Trio Guide RNA System for Large Donor DNA Integration
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Solution Overview
Problem
The CRISPR-Cas system is inefficient for integrating large exogenous DNA into target genomes, particularly for protein tagging, as it faces challenges with HDR pathways and size limitations, leading to low insertional efficiency and adverse effects on gene expression.
Innovation Solution
A trio guide RNA (gRNA) system is employed, where three gRNAs flank and target a donor sequence and a locus of interest, using RNA-guided nucleases like Cas9 to enhance integration efficacy by forming specific DNA:RNP complexes for efficient gene editing and integration of large DNA sequences.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If homology directed repair (HDR) pathway is used for targeted delivery of donor DNA, then gene knock-in can be achieved, but insertional efficiency drastically decreases
Solution Approach 1:
The patent divides the single gRNA into multiple gRNA segments (first gRNA upstream of donor sequence, second gRNA downstream of donor sequence, and third gRNA at target locus). This segmentation allows the system to overcome the inefficiency of HDR by creating multiple cleavage sites that facilitate donor DNA integration through alternative mechanisms while maintaining precise targeting capability.
2Adaptability or versatility
If large size exogenous DNA is used to detectably tag genome protein, then protein tagging capability is enhanced, but integration efficiency decreases and gene expression is adversely affected
Solution Approach 1:
The patent segments the integration process into multiple steps guided by separate gRNAs. The first and second gRNAs flank the large donor sequence to enable its integration, while the third gRNA targets the specific genomic locus. This segmentation allows efficient integration of large DNA sequences without compromising gene expression.
Solution Approach 2:
The patent introduces an intermediary mechanism where multiple gRNA-Cas9 complexes work together to facilitate the integration of large donor DNA. The flanking gRNAs create a protective framework that enables efficient integration of large sequences while the target locus gRNA ensures precise genomic placement, preventing adverse effects on endogenous gene expression.
3Ease of operation
If CRISPR-Cas system is used for genome editing, then targeted manipulation capability is achieved, but targeted delivery of donor DNA by HDR or NHEJ remains very inefficient
Solution Approach 1:
The patent divides the CRISPR-Cas system into multiple gRNA components that work in concert. The first and second gRNAs target regions flanking the donor sequence to enhance its delivery, while the third gRNA maintains targeted manipulation at the specific genomic locus. This segmentation overcomes the inefficiency of traditional single gRNA approaches for donor DNA delivery.
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 trio gRNA system significantly increases the integration efficiency of large donor DNA sequences into target loci, improving gene knock-in efficiency and minimizing aberrant recombination, enabling effective protein tagging without disrupting gene expression.
Implementation Method 1
The DNA targeting of the RNP complex is driven by about 20 nucleotides of complementary RNA-DNA base-pairing between the sgRNA and the target DNA
Implementation Method 2
targeting of genomic sequence by sgRNA-Cas9 complex leads to double-stranded DNA breaks
Implementation Method 3
The CRISPR-Cas system can be used for mutagenesis or delivery of exogenous DNA using the non-homology end joining repair (NHEJ) or homology directed repair (HDR) pathways in cells
Implementation Method 4
The CRISPR-Cas system can be used for mutagenesis or delivery of exogenous DNA using the non-homology end joining repair (NHEJ) or homology directed repair (HDR) pathways in cells
Data Source
AI summary
A new DNA knock-in approach is provided based on the usage of three single guide RNA (sgRNA) to increase the integration efficiency of donor DNA based on the CRISRP-Cas system. The approach uses a pair of universal sgRNAs complementary to the donor DNA and a single sgRNA that targets the locus of interest. In various embodiments, targeting is achieved by pre-forming a DNA:RNA:protein (DNA:RNP) complex in vitro and introducing the complex into the embryo or cells of interest either by microinjection or transfection.