Detection of genome editing
Inventors
Berman, Jennifer • Cooper, Samantha • Karlin-Neumann, George • MIYAOKA, Yuichiro • Conklin, Bruce • Shinoff, Josh
Assignees
Bio Rad Laboratories Inc • J David Gladstone Institutes
Publication Number
US-11236383-B2
Publication Date
2022-02-01
Expiration Date
2036-01-08
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Abstract
Methods, compositions, and kits are provided for quantification of genome editing.
Core Innovation
The invention provides methods, compositions, and kits for the quantification of genome editing, specifically simultaneous quantification of homology directed repair (HDR) and non-homologous end joining (NHEJ) genome editing products in samples of cells or nucleic acids extracted therefrom. The methods involve partitioning a reaction mixture containing a target genomic region, DNA-dependent DNA polymerase, amplification primers flanking the target, and detectably labeled probes including a reference probe, an HDR-specific probe, and an NHEJ drop-off probe. The target regions are amplified in partitions and the quantities of wild-type, HDR mutated, and NHEJ mutated target genomic regions are determined by detecting hybridization of labeled probes.
The problem addressed by the invention is the difficulty in accurately quantifying genome editing events, particularly the simultaneous detection of HDR and NHEJ repair products. Existing methods can quantify HDR mutations but not reliably detect or quantify the unpredictable NHEJ mutations due to variability in mutation type and location. The invention enables precise and simultaneous quantification of both HDR and NHEJ genome editing products, providing improved tools for evaluating genome editing efficiency and optimizing genome editing conditions.
Claims Coverage
The patent includes one independent claim defining a method of generating partitions containing amplified target genomic regions that may contain a mutation and quantifying wild-type and mutated target regions.
Method of partitioning and amplifying target genomic regions with mutation detection probes
A method comprising providing a sample with nucleic acid containing a target genomic region, partitioning it into multiple partitions each containing DNA polymerase, forward and reverse primers flanking the target, a detectably labeled reference probe hybridizing regardless of allele, a detectably labeled mutation probe hybridizing only to mutated target regions, and a detectably labeled drop-off probe that hybridizes to wild-type target regions but not mutated regions.
Use of a non-extendible dark probe or overlapping drop-off probe to block cross-reactivity
Incorporation of either a non-extendible dark probe that blocks cross-reactivity of the mutation probe to the wild-type sequence by competing for binding, or a drop-off probe that hybridizes to a region overlapping the mutation probe's binding site to prevent false positive signals from wild-type sequences.
Amplification and detection conditions
Amplification of target regions using PCR, optionally three-step PCR, in partitioned droplets or wells, with detection of fluorescence changes due to 5' to 3' exonuclease activity of DNA polymerase indicating hybridization of probes.
Quantitative determination of wild-type and mutated target genomic regions
Method steps to determine quantities of wild-type and mutated target genomic regions by analyzing presence or absence of fluorescent signals from reference, mutation, and drop-off probes in partitions, including calculating concentrations based on the number of positive and negative partitions.
The claims cover a method for partitioned amplification and quantification of mutated and wild-type target genomic regions using labeled oligonucleotide probes including reference, mutation-specific, drop-off, and optionally dark probes. This method enables precise detection and quantification of genomic mutations, such as those introduced by genome editing, using partitioned nucleic acid amplification and probe hybridization.
Stated Advantages
Provides simultaneous quantification of HDR and NHEJ genome editing products.
Enables precise and sensitive detection of unpredictable NHEJ mutations.
Allows optimization of genome editing conditions to improve editing efficiency and specificity.
Reduces likelihood of user error and increases assay precision by using a single reaction mixture.
Facilitates monitoring and quantitation of genome edited cells in patients or in vitro populations.
Documented Applications
Quantification of genome editing efficiency in biological samples by simultaneous detection of HDR and NHEJ products.
Identification of optimized genome editing conditions by comparing efficiencies of editing under different conditions.
Monitoring of patients treated with genome edited cells by quantifying the abundance of edited genomes in biological samples to assess treatment success or failure.
In vitro monitoring of edited cell populations to determine responses to selection pressures such as drugs or culturing.
Generation of specified doses of genome edited cells by mixing edited and wild-type cells based on quantification of edited genomes.
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