CRISPR Knock-In Editing for Higher HDR in Human Stem Cells
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Solution Overview
Problem
Existing CRISPR/Cas9 systems are inefficient for precise targeted DNA insertion, especially in human pluripotent stem cells, and lack optimized systems for human cells, including human embryonic stem cells and induced pluripotent stem cells, with low HR efficiency and high off-target effects.
Innovation Solution
Development of Cas9-mediated genome editing systems with improved homology-directed repair efficiency, including flexible donor vectors and user-friendly tools for targeted gene editing, utilizing CRISPR/Cas9 and CRISPR/Cpf1 systems for unidirectional and bidirectional knock-in, and homology-independent integration of reporter genes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If CRISPR/Cas9 system is used for targeted gene manipulation, then gene editing capability is achieved, but HDR efficiency remains low particularly in human cells
Solution Approach 1:
The patent employs multiple parameter changes to improve HDR efficiency: (1) optimizing donor DNA characteristics including size, structure (linear vs. circular), and homology arm length; (2) adjusting cell cycle timing to coincide HDR with S/G2 phases when HDR is most active; (3) modifying Cas9 and sgRNA parameters to enhance target specificity and reduce off-target effects; (4) controlling delivery parameters such as transfection timing and method. These parameter optimizations collectively achieve up to 5-fold increase in HDR efficiency in human ESCs while maintaining high gene editing productivity
Solution Approach 2:
The patent implements preliminary actions to enhance HDR efficiency: (1) pre-synchronization of cells to specific cell cycle stages before CRISPR/Cas9 delivery to ensure cells are in HDR-competent phases; (2) pre-optimization of donor DNA constructs with appropriate homology arms and selectable markers before transfection; (3) pre-screening of target sites using bioinformatics tools to identify optimal locations with high HDR potential and low off-target risk; (4) pre-treatment of cells with chemicals that temporarily enhance HDR pathway activity. These preliminary preparations enable significantly improved HDR efficiency while maintaining high productivity
2Manufacturing precision
If CRISPR/Cas9 system is used for targeted DNA insertion, then precise genomic modification is achieved, but off-target effects occur
Solution Approach 1:
The patent introduces several intermediary mechanisms to reduce off-target effects: (1) using high-fidelity Cas9 variants (e.g., eSpCas9, SpCas9-HF1) as intermediaries that maintain cutting activity at on-target sites while significantly reducing off-target cleavage; (2) employing truncated sgRNAs (17-19 nucleotides) as intermediaries that enhance mismatch discrimination and reduce off-target binding; (3) using chemical compounds as intermediaries that temporarily modulate Cas9 activity or chromatin accessibility to favor on-target editing; (4) implementing dual sgRNA systems where two guides must simultaneously bind for productive cleavage, serving as an intermediary safety check. These intermediaries preserve precise targeted DNA insertion while minimizing harmful off-target effects
3Adaptability or versatility
If CRISPR/Cas9 system is used for large DNA fragment knock-in, then genomic modification capability is achieved, but efficiency remains low especially in human pluripotent stem cells
Solution Approach 1:
The patent applies segmentation to overcome the efficiency barrier for large DNA fragment knock-in: (1) dividing large donor DNA fragments (>10 kb) into multiple smaller sub-fragments that can be more efficiently delivered and integrated; (2) using modular donor construct designs where large fragments are assembled from smaller, standardized modules with universal homology arms and selection markers; (3) implementing stepwise integration strategies where large fragments are inserted in sequential steps rather than single-event transfection; (4) segmenting the delivery process into multiple rounds of transfection and selection. These segmentation approaches enable efficient knock-in of large DNA fragments in human pluripotent stem cells while maintaining versatility for various fragment sizes
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 systems achieve up to 5-fold increase in HR efficiency in human ESCs, provide universal genomic editing, and enable precise integration of reporter genes with reduced off-target effects, suitable for various research and clinical applications.
Implementation Method 1
a new technology based on a bacterial CRISPR-associated protein-9 nuclease (Cas9) from Streptococcus pyogenes has generated considerable excitement and interest
Implementation Method 2
studies have made efforts to improve the efficiency of homology-directed repair (HDR) of CRISPR/Cas9 induced DNA cleavages for achieving precise targeted DNA insertion into genome
Implementation Method 3
Maruyama et al. in 2015 inhibited non-homologous end joining (NHEJ) pathway to increase the HDR efficiency of genome editing
Data Source
AI summary
The present invention provides novel methods and systems, including cell lines, recombinant polynucleotide constructs, compositions, and kits, for targeted yet universal genomic manipulation.