Allele-Specific Guide RNA Tuning for Precise Genome Editing
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Solution Overview
Problem
Existing genome editing techniques using CRISPR/Cas systems often induce unexpected gene mutations and low efficiency in knocking out or knocking in genes, particularly when targeting both alleles, leading to concerns about indels and mutations in normal genes.
Innovation Solution
A method for genome editing that involves using guide RNAs with added nucleotide residues at the 5′-end and spacer sequence mismatches, along with Cas proteins, to specifically edit one allele, and includes a prediction and analysis method for editing patterns.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If CRISPR/Cas system is used for genome editing, then ease of operation and productivity are improved, but manufacturing precision deteriorates due to unexpected gene mutations from NHEJ repair
Solution Approach 1:
The patent applies local quality by designing guide RNAs with specific mismatch patterns at particular positions (5' end of spacer sequence) to create allele-specific binding. This localized modification of the guide RNA structure enables differential recognition between target and non-target alleles, achieving precise control over which allele is edited while maintaining the overall simplicity of the CRISPR/Cas system.
2Reliability
If standard guide RNA is used, then on-target activity is maintained, but object-generated harmful factors increase due to off-target effects and unintended mutations
Solution Approach 1:
The patent changes the parameter of guide RNA sequence composition by introducing specific mismatches at the 5' end of the spacer sequence. This parameter modification reduces off-target binding affinity while preserving on-target activity, thereby decreasing harmful off-target effects without sacrificing reliable on-target editing.
3Manufacturing precision
If HDR is used for genome editing, then manufacturing precision is improved, but productivity deteriorates due to low probability of successful knock-in
Solution Approach 1:
The patent employs copying by introducing a donor DNA template with the desired genetic modification and utilizing the cell's HDR machinery to copy this template into the target allele. This approach enables precise knock-in of specific sequences while the allele-specific guide RNA ensures that only the intended allele is edited, improving both precision and 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
This approach allows precise editing of one allele with reduced indels and mutations, enabling accurate gene manipulation and prediction of editing outcomes.
Implementation Method 1
CRISPR consists of short conserved repeat sequences of 24 to 48 bp interspersed with unique variable DNA sequences called spacers
Implementation Method 2
The Cas9 protein, which is a nuclease that cleaves DNA, cleaves the invading exogenous DNA
Data Source
AI summary
A production method for a cell in which only one allele is genome-edited includes: a step of introducing, into a cell, (A) at least one selected from the group consisting of (a1) a guide RNA in which one or more nucleotide residues are added to a 5′-end of a spacer sequence, (a2) a guide RNA containing a spacer sequence having single-base or multiple-base mismatches with respect to a target sequence, and (a3) an expression vector that can cause the guide RNA of (a1) or (a2) to be expressed, and (B) at least one selected from the group consisting of a Cas protein and an expression vector that can cause the Cas protein to be expressed.