RGEN-CPP Complex Traverses Bacterial Membrane
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Solution Overview
Problem
Current methods for delivering protein and RNA components for CRISPR-mediated DNA targeting in bacterial cells are limited, particularly in avoiding the introduction of exogenous DNA and ensuring efficient cellular uptake.
Innovation Solution
A method involving a composition of the protein component of an RNA-guided endonuclease (RGEN) covalently or non-covalently linked with a cell-penetrating peptide (CPP), along with an associated RNA component, which traverses the bacterial cell membrane to target specific DNA sites for cleavage.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If recombinant DNA expression strategies are used to deliver Cas9 and gRNA into bacterial cells, then the DNA targeting function can be achieved, but exogenous DNA is introduced into the cell which may be undesirable
Solution Approach 1:
The invention extracts the essential DNA-cutting function by delivering only the Cas9 protein and gRNA RNA components into the cell, rather than introducing recombinant DNA constructs that would require expression. This allows the RGEN complex to perform its DNA targeting and cleavage function without the harmful side effect of introducing exogenous DNA into the bacterial genome.
Solution Approach 2:
The invention uses an mRNA intermediate to bridge the gap between having the Cas9 coding sequence and having functional Cas9 protein. By introducing mRNA that encodes Cas9 (rather than DNA that would need to be transcribed), the system delivers the protein-coding information in a form that can be directly translated by the cell's machinery, avoiding permanent DNA integration while still achieving protein delivery.
2Object-affected harmful factors
If protein and RNA components are delivered separately, then the risk of exogenous DNA introduction is avoided, but the efficiency of cellular uptake and complex formation is reduced
Solution Approach 1:
The invention merges the Cas9 protein and gRNA RNA components into a single delivered complex (the RGEN complex). By pre-assembling these components together before delivery, the system ensures they arrive at the same location simultaneously, facilitating efficient complex formation and function while maintaining the advantage of not introducing exogenous DNA.
Solution Approach 2:
The delivered mRNA encodes Cas9, which is then translated by the cell's own ribosomes and translation machinery into functional Cas9 protein. This self-service approach allows the cell to produce the protein component using its endogenous machinery, improving uptake efficiency while still avoiding exogenous DNA introduction.
3Device complexity
If RGEN components are delivered without cell-penetrating peptides, then the delivery system is simpler, but the ability to traverse the cell membrane and enter bacterial cells is limited
Solution Approach 1:
The invention uses cell-penetrating peptides (CPPs) as intermediary molecules that facilitate the entry of the RGEN complex into bacterial cells. These CPPs act as mediators that can interact with the cell membrane and transport the bulky RGEN complex across the membrane barrier, enabling reliable delivery without requiring complex alternative delivery systems.
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
Enables efficient delivery and targeting of RGEN components into bacterial cells, facilitating precise DNA editing without the need for exogenous DNA constructs, thus enhancing gene editing capabilities.
Implementation Method 1
at least one cell-penetrating peptide (CPP), wherein said protein component and CPP are covalently, or non-covalently, linked to each other in an RGEN protein-CPP complex, wherein the composition further comprises at least one RNA component... wherein said RGEN protein-CPP complex traverses (i) a cell membrane, or (ii) a cell wall and cell membrane, of the cell, thereby entering the bacterial cell
Implementation Method 2
the RNA component comprises a sequence complementary to a target site sequence on a chromosome or episome in the cell, wherein the RGEN can bind to the target site sequence
Implementation Method 3
wherein the RGEN can bind to the target site sequence, and optionally cleave one or both DNA strands at the target site sequence
Data Source
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Figure 3A~3B
AI summary
A composition is disclosed that comprises at least one protein component of an RNA-guided endonuclease (RGEN) and at least one cell-penetrating peptide (CPP), wherein the RGEN protein component and CPP are covalently or non-covalently linked to each other in an RGEN protein-CPP complex. The RGEN protein-CPP complex can traverse (i) a cell membrane, or (ii) a cell wall and cell membrane, of a cell. The RGEN protein component of an RGEN protein-CPP complex in certain embodiments can be associated with a suitable RNA component to provide an RGEN capable of specific DNA targeting. Further disclosed are compositions comprising at least one protein component of a guide polynucleotide/Cas endonuclease complex and at least one CPP, as well as methods of delivering RGEN proteins into microbial cells, as well as methods of targeting DNA with RGENs.