Nuclease-Mediated Plasmid Integration for Large DNA Fragment Insertion
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Solution Overview
Problem
Current systems for nucleic acid-guided nuclease editing face challenges in efficiently inserting large DNA fragments, such as those greater than 100 base pairs, into the genomes of live cells, particularly in achieving high editing rates for precise, targeted changes.
Innovation Solution
The use of nucleic acid-guided nuclease-mediated methods and compositions that facilitate plasmid integration into target loci in cells through sequence-directed double-stranded breaks, leveraging homology-directed repair and non-homologous end joining pathways, with the aid of CRISPR-type nucleases and editing cassettes containing gRNAs and donor DNA sequences, enabling the integration of large DNA sequences, including genes, into the cellular genome.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If nucleic acid-guided nuclease editing is used to insert large DNA fragments (>100 bp) into cell genomes, then targeted genome editing capability is achieved, but editing efficiency and integration frequency are insufficient
Solution Approach 1:
The editing system is divided into separate functional modules: a first vector containing the nuclease and gRNA, and a second vector containing the donor DNA fragment. This segmentation allows independent optimization of each component and facilitates efficient delivery and integration, resolving the contradiction between targeted precision and editing efficiency
Solution Approach 2:
A donor DNA vector serves as an intermediary carrier that delivers the large DNA fragment (>100 bp) to the target site. This intermediary approach enables efficient transfer of large sequences while maintaining precise targeting through the nuclease-gRNA complex, thereby improving both editing efficiency and integration frequency
2Reliability
If current nuclease editing systems are used for large fragment insertion, then some level of genome editing is achieved, but plasmid integration frequency remains low
Solution Approach 1:
The system optimizes multiple parameters simultaneously: the size and structure of the donor vector, the composition of the nuclease-gRNA complex, and the cellular conditions for integration. These parameter changes collectively enhance plasmid integration frequency while maintaining reliable genome editing capability
Solution Approach 2:
The donor DNA is pre-packaged in a separate vector system before delivery to cells. This preliminary preparation ensures the DNA is in an optimal state for integration, increasing integration frequency while maintaining editing reliability through pre-validated vector designs
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 allows for reliable, multiplexed, and targeted genome editing, achieving high plasmid integration frequencies, enabling the efficient insertion or substitution of large DNA sequences, including metabolic pathway genes, with enhanced precision and efficiency.
Implementation Method 1
leveraging homology-directed repair and non-homologous end joining pathways
Implementation Method 2
leveraging homology-directed repair and non-homologous end joining pathways
Data Source
AI summary
The present disclosure relates to methods and compositions for nuclease-mediated plasmid integration into the genome of a population of live cells, as well as automated multi-module instruments for performing these methods and using these compositions.