pJAT Plasmids for Efficient CRISPR HDR via Gateway Cloning
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Existing CRISPR-Cas-mediated HDR methods involve multiple cloning steps and have low efficiency, making them inefficient for precise genome modification in various species.
Innovation Solution
The Janelia Atalanta (pJAT) series of plasmids, which utilize Gateway® cloning sites for simultaneous introduction of synthesized dsDNA fragments containing gRNA sequences and homology arms, along with a unique dual selection system to enhance cloning efficiency and HDR integration efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If traditional CRISPR-Cas-mediated HDR methods are used, then genome modification can be achieved, but multiple cloning steps are required and HDR efficiency is low
Solution Approach 1:
The patent combines multiple cloning steps into a single Gateway cloning step by designing a unified plasmid platform that accommodates both gRNA and homology arm sequences. This merging of operations eliminates intermediate cloning steps and directly produces the final HDR construct, thereby increasing HDR integration efficiency while reducing process complexity
Solution Approach 2:
The pJAT plasmid platform serves multiple functions simultaneously: it acts as a cloning vector, an HDR template, and a delivery vehicle. The universal design allows the same plasmid backbone to accommodate different gRNA sequences and homology arms through standardized Gateway cloning sites, streamlining the overall process and improving efficiency across different genome modification applications
2Ease of manufacture
If multiple cloning steps are used to construct HDR plasmids, then gRNA and homology arms can be introduced, but the process becomes time-consuming and less efficient
Solution Approach 1:
The patent employs preliminary action by pre-designing the plasmid backbone with standardized Gateway cloning sites and regulatory elements in place. This allows researchers to directly clone synthesized gRNA and homology arm sequences without needing to perform intermediate cloning steps, thereby reducing both the time required and the complexity of the manufacturing process
Solution Approach 2:
The Gateway cloning system utilizes a copying mechanism where pre-assembled attL-containing fragments are recombined into the plasmid backbone. This copying approach replaces multiple traditional cloning operations with a single recombination event, significantly accelerating the construct generation process while maintaining high efficiency
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 pJAT plasmids achieve significantly higher HDR integration efficiencies, allowing for more precise and efficient genome modifications, including chromosomal inversions, in multiple species, thereby simplifying cloning processes and expanding the feasibility of genetic experiments.
Implementation Method 1
The Gateway cloning system is a site-specific recombination technology that takes advantage of the att site-specific recombination properties of bacteriophage lambda to provide a rapid and efficient way to move a gene of interest between multiple vector systems.
Implementation Method 2
Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)-Cas induced mutagenesis has revolutionized experimental approaches in model and non-model organisms. DNA cuts induced by Cas9 or other enzymes can be repaired by the non-homologous end-joining pathway to induce small deletions or, if a homologous DNA template is present, then cuts may be repaired by HDR.
Implementation Method 3
Homology-directed repair (HDR) is a powerful tool for modifying genomes in precise ways to address many biological questions. DNA cuts induced by Cas9 or other enzymes can be repaired by the non-homologous end-joining pathway to induce small deletions or, if a homologous DNA template is present, then cuts may be repaired by HDR.
Data Source
AI summary
Vectors and methods are described for efficient cloning and integration of a DNA sequence of interest into a genome of a cell. Vectors include a cassette having a nucleotide sequence having a negative selection marker that is a ccdB gene, which is flanked by non-identical attR recombination recognition sequences.