Granted Patent B2

US 12,680,131 · App. 17/259,501 · Granted Jul 14, 2026

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

Assignee: TWINSTRAND BIOSCIENCES, INC.

C12Q1/6869G16B20/20G16B30/10

Abstract

Claims (30)

1 . A method of determining if a change occurred at an off-target genomic site in a genome of a population of cells following an engineered genomic editing event directed to a targeted genomic site, the method comprising:

(a) providing a sample comprising double-stranded DNA molecules originating from the population of cells following the engineered genomic editing event, wherein the engineered genomic editing event is selected from the group consisting of: (a) a clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9)-mediated editing event, (b) a CRISPR from Prevotella and Francisella 1 (CPF1)-mediated editing event, (c) a modified CAS or CPF1-mediated event, (d) 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, (e) a polynucleotide substrate-mediated homologous recombination event, (f) an event carried out by a retrovirus or another virus, and (g) any combination of (a) to (f);

(b) generating an error-corrected sequence read for a plurality of the double-stranded DNA molecules, comprising:

ligating adapter molecules to the plurality of the double-stranded DNA molecules to generate a plurality of adapter-DNA molecules;

for any particular adapter-DNA molecule:

generating copies of an original first strand of the adapter-DNA molecule and copies of an original second strand of the adapter-DNA molecule;

sequencing one or more of the copies of the original first strand and one or more of the copies of the original second strand to provide a first strand sequence and a second strand sequence;

comparing the first strand sequence and the second strand sequence to identify one or more correspondences between the first and second strand sequences to generate the error-corrected sequence read; and

(c) comparing one or more error-corrected sequence reads comprising a sequence at the off-target genomic site to a reference genome DNA sequence,

wherein a change occurred at the off-target genomic site if the one or more error-corrected sequence reads differ from the reference genome DNA sequence.

2 . The method of claim 1 , wherein generating an error-corrected sequence read for the plurality of the double-stranded DNA molecules further comprises selectively enriching one or more genomic regions prior to sequencing to provide a plurality of enriched adapter-DNA molecules.

3 . The method of claim 2 , wherein the one or more genomic regions comprises the targeted genomic site in the genome.

4 . The method of claim 2 , wherein the one or more genomic regions comprise the off-target genomic site in the genome.

5 . The method of claim 1 , further comprising comparing one or more sequence modifications among the double-stranded DNA molecules at the targeted genomic site to an anticipated genome edited DNA sequence.

6 . The method of claim 1 , wherein the method is performed at a first time point and a second time point following the engineered genomic editing event.

7 . The method of claim 6 , wherein both the first and second time points are selected from the group consisting of within about 30 days, within about 45 days, within about 60 days, within about 75 days, and within about 90 days following the engineered genomic editing event.

8 . The method of claim 5 , wherein the one or more sequence modifications comprise an incorrect sequence modification in the sequence of the targeted genomic site.

9 . The method of claim 8 , wherein the incorrect sequence modification in the sequence of the targeted genomic site is due to a non-homologous end joining (NHEJ) event.

10 . The method of claim 1 , wherein the one or more error-corrected sequence reads comprise one or more variants as compared to the reference genome DNA sequence.

11 . The method of claim 10 , wherein the one or more variants comprise a functionally disruptive mutation.

12 . The method of claim 10 , further comprising (d) determining a frequency of the one or more variants among the plurality of double stranded DNA molecules, wherein the variant frequency of one or more variants is greater than a background variant frequency of a reference population of double-stranded DNA molecules extracted from a reference population of cells that have not undergone an engineered genomic editing event.

13 . The method of claim 1 , further comprising determining if one or more error-corrected sequence reads comprising a sequence at the targeted genomic site comprise an anticipated genome edited DNA sequence.

14 . The method of claim 13 , further comprising determining a frequency of the anticipated genome edited DNA sequence among the error-corrected sequence reads comprising the sequence at the targeted genomic site.

15 . The method of claim 13 , further comprising determining a frequency of an undesired DNA sequence among the error-corrected sequence reads comprising the sequence at the targeted genomic site.

16 . The method of claim 1 , wherein step (c) comprises comparing error-corrected sequence reads comprising sequences at a plurality of off-target genomic sites to a reference genome DNA sequence.

17 . The method of claim 1 , wherein the off-target genomic site is selected from the group consisting of a mutation-prone site, a microsatellite locus, a sequence with sequence homology to the targeted genomic site, a cancer driver, and combinations thereof.

18 . The method of claim 1 , wherein the off-target genomic site has a nucleic acid sequence that is at least partially similar to the sequence at the targeted genomic site.

19 . The method of claim 1 , wherein the off-target genomic site comprises a sequence of one or more of the following: a tumor suppressor gene, an oncogene, a proto-oncogene, and a cancer driver.

20 . The method of claim 1 , wherein a subset of cells in the population of cells has one or more pre-existing genetic mutations, and wherein following the engineered genomic editing event, the subset of cells selectively proliferate at a greater rate than other cells in the population of cells.

21 . The method of claim 1 , wherein a subset of cells in the population of cells has a pre-existing epigenetic state unique to said cells, and wherein following the engineered genomic editing event, the subset of cells selectively proliferate at a greater rate than other cells in the population of cells.

Assignments (1)

Continuity (2)

References Cited (343)