Granted Patent B2

US 12,529,101 · App. 18/495,502 · Granted Jan 20, 2026

Inventors: Jesse J. Salk (Seattle, WA); Charles Clinton Valentine, III (Seattle, WA)

Assignee: TwinStrand Biosciences, Inc.

C12Q1/6869G16B20/20G16B30/10

Abstract

Claims (15)

1 . A method for detecting and/or quantifying clonal expansion of a cell in a cell population following an engineered genomic editing event, comprising:

(a) duplex sequencing one or more target double-stranded DNA molecules originating from a cell population following the engineered genomic editing event, wherein the engineered genomic editing event is selected from the group consisting of: (i) a clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9)-mediated editing event, (ii) a CRISPR from Prevotella and Francisella 1 (CPF1)-mediated editing event, (iii) a modified Cas or CPF1-mediated event, (iv) an event carried out by a transcription activator-like effector nuclease (TALEN), a mega transcription activator-like effector (megaTAL) nuclease, a Zinc-fingered nuclease, or a homing endonuclease, (v) a polynucleotide substrate-mediated homologous recombination event, (vi) an event carried out by a retrovirus or another virus, and (vii) any combination of (i) to (vi);

(b) identifying one or more variants among the target double-stranded DNA molecules;

(c) determining a variant frequency of the one or more variants among the target double-stranded DNA molecules originating from the cell population; and

(d) comparing the variant frequency for the one or more variants to an expected variant frequency.

2 . The method of claim 1 , wherein the step of duplex sequencing comprises:

(i) preparing a sequencing library from double-stranded DNA molecules originating from the cell population following the engineered genomic editing event, wherein preparing the sequence library comprises ligating adapter molecules to the plurality of double-stranded DNA fragments to generate a plurality of adapter-DNA molecules;

for at least some of the plurality of adapter-DNA molecules:

(ii) sequencing first and second strands of the adapter-DNA molecules to provide a first strand sequence read and a second strand sequence read for the adapter-DNA molecules; and

(iii) for the adapter-DNA molecules, comparing the first strand sequence read and the second strand sequence read to identify one or more correspondences between the first and second strand sequences reads.

3 . The method of claim 1 , wherein the variant frequency of the one or more variants is determined by calculating a number of duplex sequenced target double-stranded DNA molecules having a specified variant mapping to a genomic locus of interest per a total number of duplex sequenced target double-stranded DNA molecules mapping to the genomic locus of interest.

4 . The method of claim 1 , wherein the method steps (a) to (c) are performed at a first time point after the event and at a second time point after the event, wherein the second time point is after the first time point, and wherein the variant frequency from the first time point is the expected variant frequency.

5 . The method of claim 1 , wherein the expected variant frequency is determined from a comparable cell population that has not undergone the engineered genomic editing event.

6 . The method of claim 1 , wherein the one or more variants are at one or more locations outside an intended locus for genomic editing.

7 . The method of claim 1 , wherein the one or more variants are in a sequence of a tumor suppressor gene, an oncogene, a proto-oncogene, and/or a cancer driver.

Assignments (1)

Continuity (4)

References Cited (346)