In Vivo Large Sequence Assembly via CRISPR-Cas Recombineering
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Solution Overview
Problem
Current methods for engineering long nucleic acid sequences in vivo are limited by inefficient homologous recombination and require cumbersome procedures, such as the use of selection markers and multiple plasmid curing steps, making it difficult to manipulate and integrate large DNA constructs into host genomes.
Innovation Solution
Combining recombineering technology with the CRISPR/Cas system, specifically the CRISPR/Cas9 system of Streptococcus thermophilus, to enable multi-site insertion of desired sequences up to 20 kb or more, allowing for facile selection of recombinant cells by cleaving non-recombinant cells with intact cleavage sites, thereby eliminating the need for selection markers and simplifying the assembly of large synthetic sequences.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If traditional recombineering methods (λ-red/gam or recET) are used to achieve homologous recombination, then integration of DNA constructs into host genome is possible, but the process is extremely inefficient requiring 1/10^4 cells and cumbersome procedures with selection markers and multiple plasmid curing steps
Solution Approach 1:
The patent combines CRISPR/Cas system with recombineering to create a hybrid system that merges the targeted cleavage capability of CRISPR with the homologous recombination capability of recombineering. This integration allows simultaneous achievement of precise targeting and efficient recombination, eliminating the need for separate selection markers and reducing procedural steps.
Solution Approach 2:
The patent extracts and eliminates the need for selection markers and multiple plasmid curing steps from the traditional recombineering procedure. By using CRISPR/Cas-mediated cleavage as a direct selection mechanism, the method removes unnecessary procedural elements while maintaining integration efficiency.
2Ease of manufacture
If selection markers are used in recombineering to select for recombinants, then recombinant cells can be identified, but the procedure becomes cumbersome requiring marker removal and leaves scars in the genome
Solution Approach 1:
The patent converts the potentially harmful effect of CRISPR/Cas cleavage (which would kill cells with intact cleavage sites) into a beneficial selection mechanism. Cells that fail to undergo recombination at the targeted site are cleaved and killed, while successful recombinants survive, providing direct selection without markers.
Solution Approach 2:
The patent removes selection markers from the recombineering procedure entirely. Instead of using antibiotic resistance markers or other selectable markers, the method uses CRISPR/Cas cleavage as the selection mechanism, eliminating the need for marker removal steps and preventing genomic scarring.
3Quantity of substance
If chemical DNA synthesis is used to assemble DNA segments, then DNA sequences can be generated, but the process is limited to about 1 kb in length due to low fidelity
Solution Approach 1:
The patent segments the DNA assembly process into two parts: (1) chemical synthesis of smaller, high-fidelity segments (1 kb or less), and (2) in vivo assembly of these segments into longer sequences through recombineering. This segmentation allows each step to operate within its optimal performance parameters.
Solution Approach 2:
The patent uses in vivo recombineering as an intermediary process between chemical synthesis and final assembly. Instead of attempting to synthesize long sequences directly (which fails due to low fidelity), the method synthesizes shorter segments and uses recombineering to assemble them into the final long sequence, with CRISPR/Cas providing selection.
4Measurement precision
If homologous recombination is used to integrate DNA into host genome, then integration at pre-determined sites is achieved, but the process is extremely inefficient requiring 1/10^4 cells
Solution Approach 1:
The patent performs preliminary action by introducing the CRISPR/Cas system before the recombineering event. The CRISPR/Cas system creates a double-strand break at the target site, which then facilitates efficient homologous recombination. This preliminary cleavage action dramatically increases recombination frequency from 1/10^4 to much higher levels.
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, scarless assembly of large synthetic sequences by directly selecting for recombinant cells, reducing the complexity of procedures and increasing the yield of engineered nucleic acids, allowing for the creation of long engineered sequences without the need for in vitro manipulation.
Implementation Method 1
The selected CRISPR/Cas system is used to cleave the target nucleic acids of cells in which all of the expected insertion events did not occur
Implementation Method 2
Integration into the host genome can be achieved through homologous recombination, a process by which dsDNA is integrated into the host genome at a pre-determined site by virtue of matching sequences
Data Source
AI summary
The present invention provides a method for making a large nucleic acid having a defined sequence in vivo. The method combines recombineering techniques with a CRISPR/Cas system to permit multiple insertions of defined sequences into a target nucleic acid at one time, double stranded cleavage of target nucleic acids in which the defined sequences were not successfully inserted, and selection of successful recombinant cells. The method further includes repeating the process one or more times, using a successful recombinant from one round as the host cell for the next round.