CRISPR RNP Recombination in Gram-Positive Bacteria With Protected Donor DNA
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Solution Overview
Problem
Lactic acid bacteria and Gram-positive bacteria have a complicated cell wall structure, making it difficult to introduce high concentrations of RNP for gene editing, and they lack recombinase enzymes, leading to low homologous recombination rates and donor DNA hydrolysis by nuclease, which impedes efficient genetic recombination.
Innovation Solution
A method involving the introduction of a ribonucleoprotein (RNP) complex with site-specific endonucleases, guide RNA, recombinases, and phosphorothioated donor DNA, using electroporation at 8 to 12 kV/cm, to enhance genetic recombination efficiency in these bacteria.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If electroporation is used to introduce RNP into lactic acid bacteria and Gram-positive bacteria, then gene editing can be performed, but the thick cell wall structure prevents efficient RNP introduction
Solution Approach 1:
The patent optimizes electroporation parameters (voltage, pulse duration, temperature) to enhance RNP introduction efficiency despite the cell wall barrier. By adjusting these physical parameters, the method overcomes the resistance posed by the thick cell wall structure of Gram-positive bacteria and lactic acid bacteria.
2Productivity
If donor DNA is introduced for homologous recombination, then genetic recombination can occur, but nuclease activity hydrolyzes the donor DNA
Solution Approach 1:
The patent uses phosphorothioated DNA in the donor molecule, which is resistant to nuclease hydrolysis. This modified DNA structure converts the harmful nuclease activity into a non-issue, allowing the donor DNA to survive long enough to undergo homologous recombination and integrate into the bacterial genome.
3Productivity
If plasmid expression system is used for CRISPR/Cas9, then Cas9 protein can be expressed, but off-target effects increase and antibiotic marker DNA remains
Solution Approach 1:
The patent extracts and delivers only the essential functional components (Cas9 protein and guide RNA) as a ribonucleoprotein complex, eliminating the need for plasmid-based expression systems. This removes the source of off-target effects and antibiotic marker DNA while maintaining Cas9 protein expression and function.
4Object-affected harmful factors
If high voltage electroporation is applied to overcome cell wall, then RNP can be introduced, but RNP activity is lost
Solution Approach 1:
The patent performs preliminary optimization of electroporation conditions (using lower voltages with optimized pulse parameters) before RNP introduction, and protects the RNP complex during the process. This preliminary preparation ensures that the cell wall is sufficiently permeabilized without exposing the RNP to conditions that would destroy its activity.
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 method significantly increases the efficiency of gene recombination in lactic acid bacteria and Gram-positive bacteria by stabilizing donor DNA and enhancing homologous recombination, despite their thick cell walls and nuclease activity.
Implementation Method 1
a CRISPR/Cas system for genetically recombining lactic acid bacteria and gram-positive bacteria, wherein a ribonucleoprotein (RNP) complex in which a site-specific endonuclease and a guide RNA (gRNA) which specifically binds to a target DNA and guides a cleavage site of the site-specific endonuclease are associated with each other
Implementation Method 2
introducing the RNP complex and the donor DNA into the lactic acid bacteria or the gram-positive bacteria
Implementation Method 3
they lack recombinase enzymes, leading to low homologous recombination rates
Implementation Method 4
the efficiency of a RNP recombination system using Cas proteins
Data Source
AI summary
Disclosed is a method of genetically recombining lactic acid bacteria and gram-positive bacteria using the CRISPR/Cas system, more specifically, to a method of genetically recombining lactic acid bacteria and gram-positive bacteria that is capable of recombining efficiently lactic acid bacteria and gram-positive bacteria that are difficult to recombine by increasing the efficiency of the RNP recombination system using Cas proteins. However, there is a problem in which it is difficult to recombine genes of Gram-positive bacteria and lactic acid bacteria even using the CRISPR/Cas system due to the cell wall structure thereof. On the other hand, it was found that genes of lactic acid bacteria and gram-positive bacteria that are difficult to recombine can be recombined with high efficiency by using recombinases in combination with a phosphorothioated donor DNA in an RNP recombination system using Cas protein.