Synthetic Regulatory System for CRISPR HDR Efficiency
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Solution Overview
Problem
Current CRISPR-based gene editing techniques face challenges such as low efficiency of homology-directed repair (HDR) in vivo, unwanted immune reactions, and incorrect cell targeting, which limit their therapeutic applications.
Innovation Solution
The introduction of a synthetic regulatory system comprising a multifunctional Cas nuclease and guide RNAs of varying lengths to target specific nucleotide sequences, inducing double-stranded breaks and facilitating HDR through a donor nucleic acid molecule, thereby increasing the rate of HDR compared to non-homologous end joining (NHEJ).
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If CRISPR-based gene editing is performed in vivo, then genetic therapy can be achieved, but the efficiency of homology-directed repair (HDR) is very low
Solution Approach 1:
The patent applies preliminary action by using truncated gRNAs to repress NHEJ enzyme expression before the HDR process occurs. This pre-suppression of competing repair pathways prepares the cellular environment favorably for HDR, thereby improving HDR efficiency without requiring changes to the HDR mechanism itself
Solution Approach 2:
The patent employs parameter changes by modifying gRNA length (using truncated gRNAs of 10-15 nucleotides) to specifically target and repress NHEJ enzymes. This parameter modification allows selective inhibition of competing repair pathways while maintaining HDR capability, thus resolving the efficiency contradiction
2Adaptability or versatility
If CRISPR systems are used for in vivo genetic engineering, then therapeutic applications can be developed, but unwanted immune system reactions occur
Solution Approach 1:
The patent applies the taking out principle by separating the gene editing function from the immune-triggering components. By using truncated gRNAs that specifically target NHEJ enzymes rather than using standard CRISPR Cas9 systems, the invention extracts the harmful immune-reactive elements while retaining the desired gene editing capability through alternative mechanisms
3Manufacturing precision
If CRISPR-mediated correction is performed, then genetic mutations can be corrected, but incorrect cell infection may occur
Solution Approach 1:
The patent applies the intermediary principle by introducing truncated gRNAs as mediator molecules that specifically bind to and repress NHEJ enzymes. These intermediary gRNAs act as selective agents that modulate cellular repair pathways to favor HDR in the correct cells, thereby improving both precision and reliability of gene correction
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 enhances the efficiency of CRISPR-mediated gene editing by improving HDR rates and reducing regulatory and safety hurdles, making it more suitable for clinical applications and allowing for precise genome editing.
Implementation Method 1
Clustered regularly interspaced short palindromic repeats (CRISPR), a bacterial adaptive immune system, and its CRISPR-associated protein 9 (Cas9), have gained attention for the ability to target and modify DNA sequences on demand with unprecedented flexibility and precision. The precision and programmability of Cas9 is derived from its complexation with a guide-RNA (gRNA) that is complementary to a desired genomic sequence.
Implementation Method 2
A particular concern is very low efficiency of CRISPR-mediated correction of genetic mutation using homology-directed repair (HDR) in vivo. The rate of homology-directed repair (HDR) compared with non-homologous end joining (NHEJ) can be increased.
Data Source
AI summary
Aspects of the disclosure relate to methods and synthetic regulatory systems for more efficient nuclease-mediated homology-directed repair (HDR). In particular, provided herein are methods for more efficient in vivo and in vitro HDR-based gene editing where the methods comprise introducing into a cell a synthetic regulatory system comprising Cas nuclease, guide RNAs (gRNAs) having various lengths and configured to target distinct nucleotide sequences for simultaneous transcriptional repression (or activation) and genome editing via double stranded break and use of a donor nucleic acid molecule as a template for repair.