Granted Patent B2

US 12,365,893 · App. 17/214,395 · Granted Jul 22, 2025

Inventors: Tobias Daniel Wheeler (Alameda, CA); Rajiv Bharadwaj (Pleasanton, CA); Katherine Pfeiffer (Berkeley, CA)

Assignee: 10X GENOMICS, INC.

C12N15/1096C12N15/1065C12N15/1093C12N15/111C12N2310/20

Abstract

Claims (24)

1. A method for preparing a set of nucleic acid molecules, comprising:

(a) providing a plurality of partitions comprising (i) a plurality of adapters, each comprising an adapter sequence, and (ii) a plurality of deoxyribonucleic acid (DNA) fragments, wherein said plurality of adapters comprise barcode molecules comprising a plurality of different barcode sequences, and wherein barcode molecules within a partition comprise a common barcode sequence;

(b) within said plurality of partitions, ligating said plurality of adapters and said plurality of DNA fragments to synthesize a plurality of barcoded nucleic acid molecules, wherein each barcoded nucleic acid molecule of said plurality of barcoded nucleic acid molecules comprises (i) said adapter sequence and (ii) a barcode sequence of said plurality of different barcode sequences or a complement thereof;

(c) providing a Clustered Regularly Interspaced Palindromic Repeats (CRISPR)-CRISPR associated protein (Cas) (CRISPR-Cas) complex that comprises (i) a fusion protein comprising (I) a first protein, (II) a second protein that is a site-specific endonuclease, and (III) a polypeptide linking said first protein and said second protein, wherein said polypeptide comprises a length between 150 and 700 amino acids, and (ii) a guide ribonucleic acid (RNA) capable of binding to said first protein and selectively coupling to said adapter sequence, wherein said first comprises protein is a type II or type V CRISPR-Cas RNA-guided DNA endonuclease that lacks endonuclease activity, wherein said guide RNA comprises a sequence complementary to said adapter sequence; and

(d) bringing said plurality of barcoded nucleic acid molecules in contact with said CRISPR-Cas complex under conditions sufficient for (i) said guide RNA to couple to said adapter sequence of said plurality of barcoded nucleic acid molecules and (ii) said second protein to cleave said plurality of barcoded nucleic acid molecules, thereby providing said set of nucleic acid molecules, wherein nucleic acid molecules of said set of nucleic acid molecules each (I) a barcode sequence of said plurality of different barcode sequences, or a complement thereof, and (II) a shortened DNA fragment of said plurality of DNA fragments.

2. The method of claim 1 , wherein said polypeptide comprises an alpha-helix-forming linker.

3. The method of claim 1 , wherein said plurality of DNA fragments comprises DNA fragments of varying lengths.

4. The method of claim 1 , wherein said second protein comprises a restriction endonuclease.

5. The method of claim 1 , wherein said adapter sequence comprises P5 adapter or P7 adapter.

6. The method of claim 1 , wherein said plurality of partitions are wells, microwells, tubes, vials, microcapsules, or droplets within an emulsion.

7. The method of claim 1 , wherein said plurality of partitions are aqueous droplets within a water-in-oil emulsion.

8. The method of claim 1 , wherein said guide RNA comprises a chimeric RNA that comprises a guide sequence and a tracr sequence.

9. The method of claim 1 , wherein said guide RNA comprises a sequence of 17 nucleotides to 24 nucleotides complementary to said adapter sequence.

10. The method of claim 1 , wherein said guide RNA comprises a hairpin sequence of 35 nucleotides to 50 nucleotides, wherein said hairpin binds said first protein.

11. The method of claim 1 , wherein said set of nucleic acid molecules in (d) display a tighter distribution in terms of lengths when compared with said plurality of barcoded nucleic acid molecules.

12. The method of claim 11 , wherein a largest variation in lengths among said nucleic acid molecules in said set of nucleic acid molecules in (d) is from 1 to 500 nucleotides.

13. The method of claim 1 , wherein, in (a), said plurality of adapters are coupled to a plurality of beads in said plurality of partitions.

14. The method of claim 13 , wherein said ligating in (b) comprises releasing said plurality of adapters from said plurality of beads within said plurality of partitions.

15. The method of claim 1 , wherein said polypeptide comprises an unstructured peptide.

16. The method of claim 1 , wherein said polypeptide comprises a positively charged group.

17. The method of claim 16 , wherein the positively charged group couples to a portion of a nucleic acid molecule of said nucleic acid molecules.

18. The method of claim 1 , wherein said polypeptide comprises a proline.

19. The method of claim 1 , further comprising amplifying said nucleic acid molecules of said set of nucleic acid molecules.

20. The method of claim 1 , further comprising sequencing said nucleic acid molecules of said set of nucleic acid molecules.

Assignments (1)

Continuity (4)

References Cited (400)