Methods and compositions for crispr editing of cells and correlating the edits to a resulting cellular nucleic acid profile

Patent metadata

Field	Value
Publication number	US-2021381007-A1
Application number	US-202117338603-A
Country	US
Kind code	A1
Filing date	Jun 3, 2021
Priority date	Jun 4, 2020
Publication date	Dec 9, 2021
Grant date	—

Abstract

The present disclosure provides compositions, methods and modules to edit live cells and to subsequently correlate the resulting cellular nucleic acids of the edited cells to the edits.

First claim

We claim:

1 . A method for correlating rationally-designed genome edits made in a population of cells with a cellular nucleic acid profile from individual cells comprising:
designing and synthesizing a library of editing cassettes comprising repair templates and gRNAs; inserting the library of editing cassettes in a vector backbone resulting in a library of editing vectors; transforming the population of cells with the library of editing vectors to produce transformed cells; allowing nucleic acid-guided nuclease or nickase fusion editing to take place in the transformed cells to produce edited cells; singulating the edited cells into partitions; lysing the edited cells; adding barcoded product capture primers and barcoded cassette capture primers to each partition, wherein the barcodes used in the barcoded product capture primers and barcoded cassette capture primers in a same partition are a same barcode and wherein the barcodes used in the barcoded product capture primers and barcoded cassette capture primers in a different partition are different from barcodes used in other partitions; creating DNA copies and/or cDNAs from cellular nucleic acids in the edited cells using the barcoded product capture primers; creating DNA copies and/or cDNAs from the editing cassettes in the edited cells using the barcoded cassette capture primers; pooling the DNA copies and/or cDNAs from the partitions; sequencing the DNA copies and/or cDNAs; and correlating sequences from the DNA copies and/or cDNAs from cellular nucleic acids with sequences from the DNA copies and/or cDNAs from the editing cassettes for each cell.

2 . The method of claim 1 , wherein the barcoded product capture primers comprise product capture primers and first barcoded template switching oligonucleotides.

3 . The method of claim 2 , wherein the barcoded cassette capture primers comprise cassette capture primers and second barcoded template switching oligonucleotides.

4 . The method of claim 1 , wherein the adding step is performed before singulating the edited cells into the partitions.

5 . The method of claim 1 , wherein the adding step is performed after singulating the edited cells into the partitions.

6 . The method of claim 1 , wherein the editing is nucleic acid-guided nuclease editing.

7 . The method of claim 1 , wherein the editing is nickase fusion editing.

8 . The method of claim 1 , wherein the population of cells is a population of mammalian cells.

9 . The method of claim 8 , wherein the vector backbone is a viral vector backbone.

10 . The method of claim 9 , wherein the viral vector backbone is a lentiviral vector backbone.

11 . The method of claim 9 , wherein the transforming step comprises transduction.

12 . The method of claim 1 , wherein the vector backbone further comprises a selection marker and further comprising a selecting step to select for the selection marker after the transforming step.

13 . The method of claim 1 , wherein the partitions are droplets.

14 . The method of claim 1 , wherein the partitions are gel beads.

15 . The method of claim 1 , wherein the partitions are wells.

16 . A method for correlating rationally-designed genome edits made in a population of cells with a cellular nucleic acid profile from individual cells comprising:
transforming the population of cells with a coding sequence for a nucleic acid-guided nuclease or a nickase fusion enzyme or a nucleic acid-guided nuclease or a nickase fusion enzyme; designing and synthesizing a library of editing cassettes comprising repair templates and gRNAs; inserting the library of editing cassettes in a vector backbone resulting in a library of editing vectors; transforming the population of cells with the library of editing vectors to produce transformed cells; allowing nucleic acid-guided nuclease or nickase fusion editing to take place in the transformed cells to produce edited cells; singulating the edited cells into partitions; lysing the edited cells; adding barcoded product capture primers and barcoded cassette capture primers to each partition, wherein the barcodes used in the barcoded product capture primers and barcoded cassette capture primers in a same partition are a same barcode and wherein the barcodes used in the barcoded product capture primers and barcoded cassette capture primers in a different partition are different from barcodes used in other partitions; creating DNA copies and/or cDNAs from cellular nucleic acids in the edited cells using the barcoded product capture primers; creating DNA copies and/or cDNAs from the editing cassettes in the edited cells using the barcoded cassette capture primers; pooling the DNA copies and/or cDNAs from the partitions; sequencing the DNA copies and/or cDNAs; and correlating sequences from the DNA copies and/or cDNAs from cellular nucleic acids with sequences from the DNA copies and/or cDNAs from the editing cassettes for each cell.

17 . The method of claim 16 , wherein the barcoded product capture primers comprise product capture primers and first barcoded template switching oligonucleotides.

18 . The method of claim 17 , wherein the barcoded cassette capture primers comprise cassette capture primers and second barcoded template switching oligonucleotides.

19 . The method of claim 17 , wherein the adding step is performed before singulating the edited cells into the partitions.

20 . The method of claim 16 , wherein the adding step is performed after singulating the edited cells into the partitions.

21 . The method of claim 16 , wherein the editing is nucleic acid-guided nuclease editing.

22 . The method of claim 16 , wherein the editing is nickase fusion editing.

23 . The method of claim 16 , wherein the population of cells is a population of mammalian cells.

24 . The method of claim 23 , wherein the vector backbone is a viral vector backbone.

25 . The method of claim 24 , wherein the viral vector backbone is a lentiviral vector backbone.

26 . The method of claim 9 , wherein the transforming step comprises transduction.

27 . The method of claim 1 , wherein the vector backbone further comprises a selection marker and further comprising a selecting step to select for the selection marker after the transforming step.

28 . The method of claim 1 , wherein the partitions are droplets.

29 . The method of claim 1 , wherein the partitions are gel beads.

30 . The method of claim 1 , wherein the partitions are wells.

Assignees

• Inscripta Inc

Inventors

• Mok Janine
• Gander Miles
• Garst Andrew
• Dura Burak
• Church Deanna

Classifications

• C12N15/905 Primary

  in yeast · CPC title

• C12N2310/20

  involving clustered regularly interspaced short palindromic repeats [CRISPR] · CPC title

• C12N15/1075

  by coupling phenotype to genotype, not provided for in other groups of this subclass · CPC title

• C12N15/1065

  Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags · CPC title

• C12N9/22 Primary

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title