Codon-Optimized CRISPR II System for Mammalian Genome Editing
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Solution Overview
Problem
Current methods for targeted genomic modification in mammalian cells are not efficient or versatile enough for RNA-programmed genome engineering, limiting their application in both basic research and therapeutic interventions.
Innovation Solution
Development of an all-in-one CRISPR II system comprising a human codon-optimized S. pyogenes Cas9 protein with an N-terminal Myc tag and two nuclear localization signals, combined with guide-RNAs for RNA-guided genome engineering in human or mouse cells, enabling efficient and flexible genomic targeting and modification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If current methods for targeted genomic modification are used, then basic research and therapeutic interventions can be performed, but the efficiency and versatility are insufficient
Solution Approach 1:
The CRISPR-Cas9 system provides a universal platform for genomic modification that can be programmed to target any DNA sequence through the use of customizable guide RNAs. The system combines the Cas9 endonuclease activity with programmable RNA guidance, enabling a single system to perform multiple genomic editing functions across different research and therapeutic applications.
Solution Approach 2:
The system utilizes parameter changes in the guide RNA sequence to achieve different targeting specificities. By modifying the 20-nucleotide spacer sequence in the guide RNA, the system can be reprogrammed to recognize and cleave different target DNA sequences, thereby achieving high versatility without changing the core Cas9 protein.
2Manufacturing precision
If complex molecular cloning procedures are required, then precise genomic editing can be achieved, but the ease of use decreases for researchers without extensive experience
Solution Approach 1:
The system divides the genomic editing function into separate modular components: the Cas9 endonuclease protein and the guide RNA. This segmentation allows the complex editing function to be delivered as pre-assembled, ready-to-use components rather than requiring researchers to perform complex molecular cloning procedures to assemble them.
Solution Approach 2:
The Cas9 protein and guide RNA are prepared and validated in advance by the developers, with the Cas9 protein being codon-optimized for mammalian expression and the guide RNA sequences being pre-designed for specific targets. This preliminary preparation eliminates the need for end-users to perform time-consuming molecular cloning and optimization procedures.
3Reliability
If bacterial CRISPR-Cas systems are used directly, then the natural immune mechanism is preserved, but the expression efficiency in mammalian cells decreases
Solution Approach 1:
The Cas9 gene sequence was modified by changing the codon usage to match the preferences of mammalian cells. This codon optimization changes the nucleotide sequence parameters while preserving the amino acid sequence, thereby improving translation efficiency and protein expression levels in mammalian systems without altering the functional properties of the Cas9 protein.
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 CRISPR II system achieves precise and efficient genomic editing, allowing for multiplex genome editing and various applications across basic sciences and biomedicine, with high transformation efficiency and ease of use, even for researchers without extensive molecular cloning experience.
Implementation Method 1
crRNA-Cas protein complexes recognize and degrade foreign DNAs or RNAs
Implementation Method 2
a short fragment of foreign DNA (protospacer) is acquired from the invader and integrated into the host CRISPR locus
Data Source
AI summary
The invention relates to engineered CRISPR/Cas9 systems for genomic modification in mammalian cells. The present specification describes the design and testing of a polynucleotide encoding the Streptococcus pyogenes (S. pyogenes) Cas9 protein, where the nucleotide sequence has been optimized for expression in mammalian cells. The specification also describes all-in-one systems for RNA-guided genome engineering in mammalian cells, including human cells.