Dual-Guide CRISPR-Cas Transgene Insertion With High Cell Viability
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Solution Overview
Problem
Existing CRISPR-Cas systems fail to efficiently integrate and express transgenes with high integration and expression efficiency in eukaryotic cells, particularly in human immune or stem cells, and often compromise cell viability.
Innovation Solution
The development of engineered non-naturally-occurring dual guide CRISPR-Cas systems, including optimized compositions and methods for genome engineering, utilizing guide nucleic acids, nucleases, RNP stabilizers, and NHEJ inhibitors to enhance transgene insertion and expression efficiency while maintaining cell viability.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional CRISPR-Cas systems are used for transgene insertion, then genome editing can be performed, but integration efficiency and transgene expression are insufficient
Solution Approach 1:
The patent modifies the CRISPR-Cas system parameters by introducing engineered nucleases with altered PAM recognition specificities and optimized guide RNA sequences. These parameter changes enable the system to target specific genomic loci with higher precision and efficiency, thereby improving transgene integration and expression while maintaining cell viability.
Solution Approach 2:
The patent employs engineered nucleic acid-guided nucleases as intermediary molecules that facilitate precise genome targeting. These nucleases act as mediators between the guide RNA and the target DNA, enabling efficient transgene insertion through controlled double-strand breaks and subsequent homology-directed repair, thereby resolving the contradiction between integration efficiency and expression reliability.
2Productivity
If conventional CRISPR-Cas systems are used for genome engineering, then editing can be achieved, but cell viability is compromised
Solution Approach 1:
The patent applies local quality by designing nucleic acid-guided nucleases with specific PAM recognition properties that enable targeted editing at predetermined loci while minimizing off-target effects. This localized approach ensures that genome editing occurs only at the intended sites, thereby maintaining overall cell viability while achieving efficient editing.
Solution Approach 2:
The patent converts the potentially harmful effect of CRISPR-Cas induced DNA breaks into a beneficial process by leveraging homology-directed repair mechanisms. The engineered nucleases create controlled double-strand breaks that trigger cellular repair pathways, which can be harnessed to insert transgenes with high efficiency while maintaining cell viability through precise repair mechanisms.
3Ease of manufacture
If standard CRISPR-Cas systems are used, then transgene insertion can be performed, but integration and expression efficiency remain insufficient
Solution Approach 1:
The patent segments the CRISPR-Cas system into distinct functional components: engineered nucleases with specific PAM recognition, guide RNAs with optimized sequences, and donor templates with homology arms. This segmentation allows each component to be independently optimized for its specific function, thereby improving overall transgene insertion ease and expression efficiency.
Solution Approach 2:
The patent creates a composite system combining engineered nucleases, guide RNAs, and donor templates into a coordinated genome editing platform. This composite approach integrates multiple functional elements that work synergistically to achieve high transgene insertion efficiency and expression, resolving the contradiction between ease of manufacture and productivity.
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 engineered systems achieve high integration and expression efficiency of transgenes in eukaryotic cells, particularly human immune and stem cells, with improved post-transfection cell viability and targeted editing.
Implementation Method 1
a nuclease complex capable of binding to and cleaving a target polynucleotide at a specific location
Implementation Method 2
followed by homology-directed repair using a donor polynucleotide template to insert the transgene at the location of the cleavage
Data Source
AI summary
CRISPR-Cas systems have been engineered for various purposes, such as genomic DNA cleavage, base editing, epigenome editing, and genomic imaging. Although significant developments have been made, there still remains a need for new and useful CRISPR-Cas systems as powerful precise genome targeting tools. The invention disclosed herein comprises CRISPR-Cas based methods for high integration and expression efficiency of transgenes together with high post-transfection cell viability in eukaryotic cells.