Multi-Gene Base Editing Vector Without Double-Strand Breaks
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Solution Overview
Problem
Current gene knockout methods, particularly for multi-gene editing, face challenges in efficiency and safety, as they often cause double-strand breaks that can activate proto-oncogenes and inhibit tumor suppressor genes, and are not optimized for homozygous cell lines.
Innovation Solution
A vector system using a cytosine base editing system with a replicon element, U6 promoter, and sgRNA for each target gene, linked in series, to mutate exons and introduce premature termination codons, thereby silencing multiple genes without causing double-strand breaks.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If the CRISPR/Cas9 system is used to cleave target genes for knockout, then gene editing can be achieved, but double-strand breaks are caused which activate proto-oncogenes and inhibit tumor suppressor genes
Solution Approach 1:
The patent changes the editing mechanism from double-strand break (DSB) based Cas9 to base substitution using cytosine base editors. This parameter change in the editing mode eliminates DSB formation while achieving gene knockout through precise base conversion (C•G to T•A) followed by premature stop codon introduction, thereby resolving the safety issue without sacrificing editing capability
Solution Approach 2:
The patent extracts and removes the problematic DSB formation step from the gene editing process. By using cytosine base editors that perform direct base substitution without requiring DSB, the harmful double-strand break event is completely eliminated while the desired gene knockout function is preserved through alternative molecular mechanisms
2Productivity
If multiple genes are edited simultaneously to achieve multi-gene knockout, then breeding efficiency is improved, but the complexity of the editing system increases
Solution Approach 1:
The patent merges multiple sgRNA expression units into a single plasmid vector, with each unit containing a U6 promoter driving a specific sgRNA. This consolidation allows simultaneous delivery and expression of multiple guide RNAs for multi-gene editing in one transformation event, improving productivity while managing system complexity through integrated vector design
Solution Approach 2:
The patent segments the multi-gene editing system into modular units, where each target gene has its own U6 promoter-sgRNA pair. This segmentation allows independent optimization of each sgRNA while maintaining coordinated expression, facilitating systematic design and troubleshooting of multi-gene editing experiments
3Ease of manufacture
If the CRISPR/Cas9 system is used for gene knockout, then the operation is simple and cost is low, but the editing efficiency for homozygous cell lines is insufficient
Solution Approach 1:
The patent changes the editing mechanism from non-homologous end joining (NHEJ) dependent DSB repair to base substitution followed by homology-directed repair. The cytosine base editor introduces precise C•G to T•A transitions that create premature stop codons, which are then fixed into homozygous mutations through selective breeding or additional editing rounds, achieving high precision while maintaining operational simplicity
Solution Approach 2:
The patent performs preliminary base substitution editing to introduce premature stop codons before final homozygous knockout is achieved. This preliminary action creates intermediate mutant alleles that can be selectively amplified and fixed, improving overall editing efficiency for homozygous cell line generation while maintaining the simplicity of the CRISPR platform
Data Source
AI summary
Provided are a vector for multi-gene base editing, an editing method, and a use, and belongs to the technical field of gene editing. This application provides a vector for multi-gene base editing. In the vector, a replicon element, U6, and sgRNA are linked in series to allow a plasmid carrying the replicon element to persist in a cell, such that other elements on the plasmid can be continuously expressed in the cell, which improves the efficiency of multi-gene silencing. This application also provides a tandem vector, which is obtained by removing an epi element from the vector above. The two multi-gene editing vectors constructed in the embodiments of the present disclosure both have a silencing editing effect for target loci. The vector including the epi element has a higher editing efficiency than the vector without the epi element.