Targeted Genome Modification With Circular ssDNA Donors for Longer Inserts
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Solution Overview
Problem
Existing genome editing technologies using linear single-stranded DNA (LiSSD) templates are limited by their error-prone, inefficient, and costly methods, which restrict them to carrying short DNA sequences of less than 2 kb, failing to meet the need for longer sequences in cellular engineering.
Innovation Solution
The use of circular single-stranded DNA (CiSSD) as donor templates for targeted genome modification, which includes transferring CiSSD with a DNA insert and homology arms to cells, inducing a nucleotide break, and inserting the DNA insert into the target region of genomic DNA, thereby generating genetically modified cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Length of moving object
If linear single-stranded DNA (LiSSD) templates are used for targeted genome modification, then the method is simple to implement, but the template is limited to carrying short DNA sequences of less than 2 kb and has high error rates
Solution Approach 1:
The patent applies the curvature principle by transitioning from linear single-stranded DNA (LiSSD) to circular single-stranded DNA (CiSSD) templates. The circular configuration eliminates the terminal ends of linear DNA that are susceptible to degradation and errors, thereby increasing template stability and reliability. This structural transformation enables the template to carry longer DNA sequences (exceeding 2 kb) while maintaining low error rates, as the circular topology provides continuous protection against exonuclease activity and improves overall template integrity during genome editing processes.
2Productivity
If linear single-stranded DNA (LiSSD) templates are used for targeted genome modification, then the production method is straightforward, but the process is inefficient and expensive
Solution Approach 1:
The circular configuration of CiSSD templates fundamentally improves integration efficiency compared to linear templates. The circular topology enables more effective cellular uptake and sustained presence within the cell nucleus, leading to enhanced homology-directed repair (HDR) efficiency. Additionally, the circular structure allows for more efficient in vitro synthesis methods, such as rolling circle amplification, which can produce high yields of uniform CiSSD templates at lower costs, thereby resolving the contradiction between productivity and ease of manufacture.
3Reliability
If linear single-stranded DNA (LiSSD) templates are used for targeted genome modification, then the template structure is simple, but the cytotoxicity is high and specificity is reduced
Solution Approach 1:
The circular topology of CiSSD templates significantly reduces cytotoxicity compared to linear templates. The closed circular structure prevents degradation by exonucleases that target linear DNA ends, reducing the accumulation of toxic DNA fragments in cells. This structural advantage also enhances specificity by ensuring that only the intended circular template is efficiently incorporated into the genome editing process, minimizing off-target effects and improving the overall reliability of the genome modification.
Data Source
AI summary
The present invention is directed to methods for generating one or more genetically modified cells by using a circular single stranded DNA (CiSSD) as a donor template and targeting genome modification. These methods include transferring one or more DNA polynucleotides into the cell for site-specific nuclease-mediated DNA repair and selecting one or more cells having the transferred DNA incorporated into the cell's genome.