Scarless genome editing through two-step homology directed repair
Inventors
Ikeda, Kazuya • PORTEUS, Matthew H.
Assignees
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Publication Number
US-12442016-B2
Publication Date
2025-10-14
Expiration Date
Abstract
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.
Core Innovation
The disclosure describes scarless genome editing using a two-step Cas9-mediated homology directed repair workflow. A first HDR step integrates an intended edit flanked by left and right homology arms together with an expression cassette encoding a selection marker, and cells carrying the integrated marker are isolated by positive selection.
A second HDR step generates a non-gene destructive double-stranded break at a target sequence and performs HDR using a second donor and guide RNA. This second HDR replaces the integrated locus with the intended edit while deleting the selection marker cassette, and final scarless outcomes are obtained by negative selection using deletion of the at least one selection marker.
The document further covers donor, guide, Cas9 vector delivery and selection marker classes including fluorescent markers, cell-surface markers, drug resistance genes, reporter genes, and suicide genes. It also describes negative selection based on fluorescence intensity and/or marker sequence absence, and embodiments using shRNA-based counterselection to reduce random integration, including pairing of edits to enable large insertions or deletions.
Claims Coverage
Independent claim clm-00001 covers a genome editing method with three main inventive aspects: performing a non-gene destructive double-stranded break at a target sequence, using HDR-driven removal of at least one selection marker at the target sequence, and applying negative selection for the selection marker. The dependent claims refine the break generation, marker categories, and the negative-selection readout, and they specify donor architecture with left and right homology arms flanking an intended edit sequence.
Non-gene destructive DSB at the target sequence with HDR-driven selection marker removal
Performing a combination of a double-stranded break at a target sequence in the genome of the cell, and homology directed repair-driven removal of at least one selection marker at the target sequence, wherein the double-stranded break is a non-gene destructive double-stranded break.
Non-gene destructive DSB generated by a sequence-specific nuclease
The non-gene destructive double-stranded break is produced by a sequence-specific nuclease to stimulate homology directed repair-driven removal.
Cas9 for the sequence-specific nuclease
The sequence-specific nuclease is Cas9.
Negative selection based on deletion of the selection marker
Negative selection for the at least one selection marker to select for cells in which the at least one selection marker is removed.
Fluorescence-intensity-based negative selection
Negative selection comprises measuring fluorescence intensity of the at least one selection marker to determine if the at least one selection marker is deleted.
Donor polynucleotide with left and right homology arms flanking an intended edit
Introducing a donor polynucleotide into the cell, where the donor polynucleotide comprises a left homology arm and a right homology arm flanking a sequence comprising an intended edit to the target sequence in the genome of the cell.
Selection marker selection from fluorescent, cell-surface, drug resistance, reporter, or suicide categories
The selection marker is selected from fluorescent markers, a cell surface marker, a drug resistance gene, a reporter gene, and a suicide gene.
Across the independent claim and its dependents, the coverage centers on scarless selection marker removal by using a non-gene destructive DSB at a target sequence followed by HDR removal of the marker and negative selection for marker deletion, with refinements specifying Cas9, fluorescence-intensity-based negative selection, and donor polynucleotide architecture using left and right homology arms flanking an intended edit.
Stated Advantages
Scarless genome editing outcomes.
Removal of at least one selection marker at the target sequence using homology directed repair followed by negative selection.
Selection marker deletion can be determined using fluorescence intensity measurement.
Documented Applications
Point mutation correction in human ESCs/iPSCs (TBX1).
Reporter knock-in in human ESCs/iPSCs using RUNX1-mOrange.
Reporter and disease-relevant edits in human ESCs/iPSCs including GFI1 and B2M/HLA-A*24.
Research and regenerative/therapeutic uses.
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