Cas9 Ribonucleoprotein Delivery to Primary Hematopoietic Cells
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Solution Overview
Problem
Current methods for delivering nucleic acids and proteins to primary hematopoietic cells or stem cells are inefficient, limiting genome editing and gene regulation in these cells.
Innovation Solution
A method involving a reaction mixture with a Cas9 ribonucleoprotein complex, guide RNA, and a double or single-stranded oligonucleotide DNA template is introduced into primary hematopoietic cells or stem cells via electroporation, achieving high efficiency genome editing by targeting specific genomic regions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional delivery methods are used for nucleic acids and proteins to primary hematopoietic cells, then the delivery process is simpler, but the delivery efficiency is low
Solution Approach 1:
The patent segments the delivery system into separate components: purified Cas9 protein and guide RNA are delivered independently rather than as a single complex. This segmentation allows each component to be optimized separately and improves overall delivery efficiency to primary hematopoietic cells while maintaining operational simplicity
Solution Approach 2:
The patent uses an intermediary delivery mechanism involving electroporation or microinjection to facilitate the entry of Cas9 protein and guide RNA into primary hematopoietic cells. This intermediary method overcomes the natural resistance of these cells to conventional delivery, achieving high efficiency without requiring complex viral vectors
2Reliability
If Cas9 ribonucleoprotein complex is delivered to primary hematopoietic cells, then genome editing efficiency improves, but the complexity of the delivery system increases
Solution Approach 1:
The patent applies preliminary action by pre-forming the Cas9- guide RNA complex outside the cell before delivery. This pre-assembled ribonucleoprotein complex is then introduced into primary hematopoietic cells using electroporation, ensuring that the editing machinery is ready to function immediately upon entry, thereby提高ing genome editing efficiency while avoiding the complexity of intracellular assembly
Solution Approach 2:
The patent employs disposable purified Cas9 protein and guide RNA that are delivered in a single-use format for each editing experiment. These components are not integrated into the cell genome permanently but function transiently to achieve the editing goal, then are degraded naturally, simplifying the overall system compared to permanent genetic modifications
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
This approach enables efficient genome editing in primary hematopoietic cells, with editing efficiencies ranging from 20% to 80%, facilitating applications in cancer therapy, infectious disease treatment, and autoimmune disease management by modifying T cells and other immune cells.
Implementation Method 1
introducing the Cas9 ribonucleoprotein complex and the DNA template inside the cell via electroporation
Data Source
Figure 1A
Figure 1B~1D
Figure 1E
AI summary
Methods and compositions are provided for highly efficient delivery of Cas9 and Cas9 ribonucleoproteins to cells, including primary hematopoietic cells and primary hematopoietic stem cells.