Targeted CRISPR Delivery with Truncated sgRNA for Precise Editing
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Solution Overview
Problem
Existing CRISPR-Cas9 systems face challenges in achieving specific and accurate editing of a wide range of target sites, particularly when combined with reliable nucleic acid delivery platforms, and there is a need for improved Cas9 and sgRNA sequences to enhance genome editing precision and reduce off-target effects.
Innovation Solution
The use of Neisseria meningitidis Cas9 systems, specifically Nme1Cas9 and Nme2Cas9, with truncated guide RNA sequences and adeno-associated viral vectors for in vivo administration, along with Type II-C Cas9 orthologs that target protospacer adjacent motif sequences limited to one to four required nucleotides, to enhance editing accuracy and compatibility with nucleic acid delivery.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional CRISPR-Cas9 systems are used, then genome editing can be performed, but editing accuracy is insufficient and off-target effects occur
Solution Approach 1:
The patent applies parameter changes by modifying the PAM recognition requirement from traditional 3-4 nucleotide sequences to a reduced 1-4 nucleotide requirement, and by optimizing sgRNA sequence parameters (truncating stem 2 region, adjusting repeat-anti-repeat structure) to enhance Cas9 binding specificity and reduce off-target effects while maintaining editing accuracy
Solution Approach 2:
The patent replaces the traditional mechanical delivery method (plasmid transfection) with a viral vector-based delivery system (AAV) that directly packages and delivers the CRISPR-Cas9 ribonucleoprotein complex to target cells in vivo, enabling more precise and controlled genome editing
2Reliability
If full-length guide RNA sequences are used, then Cas9 binding is stable, but the system is not compatible with in vivo administration
Solution Approach 1:
The patent extracts and removes the problematic stem 2 region from the full-length sgRNA sequence, retaining only the essential repeat-anti-repeat structure and guide sequence. This truncated sgRNA maintains sufficient binding stability while reducing the overall size and complexity, making it compatible with in vivo delivery via viral vectors
Solution Approach 2:
The patent segments the sgRNA into functional regions: the repeat-anti-repeat structure for Cas9 binding and the guide sequence for target recognition. By optimizing these segmented regions independently, the patent achieves both stable binding and in vivo compatibility
3Adaptability or versatility
If traditional Cas9 systems are used, then a wide range of target sites can be accessed, but specific and accurate editing is difficult to achieve
Solution Approach 1:
The patent changes the PAM recognition parameter to require only 1-4 nucleotides instead of traditional 3-4 nucleotides, expanding the range of accessible target sites. Simultaneously, by optimizing the sgRNA sequence parameters (truncating stem 2, adjusting repeat-anti-repeat structure), the patent enhances editing precision at each target site
Data Source
AI summary
The present invention is related to compositions and methods for gene therapy. Several approaches described herein utilize the Neisseria meningitidis Cas9 system that provides a hyperaccurate CRISPR gene editing platform. Furthermore, the invention incorporates full length and truncated single guide RNA sequences that permit a complete sgRNA-Nme1Cas9 vector to be inserted into an adeno-associated viral plasmid that is compatible for in vivo administration. Furthermore, Type II-C Cas9 orthologs have been identified that target protospacer adjacent motif sequences limited to between one-four required nucleotides.