Linear Double-Stranded Donor DNA for CRISPR Genome Editing
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Solution Overview
Problem
Current genome editing techniques face inefficiencies in introducing precise changes, particularly with large edits, requiring long homology arms or selection markers, and are prone to imprecise repair mechanisms.
Innovation Solution
The use of linear, double-stranded donor DNAs with short homology arms (35-60 bp) for CRISPR-based genome editing, allowing for efficient integration of edits up to 1 kb without cloning or selection, and employing PCR fragments as donors to facilitate precise genome editing in mammalian cells and mouse embryos.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If long homology arms (>500 nt) are used for HDR, then large edits (such as GFP knock-ins) can be recovered, but the editing efficiency remains low and requires selection markers
Solution Approach 1:
The patent changes the critical parameter of homology arm length from conventional long arms (>500 nt) to short arms (35-60 bp), which fundamentally alters the HDR mechanism to enable efficient editing without selection markers while maintaining precision for large inserts
2Productivity
If short homology arms (35-60 bp) are used, then editing efficiency is significantly enhanced and selection markers are minimized, but the ability to recover large edits was previously thought to be compromised
Solution Approach 1:
The patent demonstrates that changing the homology arm length parameter to 35-60 bp enables both high efficiency and high precision for large edits, overturning the conventional wisdom that short arms compromise precision
3Manufacturing precision
If ssODNs are used for editing, then short edits (10 bp) can be introduced, but recovery frequency is low
Solution Approach 1:
The patent changes the donor DNA structure from single-stranded to linear double-stranded format with short homology arms, which significantly improves recovery frequency while maintaining the ability to introduce precise edits
4Manufacturing precision
If large plasmid donors with long homology arms are used, then large insertions can be obtained through HDR, but the process requires cloning and selection markers increasing complexity
Solution Approach 1:
The patent extracts only the essential functional elements (short homology arms of 35-60 bp) needed for efficient HDR, eliminating the need for cloning procedures and selection markers while maintaining the ability to achieve precise large insertions
Solution Approach 2:
The patent changes the homology arm length parameter to 35-60 bp, which enables efficient HDR with simplified donor DNA constructs, eliminating the need for complex plasmid cloning and selection marker systems
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 significantly enhances the efficiency and precision of genome editing by reducing the length of homology arms required, improving the recovery of edits, and minimizing the need for selection markers, while maintaining high accuracy and efficiency in human cells and mouse embryos.
Implementation Method 1
CRISPR-Cas9, a programmable DNA endonuclease that can be targeted to a specific DNA sequence by a small 'guide' RNA (crRNA)
Implementation Method 2
HDR utilizes DNAs that contain homology to sequences flanking the DSB (termed homology arms) to template the repair
Data Source
AI summary
The present invention relates to the field of genome editing. More specifically, the present invention provides compositions and methods useful in clustered regularly interspaced short palindromic repeats (CRISPR)-based techniques. In one embodiment, the present invention provides a double-stranded, linear donor polynucleotide comprising a template polynucleotide flanked by a first homology arm and a second homology arm, wherein the homology arms are between 30-35 bases in length.