Cps1 Cps2 CRISPR Endonucleases Eliminate tracrRNA Requirement
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Solution Overview
Problem
Current CRISPR-Cas systems have limitations, such as requiring trans-activating crRNA for target DNA recognition and cleavage, which complicates genome editing applications, and there is a need for novel RNA-guided DNA endonucleases with distinct characteristics to overcome issues like pre-existing immune responses in humans and expand genome editing capabilities.
Innovation Solution
Identification and characterization of novel CRISPR-Cas endonucleases, specifically Cps1 and Cps2, which do not require trans-activating crRNA and possess unique properties like a RuvC domain for targeted DNA cleavage, guided by specific guide RNAs and Protospacer Adjacent Motif (PAM) sequences, enabling efficient genome editing without the need for additional cellular nucleases.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If Class 2 Cas proteins (Cas9, Cpf1, Cms1) are used for genome editing, then targeted DNA cleavage can be achieved, but the requirement for trans-activating crRNA (tracrRNA) and additional cellular nucleases complicates the system
Solution Approach 1:
The patent extracts and removes the tracrRNA requirement from the CRISPR-Cas system by identifying and characterizing novel CRISPR-Cas endonucleases (Cps1 and Cps2) that function without this additional RNA component, thereby simplifying the genome editing machinery while retaining targeted DNA cleavage capability
Solution Approach 2:
The patent segments the CRISPR-Cas system into essential and non-essential components, identifying that the novel Cps1 and Cps2 endonucleases can operate with only the crRNA guide component, separating the necessary functional elements from the optional tracrRNA element that complicates other Class 2 systems
2Productivity
If established CRISPR-Cas systems are applied in humans, then genome editing can be performed, but pre-existing immune responses may occur
Solution Approach 1:
The patent changes the biological parameters by introducing novel CRISPR-Cas endonucleases (Cps1 and Cps2) with distinct amino acid sequences and structural characteristics that differ from previously used Cas9, Cpf1, and Cms1 systems, thereby potentially evading pre-existing human immune responses while maintaining genome editing functionality
3Device complexity
If novel CRISPR-Cas endonucleases (Cps1, Cps2) are used, then the need for additional cellular nucleases is eliminated, but the enzymes require identification and characterization from metagenome
Solution Approach 1:
The patent performs preliminary action by conducting metagenomic sequencing and bioinformatic analysis to identify and characterize novel CRISPR-Cas endonucleases (Cps1 and Cps2) before their application in genome editing, thereby preparing and validating these simplified enzymes in advance for future use
Solution Approach 2:
The patent uses metagenomic databases and bioinformatic tools as intermediaries to bridge the gap between environmental DNA samples and the discovery of functional CRISPR-Cas endonucleases, facilitating the identification and characterization of Cps1 and Cps2 without direct cultural isolation of the source organisms
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
Cps1 and Cps2 demonstrate effective genome editing capabilities, as shown by repairing a mutant beta-glucuronidase gene in wheat embryos, expanding the repertoire of CRISPR-Cas systems for diverse genetic backgrounds and potentially overcoming immune response challenges, with the ability to introduce targeted double-strand breaks without additional cellular processing.
Implementation Method 1
Cps1 is a type V class 2 CRISPR nuclease that, together with a guide RNA, is capable of binding to a target DNA sequence and introducing a double-strand break at a defined location
Implementation Method 2
Cps2 is a type V class 2 CRISPR nuclease that, together with a guide RNA, is capable of binding to a target DNA sequence and introducing a double-strand break at a defined location
Data Source
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AI summary
The present invention relates to a nucleic acid molecule encoding an RNA-guided DNA endonuclease, which is (a) a nucleic acid molecule encoding the RNA-guided DNA endonuclease comprising or consisting of the amino acid sequence of SEQ ID NO: 1 or 3; (b) a nucleic acid molecule comprising or consisting of the nucleotide sequence of SEQ ID NO: 2 or 4; (c) a nucleic acid molecule encoding a RNA-guided DNA endonuclease the amino acid sequence of which is at least 70 % identical to the amino acid sequence of (a); preferably at least 80 % identical, more preferably at least 90 % identical, and most preferred at least 95% identical; (d) a nucleic acid molecule comprising or consisting of a nucleotide sequence which is at least 70 % identical to the nucleotide sequence of (b), preferably at least 80 % identical, more preferably at least 90 % identical, and most preferred at least 95% identical; (e) a nucleic acid molecule which is degenerate with respect to the nucleic acid molecule of (d); or (f) a nucleic acid molecule corresponding to the nucleic acid molecule of any one of (a) to (d) wherein T is replaced by U.