CRISPR-Cas Sigma Enzyme With Single-Guide RNA and Simpler PAM Recognition
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Solution Overview
Problem
Current CRISPR/Cas systems have 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 having specific amino acid sequences and derivatives, capable of binding to guide RNA and cutting target sequences efficiently.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If a new CRISPR/Cas system is developed to improve transfection efficiency and PAM recognition, then delivery efficiency and editing capability are improved, but system complexity and development difficulty increase
Solution Approach 1:
The CRISPR system is divided into distinct functional modules: guide RNA molecules with specific sequences, Cas effector proteins with defined PAM recognition domains, and modular fusion protein constructs. This segmentation allows independent optimization of each component's function while maintaining overall system efficiency.
Solution Approach 2:
The developed Cas effector proteins are designed to recognize multiple PAM sequence variants (including 5'-TTN, 5'-ANT, 5'-ATN motifs), enabling a single system to target diverse genomic locations. Fusion proteins combine multiple functional domains to achieve both cleavage and anti-crisp functions simultaneously.
2Manufacturing precision
If existing CRISPR/Cas systems are used, then PAM sequence complexity is reduced, but off-target effects increase and editing precision decreases
Solution Approach 1:
The guide RNA molecules are designed with localized sequence features including specific 5'-end modifications and optimized spacer regions that enhance target specificity. The Cas effector proteins possess localized PAM recognition domains that precisely distinguish on-target from off-target sequences through localized molecular interactions.
Solution Approach 2:
Guide RNA molecules serve as intermediaries between the Cas effector protein and the target DNA sequence, providing an additional layer of specificity through RNA-DNA hybridization. The guide RNA's sequence complementarity and structural features mediate precise target recognition while preventing off-target binding.
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 enhances gene editing efficiency and specificity by utilizing a single guide RNA and simplified PAM sequences, reducing off-target effects.
Implementation Method 1
specifically binding to target sequences on the genome through RNA guidance
Implementation Method 2
cutting the DNA to produce double-strand breaks
Data Source
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.