Virus-Mediated Gene Insertion Without Homologous Arms
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Solution Overview
Problem
Current gene editing technologies using viruses are limited by the size of nucleic acid packaging capacity and the requirement for homologous arms in HDR-based methods, making it impractical for inserting long sequences.
Innovation Solution
A method involving the generation of a DNA double-strand break at a target genome location and introduction of a virus with a nucleic acid containing the polynucleotide sequence or its complementary sequence, without homologous arms, using site-specific nucleases like CRISPR-Cas, TALEN, or ZFN, to facilitate insertion through the NHEJ pathway.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If HDR-based gene editing with homologous arms is used, then precise site-specific gene insertion is achieved, but the insertion fragment size is limited due to virus packaging capacity
Solution Approach 1:
The invention extracts and removes the requirement for long homologous arms from the gene editing process. By using microhomologous arms (5-25 bp) instead of traditional long homologous arms, the patent eliminates the packaging size constraint while maintaining precise site-specific insertion capability through the NHEJ pathway
Solution Approach 2:
The invention changes the parameter of homologous arm length from traditional long sequences to microhomologous arms (5-25 bp). This parameter change enables the use of viruses with limited packaging capacity to deliver larger polynucleotide sequences while still achieving precise genomic insertion
2Productivity
If viruses with limited packaging capacity are used, then gene delivery is efficient, but long sequence insertion becomes impractical
Solution Approach 1:
The invention extracts the homologous arm requirement from the delivery payload, allowing viruses to carry larger polynucleotide sequences without needing to include long homologous arms. The microhomologous arms (5-25 bp) are sufficient for NHEJ-mediated insertion, greatly expanding the usable packaging capacity
Solution Approach 2:
The invention uses microhomologous arms as intermediaries that bridge the virus-delivered polynucleotide sequence and the target genomic location. These short sequences (5-25 bp) are sufficient to mediate precise insertion through NHEJ without requiring the virus to package large homologous arm sequences
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 allows for efficient insertion of larger polynucleotide sequences into genomes without the need for homologous arms, enhancing gene editing capabilities and increasing the size of the transgene that can be inserted, as demonstrated by successful knock-in of the BDD-F8 transgene into the Alb locus in mouse liver cells and functional recovery in a hemophilia mouse model.
Implementation Method 1
generating a DNA double-strand break at a target location of the genome
Data Source
AI summary
Provided is a method of inserting a polynucleotide sequence into a genome of a cell. The method comprises: generating a double-strand break at a target site of the genome; and introducing into the cell a virus. The virus comprises a nucleic acid comprising the polynucleotide sequence to be inserted or the complementary sequence thereof. The nucleic acid does not comprise a homologous arm or comprises very short (5˜25 bp) homologous arms corresponding to the target site. Also provided herein is a composition for inserting a polynucleotide sequence into a genome of a cell. The composition comprises a site-specific nuclease capable of generating a DNA double-strand break at a target site of the genome and a virus comprising a nucleic acid comprising the polynucleotide sequence or the complementary sequence thereof.