Two-Step HDR Genome Editing Without Residual Selection Markers
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Solution Overview
Problem
Existing genome editing methods in human pluripotent stem cells (hPSCs) face challenges in achieving scarless editing, including the need for highly active nucleases, preventing indels on untargeted alleles, and avoiding re-cutting of HDR-modified alleles, while also being limited in efficiency and versatility, especially for large edits and target site availability.
Innovation Solution
A two-step homology directed repair (HDR) method using Cas9-mediated integration of donor polynucleotides with selection markers, followed by removal of the marker through a second HDR step, ensuring no residual editing remnants.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If selection markers are used to identify and purify genome edited cells, then clone identification efficiency is improved, but residual markers remain in the genome interfering with transcriptional regulation
Solution Approach 1:
The editing process is divided into two distinct phases: first integrating the selection marker to enable efficient clone identification, then removing the marker in a second step. This segmentation allows the marker to serve its identification function temporarily without permanently remaining in the genome to cause transcriptional interference.
Solution Approach 2:
The selection marker is introduced preliminarily to facilitate clone identification and purification, but is then removed in a subsequent action. This preliminary use of the marker enables efficient selection without the permanent presence that would cause harmful transcriptional interference.
2Productivity
If highly active nucleases are used to achieve high frequency editing, then editing efficiency is improved, but re-cutting of HDR-modified alleles occurs
Solution Approach 1:
The editing process is segmented into two steps: first performing HDR-mediated editing with high activity nuclease, then performing a second editing step to remove the selection marker. This segmentation allows high efficiency editing initially, then stabilizes the allele by removing the marker that would otherwise be re-cut.
Solution Approach 2:
The selection marker, which is the target of the high activity nuclease and causes re-cutting problems, is extracted or removed from the genome in the second editing step. This extraction eliminates the source of re-cutting while preserving the desired editing outcome.
3Reliability
If silent mutations are introduced to block re-cutting, then allele stability is improved, but splicing abnormalities and protein structure alterations occur
Solution Approach 1:
Instead of introducing silent mutations that could alter protein structure or splicing, the selection marker is simply removed in the second editing step. This extraction approach ensures allele stability without risking any changes to the coding sequence that could affect protein structure or function.
4Manufacturing precision
If Cas9-mediated HDR is used for scarless editing, then residual editing remnants are eliminated, but the process complexity increases
Solution Approach 1:
The scarless editing process is segmented into two Cas9-mediated HDR steps: first integrating the selection marker precisely, then removing it precisely. This segmentation achieves scarless editing by using controlled, precise molecular operations rather than leaving residual markers, though it does increase process complexity.
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
Enables efficient, scarless genome editing at any location, allowing for large edits and high frequencies of desired edits without leaving selection markers or silent mutations, with improved clone identification and reduced analysis burden.
Implementation Method 1
a guide RNA and Cas9 into the cell, wherein the Cas9 forms a complex with the guide RNA, the guide RNA being complementary to the target sequence in the genomic DNA of the cell, the guide RNA directing the complex to the target sequence
Implementation Method 2
The HDR path of repair can be divided into two mechanistically distinct strategies: classic gene targeting using the Rad51 dependent homologous recombination pathway (HR)
Data Source
AI summary
A method for scarless genome editing is disclosed. In particular, the method provides scarless genome modification by using homology directed repair (HDR) steps to genetically modify cells and remove unwanted sequences. This method can be used for genome editing, including introducing mutations, deletions, or insertions at any position in the genome without leaving silent mutations, selection marker sequences, or other additional undesired sequences in the genome.