiple PAM sequence variants (including 5'-TTN and other motifs) while maintaining the high precision editing capability of C2c1. This multi-functional PAM recognition expands target site availability without sacrificing editing precision, allowing the system to adapt to diverse genomic locations
3Productivity
If Cas9, C2c1, or CasX is used for gene editing, then editing functionality is achieved, but the protein size is large (980-1300 amino acids) affecting delivery efficiency
Solution Approach 1:
The patent extracts only the essential catalytic core of Cas proteins, creating truncated Casδ-1 and Casδ-2 variants with reduced amino acid sequences. These truncated proteins retain full gene editing functionality while significantly reducing protein size compared to full-length Cas9 (1300 aa), C2c1 (1300 aa), or CasX (980 aa), thereby improving delivery efficiency to target cells
Solution Approach 2:
The patent employs smaller, more transient Casδ protein constructs that can be efficiently delivered and function temporarily to perform the editing task. These reduced-size proteins are easier to package in viral vectors or deliver as mRNA, and their temporary presence in cells minimizes immune response and off-target effects while maintaining productive editing
4Productivity
If conventional CRISPR/Cas systems are used, then gene cutting capability is achieved, but off-target effects occur due to complex and diverse PAM sequences
Solution Approach 1:
The patent enhances the specificity of PAM recognition by the Casδ system, creating localized high-fidelity binding at the intended target site. The truncated Casδ proteins exhibit stricter PAM matching requirements at the cutting site while maintaining efficient on-target activity, thereby reducing off-target effects through improved local recognition quality rather than relying on complex global PAM diversity
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 new CRISPR/Cas system provides enhanced gene editing capabilities with improved specificity and efficiency, allowing for targeted DNA or RNA sequence modifications without significant off-target effects.
Implementation Method 1
specifically binding to target sequences on the genome through RNA guidance
Implementation Method 2
the activity of endonuclease, and the activity of binding to and cutting a specific site of a target sequence
Data Source
Figure 1A~1B
Figure 2~3
Figure 4A~4B
AI summary
The present invention relates to the field of nucleic acid editing, in particular to the field of clustered regularly interspaced short palindromic repeat (CRISPR) technology. Specifically, the present invention relates to Cas effector proteins, fusion proteins comprising such proteins, and nucleic acid molecules encoding them. The present invention also relates to complexes and compositions for nucleic acid editing (e.g., gene or genome editing), which comprise the proteins or fusion proteins of the present invention, or nucleic acid molecules encoding them. The present invention also relates to a method for nucleic acid editing (e.g., gene or genome editing), which uses the proteins or fusion proteins comprising the present invention.