RNP Base Editor Reduces Off-Target Effects in Gene Correction
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Solution Overview
Problem
Current gene-editing technologies, such as CRISPR/Cas9, suffer from off-target effects and inefficiencies in gene correction, particularly in applications like treating retinal degenerative diseases.
Innovation Solution
A base editor in the form of a ribonucleoprotein (RNP) complex, comprising a fusion protein with a Cas9 domain and an adenine or cytidine deaminase domain, is used for precise gene correction by reducing off-target effects.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If base editors are delivered using bacterial plasmids or viral vectors, then base editing function is achieved, but off-target effects increase due to continuous production of exogenous base editors
Solution Approach 1:
The patent uses ribonucleoprotein (RNP) complexes consisting of Cas9 protein and guide RNA that are transient and non-integrating. These RNPs are delivered directly into cells and degrade naturally after performing their editing function, avoiding continuous production and accumulation that causes off-target effects. This disposable approach contrasts with plasmid/viral delivery where base editors are continuously expressed
Solution Approach 2:
The patent extracts and delivers only the essential functional components (Cas9 protein and guide RNA as RNP complex) without delivering the genetic material that would lead to continuous expression. By taking out just the active editing machinery and delivering it in a transient form, the system achieves editing function while eliminating the source of continuous production that causes off-target effects
2Object-affected harmful factors
If base editors are delivered using RNP complexes, then off-target effects are reduced, but manufacturing difficulty increases due to challenges in producing high purity base editor proteins
Solution Approach 1:
The patent segments the base editor into two separate components: the Cas9 protein and the guide RNA. These can be produced independently using established methods (Cas9 via bacterial expression, gRNA via in vitro transcription), then assembled into RNP complexes. This segmentation allows each component to be optimized and purified separately, simplifying the overall manufacturing process while maintaining high purity
Solution Approach 2:
The patent uses an intermediary assembly step where pre-purified Cas9 protein and synthesized guide RNA are combined to form RNP complexes. This intermediary step allows for quality control and purification of each component before final assembly, facilitating high purity production without requiring complex direct expression systems
3Reliability
If CRISPR/Cas9 nuclease is used for gene correction, then DNA cleavage and repair pathways are activated, but DNA double-strand breaks cause unwanted insertions or deletions
Solution Approach 1:
The patent extracts and removes the DNA cleavage function from the CRISPR/Cas9 system by using a catalytically inactive Cas9 (dCas9) or Cas9 nickase (nCas9) variant. Only the genome-targeting and base modification functions are retained, while the harmful double-strand break activity is taken out and eliminated, preventing unwanted indels
Solution Approach 2:
The patent converts the potentially harmful DNA cleavage activity into a beneficial non-cleaving base editing function. By modifying Cas9 to be catalytically inactive or have reduced activity, the system transforms from a DNA-cutting tool that causes indels into a precise base substitution tool that leaves DNA integrity intact while still achieving 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
The RNP complex-mediated base editing significantly reduces off-target effects, enabling effective in vivo gene correction, particularly in retinal degenerative diseases, with improved purity and yield of the base editor proteins.
Implementation Method 1
a fusion protein with a Cas9 domain and an adenine or cytidine deaminase domain
Data Source
AI summary
Provided are a base editor, specifically, a base editor in the form of a ribonucleoprotein (RNP) complex, and uses in gene correction in vivo using the same. When using the base editor in the form of an RNP complex of the present disclosure, target genes can be effectively corrected by reducing off-target effects.