Fusion Proteins for Precise Gene Targeting
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Solution Overview
Problem
Current genetic modification techniques, such as CRISPR-Cas9 and CRISPR-Cpf1, rely heavily on the error-prone NHEJ pathway for DNA repair, leading to 'indels' and off-site breaks, and face challenges in efficiently inserting longer DNA sequences due to the rapid repair via NHEJ over HDR, necessitating more accurate and reliable methods for sequence-specific modification.
Innovation Solution
The development of fusion proteins combining a 5' to 3' DNA exonuclease domain with an RNA binding domain, an endonuclease domain fused to a replication initiation complex component, a recombination inducing domain, a mismatch repair inhibitor, and a Holliday junction resolvase, which inhibit NHEJ, promote HDR, and enhance donor DNA accumulation near the target locus, facilitating precise genetic modifications.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If CRISPR-Cas9 or CRISPR-Cpf1 systems are used to create DNA double strand breaks, then sequence-specific DNA cutting is achieved, but off-site breaks occur and indels are generated due to error-prone NHEJ repair
Solution Approach 1:
The patent introduces a donor DNA template as an intermediary molecule that mediates the repair process. Instead of relying on the error-prone NHEJ pathway alone, the donor template provides a correct sequence blueprint for HDR, reducing indels and off-site breaks while maintaining sequence-specificity through guide RNA-directed targeting.
Solution Approach 2:
The patent changes the repair pathway parameter by shifting from NHEJ-dominated repair to HDR-dominated repair. This is achieved by optimizing the timing and concentration of donor DNA delivery, and by using modified CRISPR systems that enhance HDR efficiency, thereby improving repair accuracy while maintaining targeting precision.
2Productivity
If NHEJ pathway is used for DNA repair, then rapid and efficient repair is achieved, but insertion or deletion of nucleotides (indels) occurs at the break site
Solution Approach 1:
The donor DNA template serves as an intermediary that redirects the repair process from NHEJ to HDR. The template provides a precise nucleotide sequence that guides accurate repair, eliminating indels while the CRISPR system maintains rapid targeting. The intermediary template thus reconciles speed with precision.
Solution Approach 2:
The patent applies preliminary action by pre-delivering the donor DNA template to the cell before or concurrent with CRISPR-mediated DSB creation. This ensures the template is available when HDR is initiated, allowing accurate nucleotide incorporation to occur rapidly alongside the fast CRISPR targeting, thus maintaining both speed and precision.
3Manufacturing precision
If HDR pathway is used for DNA repair, then accurate repair using donor DNA sequence is achieved, but the efficiency is low compared to NHEJ
Solution Approach 1:
The patent enhances the HDR pathway by making it multi-functional. The donor DNA template not only provides the sequence for accurate repair but also incorporates elements that enhance its own delivery and retention. Additionally, the CRISPR system is optimized to simultaneously create DSBs and promote HDR, making the overall system both accurate and efficient.
Solution Approach 2:
The patent applies preliminary action by pre-delivering and pre-positioning the donor DNA template in the cell nucleus before CRISPR-mediated DSB creation. This preliminary positioning ensures high local concentration of the template at the target site when the DSB occurs, dramatically increasing HDR efficiency while maintaining its high accuracy advantage over NHEJ.
4Adaptability or versatility
If donor DNA is provided for HDR, then specific modifications and sequence insertions are enabled, but the efficiency remains low due to rapid NHEJ repair
Solution Approach 1:
The patent applies preliminary action by delivering donor DNA templates with enhanced stability and nuclear retention properties before CRISPR treatment. The templates are pre-positioned at the target loci using targeted delivery methods, ensuring they are ready to be used by HDR machinery immediately when DSBs occur, thus enabling versatile modifications with high efficiency.
Solution Approach 2:
The patent uses composite donor DNA constructs that combine multiple functional elements: homology arms for HDR targeting, selectable markers for verification, and sequences that enhance nuclear retention and stability. This composite design makes the donor DNA both versatile for different modification types and highly efficient in promoting HDR over NHEJ.
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 a 5′ to 3′ DNA exonuclease domain and an RNA binding domain is described. Also provided are methods, reagents and compositions for in vivo genetic modification of the genome of a human or animal cell. Furthermore, the present application relates to uses of the said methods, reagents and compositions in the treatment of disease and production of transgenic animals.