CRISPR-Cas Delta Enzyme With Single-Guide RNA and Flexible PAM Targeting
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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 (SEQ ID NOs: 1, 2, and 3) and truncated variants, along with fusion proteins and nucleic acid molecules for enhanced gene editing capabilities.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If Cas9, C2c1, or CasX are used for gene editing, then gene editing function is achieved, but multiple guide RNAs are required which increases system complexity
Solution Approach 1:
The invention extracts and eliminates the requirement for multiple RNA components (tracrRNA and other auxiliary RNAs) from the CRISPR system, retaining only the essential guide RNA that contains both the spacer and repeat sequences. This simplification maintains the gene editing function while reducing the number of required components from two or more RNAs to a single guide RNA molecule.
2Device complexity
If Cpf1 is used which requires only one guide RNA, then RNA complexity is reduced, but the PAM sequence recognition is limited
Solution Approach 1:
The invention changes the PAM recognition parameter by designing a guide RNA with a specific repeat sequence that recognizes a broader range of PAM sequences (5'-TTN, 5'-TAN, 5'-TGN, 5'-TYN where N is any nucleotide and Y is pyrimidine). This expands the adaptability of the system compared to Cpf1's more restricted PAM recognition, allowing the system to target diverse genomic locations while maintaining single guide RNA simplicity.
3Weight of moving object
If CasX with 980 amino acids is used, then protein size is reduced, but it still requires multiple RNAs and has complex PAM requirements
Solution Approach 1:
The invention merges the guide RNA and repeat sequence recognition functions into a single integrated guide RNA molecule that combines both elements. This consolidation allows the CasX protein (980 amino acids) to function with simplified RNA requirements, eliminating the need for separate tracrRNA and auxiliary RNA components, thereby reducing overall system complexity while maintaining the compact protein size advantage.
4Reliability
If C2c1 with rigorous 5'-TTN PAM recognition is used, then off-target effects are reduced, but target site prediction is limited
Solution Approach 1:
The invention creates a multi-functional PAM recognition capability where the guide RNA repeat sequence can recognize multiple PAM sequence variants (5'-TTN, 5'-TAN, 5'-TGN, 5'-TYN). This universality allows the system to maintain the high specificity of C2c1 for reducing off-target effects while expanding the range of acceptable PAM sequences, thereby increasing the number of available target sites across the genome.
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 improved efficiency and specificity in gene editing by utilizing a single guide RNA and offering flexible protein configurations for targeted DNA or RNA manipulation.
Implementation Method 1
CRISPR/Cas technology is a widely used gene editing technique that utilizes biological non-homologous end joining or homologous recombination to perform site-directed gene editing by specifically binding to target sequences on the genome through RNA guidance
Implementation Method 2
CRISPR/Cas9 system is the most commonly used type II CRISPR system, which recognizes 3′-NGG PAM motifs and performs blunt-end cutting on the target sequences
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.