Fusion Protein Targeting for Accurate Gene Editing
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Solution Overview
Problem
Current genetic modification techniques, such as CRISPR-Cas9, face challenges in achieving accurate and reliable modifications, particularly due to off-site breaks and the error-prone nature of the NHEJ pathway, which limits the efficiency and accuracy of HDR.
Innovation Solution
The use of fusion proteins that combine an endonuclease or bacteriophage coat protein domain with a binding domain for an origin of replication, along with the production of single-stranded DNA in vivo, enhances the targeting and accumulation of donor DNA near the modification site, promoting HDR and reducing off-site breaks.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If CRISPR-Cas9 or other protein complexes are used to make DNA double strand breaks, then genetic modification can be achieved, but off-site breaks occur reducing accuracy
Solution Approach 1:
The patent uses a small RNA molecule as an intermediary to guide the Cas9 endonuclease to the specific target sequence. The RNA guide forms a complex with Cas9, enabling sequence-specific recognition and binding only at the intended genomic location, thereby preventing off-site breaks while maintaining the ability to induce precise double-strand breaks for genetic modification.
2Productivity
If NHEJ pathway is used to repair DNA double strand breaks, then repair is efficient and rapid, but errors occur reducing modification accuracy
Solution Approach 1:
The patent introduces a donor DNA template with homology arms flanking the desired modification sequence before inducing the double-strand break. This preliminary preparation of the correct repair template ensures that when HDR occurs, the modification is accurate. The homology arms guide the repair machinery to use the correct sequence, preventing errors that would occur with NHEJ.
Solution Approach 2:
The patent changes the repair pathway parameter by providing conditions that favor HDR over NHEJ. This is achieved by introducing a donor template with extended homology regions and potentially modulating cellular conditions to promote homology-directed repair, thereby shifting the balance from error-prone NHEJ to accurate HDR while maintaining efficient repair.
3Manufacturing precision
If HDR is used to enable larger genetic modifications, then accuracy improves, but efficiency decreases compared to NHEJ
Solution Approach 1:
The patent performs preliminary actions to enhance HDR efficiency by: (1) introducing a donor DNA template with optimized homology arm lengths and sequences before break induction, (2) potentially pre-conditioning cells to be more receptive to HDR, and (3) designing the donor template to include all necessary modification elements in the correct configuration, thereby facilitating efficient and accurate HDR simultaneously.
Solution Approach 2:
The patent creates a universal system that can achieve both accurate HDR and efficient modification by using a modular donor template design. The donor template can accommodate various types of modifications (insertions, deletions, replacements) while maintaining the same homology arm structure, making the system universally applicable for different genetic engineering goals with consistent high efficiency and accuracy.
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 significantly improves the accuracy and efficiency of genetic modifications by enhancing the transformation efficiency, promoting HDR, and reducing the occurrence of off-site breaks and errors associated with NHEJ.
Implementation Method 1
an endonuclease or bacteriophage coat protein domain
Implementation Method 2
a binding domain for an origin of replication
Implementation Method 3
the production of single-stranded DNA in vivo
Implementation Method 4
the efficiency of genetic modification using homology-dependent recombination (HDR)
Data Source
AI summary
Methods, reagents and compositions for providing more accurate and reliable genetic modification are provided. In particular a nucleic acid encoding a fusion protein comprising an endonuclease domain and a binding domain for an origin of replication is described. Also provided are methods, reagents and compositions for in vivo genetic modification of the genome of a non-animal cell or organism. Furthermore, the present application relates to uses of the said methods, reagents and compositions for introducing desirable traits to non-animal organisms or ameliorating or removing non-desirable traits in these organisms including in the treatment of disease.