Prime Editor Composition with Chromatin Modulation
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Solution Overview
Problem
Current prime editing technologies face limitations in gene editing efficiency and target specificity, particularly due to low efficiency of intracellular homology-directed repair and issues with off-target effects and insertion/deletion of additional base sequences.
Innovation Solution
A gene editing composition incorporating a fusion protein comprising CRISPR/Cas9 and reverse transcriptase, along with prime editing guide RNA (pegRNA) and dead single guide RNA (dsgRNA), and optionally chromatin-modulating peptides, to enhance editing efficiency and specificity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If prime editing is used to generate target mutations, then substitution and small insertion/deletion can be achieved without double-stranded DNA cleavage, but off-target effects and additional insertion/deletion occur reducing accuracy
Solution Approach 1:
The guide RNA is divided into two separate functional components: pegRNA for prime editing and dsgRNA for chromatin modulation. This segmentation allows each component to perform its specific function independently, improving overall editing accuracy while reducing unwanted side effects
Solution Approach 2:
dead single guide RNA (dsgRNA) acts as an intermediary that modulates chromatin structure at the target site, making it more accessible to the prime editor without introducing harmful off-target effects or additional insertions/deletions
2Productivity
If currently developed prime editors are used, then genome editing can be performed, but editing efficiency remains very low at 20 to 50%
Solution Approach 1:
The invention creates a composite gene editing system combining prime editor (PE) with dead single guide RNA (dsgRNA) and chromatin-modulating peptides. This composite approach synergistically enhances editing efficiency while maintaining reliability, overcoming the limitations of individual components
3Manufacturing precision
If intracellular homology-directed repair is used for gene editing, then target mutation can be generated, but the process has low efficiency limiting accurate mutation generation
Solution Approach 1:
The dsgRNA performs preliminary action by pre-modulating the chromatin structure at the target site before the prime editor arrives. This preliminary chromatin relaxation facilitates more efficient homology-directed repair and improves overall mutation generation efficiency
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
Significantly improved genome editing efficiency and target specificity are achieved, allowing for accurate and efficient generation of target mutations, including insertions and deletions, with reduced off-target effects, as demonstrated by enhanced editing performance in mammalian cells and successful transmission of mutations to the next generation.
Implementation Method 1
the reverse transcriptase synthesizes a new DNA strand including a base sequence edited based on the RT template strand of the pegRNA
Implementation Method 2
The Cas9 nickase of the prime editor cleaves the non-target strand of the target gene
Data Source
AI summary
The present invention relates to a prime editing-based gene editing composition with enhanced editing efficiency, a method for gene editing by using the composition, a gene editing kit, and a method for construction of a gene-modified mammal. The prime editor developed in the present invention exhibits remarkably enhanced genome editing efficiency and target specificity and mutant animal models constructed by using same were observed to perform mutation transmission to the next generation and a phenotype change in the next generation. Thus, the enhanced prime editor or a gene editing composition comprising same can find advantageous applications in various purposes, such as the construction and research of humanized animal models, the genetic engineering technical field, and therapeutic means for genetic diseases.