Two-Step HDR Genome Editing for Scarless Marker Removal
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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 unwanted insertions or deletions (INDELs), and efficiently removing selection markers without leaving genetic remnants, while maintaining the ability to perform HDR-mediated edits.
Innovation Solution
A two-step homology directed repair (HDR) method involving the integration and removal of selection markers using donor polynucleotides and guide RNAs with Cas9, allowing for scarless genome modifications by positive and negative selection to isolate desired edits.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If selection markers are used to identify and purify genome edited cells, then the ability to identify and purify edited cells is improved, but genetic remnants interfere with transcriptional regulation and are incompatible with clinical translation
Solution Approach 1:
The selection marker is incorporated into the donor template before the editing process, allowing cells to be selected during or immediately after HDR. This preliminary selection ensures that only cells with the desired edit are identified, and the marker can then be removed in a second step, eliminating the need for persistent marker activity and avoiding transcriptional interference while maintaining reliable identification of edited cells
Solution Approach 2:
The selection marker serves a temporary function during the editing process to identify and purify edited cells, then is deliberately removed in a second HDR step. This discarding of the marker after it has served its purpose eliminates genetic remnants that would otherwise interfere with transcriptional regulation, while the marker's temporary presence ensures reliable identification and purification of edited cells
2Reliability
If silent mutations are introduced to protect edited alleles from persistent nuclease activity, then the ability to prevent recutting is improved, but splicing abnormalities and altered mRNA or protein structure occur
Solution Approach 1:
The selection marker is placed at the target site before editing, and cells are selected while the marker is present. This preliminary selection occurs before any potential recutting could occur, eliminating the need for silent mutations to block nuclease activity. The marker's presence during selection provides protection without requiring genetic changes that would cause splicing abnormalities or alter protein structure
Solution Approach 2:
The selection marker acts as an intermediary element that temporarily occupies the target site, preventing persistent nuclease activity from recutting the edited allele. This intermediary marker provides the protective function that silent mutations would otherwise need to provide, but without causing splicing abnormalities or altering mRNA and protein structure, since the marker can be cleanly removed after selection
3Measurement precision
If extensive clone screening is performed to identify scarless edits, then the ability to find desired edits is improved, but time and resource consumption increase
Solution Approach 1:
The selection marker is incorporated into the donor template and used to select cells during or immediately after HDR. This preliminary selection enriches for cells with the desired edit before any screening is needed, dramatically reducing the number of clones that require detailed analysis. The marker's presence during selection ensures that only cells with successful editing are considered, eliminating the need for extensive screening of numerous random clones
Solution Approach 2:
The selection marker provides immediate feedback during the editing process by enabling identification of successfully edited cells. This feedback mechanism allows researchers to know early which cells have the desired edit, eliminating the need for extensive clone screening. The marker's presence creates a selection pressure that enriches for correct edits, providing real-time information about editing success without requiring time-consuming analysis of many clones
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, including large insertions or deletions, without leaving silent mutations or selection markers, and reduces the need for extensive clone screening.
Implementation Method 1
The advent of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9/guide RNA system (Cas9/gRNA), however, has enabled targeted cleavage with unprecedented ease of use, efficiency, and specificity
Implementation Method 2
when a homology donor is supplied, HDR enables the introduction of precise insertions of single nucleotides to large gene cassettes, deletions, or substitutions
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.