CRISPR/Cas9 Genome Editing Efficiency in Human Stem Cells
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Solution Overview
Problem
Current CRISPR/Cas systems for genome editing in mammalian cells have low efficiency, with only 2%-4% allele targeting in human stem cells, limiting their therapeutic applications.
Innovation Solution
Improved methods for allele targeting using CRISPR/Cas systems, including efficient delivery to human stem cells, identification of specific RNA guide sequences, and methods for treating disorders like severe combined immunodeficiency, sickle cell disease, and beta thalassemia by altering target polynucleotide sequences with high efficiency up to 80%, using Cas9 protein complexed with ribonucleic acids that direct the protein to specific motifs in the genome.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If CRISPR/Cas systems are used for genome editing in human stem cells, then gene targeting capability is achieved, but editing efficiency is low (2%-4%)
Solution Approach 1:
The patent optimizes multiple parameters including Cas9 protein concentration, guide RNA design, electroporation voltage and pulse duration, and culture conditions to achieve up to 80% editing efficiency in human stem cells, resolving the contradiction between achieving gene targeting capability and improving editing efficiency
Solution Approach 2:
The patent employs preliminary selection of optimal guide RNA sequences and pre-optimization of delivery conditions before actual genome editing, which enables high efficiency editing (up to 80%) while maintaining reliable gene targeting capability
2Reliability
If CRISPR/Cas systems are delivered to human stem cells, then genome editing capability is achieved, but delivery efficiency limits therapeutic application
Solution Approach 1:
The patent uses electroporation as an intermediary method to deliver Cas9 protein and guide RNA into human stem cells, achieving efficient delivery and genome editing capability that overcomes the limitations of other delivery methods
Solution Approach 2:
The patent optimizes electroporation parameters including voltage, pulse duration, and cell density to maximize delivery efficiency while maintaining cell viability, enabling therapeutic applications of CRISPR/Cas systems in human stem cells
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 methods enable effective genome editing with efficiencies up to 80%, making CRISPR/Cas systems suitable for therapeutic purposes by specifically targeting and altering disease-associated genes, thereby treating severe combined immunodeficiency, sickle cell disease, and beta thalassemia.
Implementation Method 1
the ribonucleic acids direct Cas protein to and hybridize to a target motif of the target SCID-associated polynucleotide sequence
Implementation Method 2
wherein the target SCID-associated polynucleotide sequence is cleaved
Data Source
AI summary
Disclosed herein are methods, compositions, and kits for high efficiency, site-specific genomic editing of cells for treating or preventing genetic blood disorders.