CRISPR DNA Template for Precise Targeted Insertion
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Solution Overview
Problem
Current CRISPR/Cas systems face challenges in achieving high-frequency, precise targeted insertions into DNA, with homology-directed repair being inefficient and non-homologous end joining being error-prone, limiting the ability to insert desired sequences at specific sites.
Innovation Solution
A polynucleotide composition comprising an RNA guide sequence, a Cas-binding region, and a DNA template sequence, where the DNA template is positioned at the 3' end, incorporating modified nucleotides, non-B DNA structures, and recruitment moieties for DNA polymerase and ligase, facilitates targeted insertions by guiding Cas nucleases to specific DNA sites and enabling precise integration of sequences of interest.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If homology-directed repair (HDR) is used for template-dependent repair, then insertion precision is improved, but repair frequency is reduced
Solution Approach 1:
The patent introduces a single-stranded DNA template as an intermediary molecule that mediates the repair process. This template is designed with specific features (modified nucleotides, secondary structures) to facilitate its own integration into the break site, serving as both the repair template and the integrated sequence, thereby resolving the contradiction between precision and frequency
Solution Approach 2:
The patent modifies the physical and chemical parameters of the DNA template by incorporating modified nucleotides (e.g., phosphorothioate bonds, LNA, PNA) and creating non-B DNA structures (hairpins, G-quadruplexes). These parameter changes enhance the template's stability, binding affinity, and integration efficiency, enabling high-frequency repair while maintaining precision
2Productivity
If non-homologous end joining (NHEJ) is used for template-independent repair, then repair frequency is improved, but insertion precision is reduced due to error-prone repair
Solution Approach 1:
The single-stranded DNA template acts as an intermediary that directs the NHEJ process. By designing the template with specific secondary structures and modified nucleotides, it becomes preferentially integrated into the break site through NHEJ, converting an error-prone process into a precise insertion method
Solution Approach 2:
The patent performs preliminary design of the DNA template structure before the repair process. The template is pre-configured with hairpins, G-quadruplexes, or other secondary structures that facilitate its own recognition and integration by the NHEJ machinery, ensuring precise insertion before the repair occurs
Data Source
AI summary
The present disclosure provides a polynucleotide comprising an RNA guide sequence, a Cas-binding region, and a DNA template sequence. The disclosure also provides compositions comprising a Cas nuclease or a Cas nickase and one or more polynucleotides comprising a guide sequence, a Cas binding region, and a DNA template sequence. The disclosure further provides a fusion protein comprising a Cas nuclease or a Cas nickase and a DNA polymerase recruitment moiety. Also provided are methods for providing a targeted insertion in a target DNA of a cell.