Nucleic acid-guided editing of exogenous polynucleotides in heterologous cells

Patent metadata

Field	Value
Publication number	US-11332850-B2
Application number	US-202117554373-A
Country	US
Kind code	B2
Filing date	Dec 17, 2021
Priority date	Apr 24, 2018
Publication date	May 17, 2022
Grant date	May 17, 2022

Abstract

The present disclosure provides shuttle vectors for editing exogenous polynucleotides in heterologous live cells, as well as automated methods, modules, and multi-module cell editing instruments and systems for performing the editing methods.

First claim

We claim:

1. A method of nucleic acid-guided nuclease editing of five hundred or more exogenous polynucleotides from mammalian cells within heterologous editing cells in a single multiplexed automated operation, comprising:
inserting the five hundred or more different target polynucleotides from the mammalian cells into synthetic chromosomes to form a library of synthetic chromosomes, wherein the synthetic chromosomes are produced by a top down method, a bottom up method, engineering of minichromosomes, or induced de novo chromosome generation;
transferring the library of synthetic chromosomes to a first receptacle;
providing heterologous editing cells in a second receptacle;
providing one or more editing vectors in a third receptacle, wherein the editing vectors comprise a coding sequence for a nuclease, a guide nucleic acid and a DNA donor sequence;
moving the library of synthetic chromosomes from the first receptacle to a transformation module;
moving the heterologous editing cells from the second receptacle to the transformation module;
moving the one or more editing vectors from the third receptacle to the transformation module;
transforming the heterologous editing cells with the library of synthetic chromosomes and the editing vectors to produce transformed heterologous editing cells;
transferring the transformed heterologous editing cells from the transformation module to an editing module;
allowing editing to take place in the editing module under conditions that allow the editing vectors to edit the synthetic chromosomes thereby forming edited synthetic chromosomes;
isolating the edited synthetic chromosomes;
wherein the liquid handling system moves the library of synthetic chromosomes from the first receptacle to the transformation module, the heterologous editing cells from the second receptacle to the transformation module, the editing vectors from the third receptacle to the transformation module, and the transformed heterologous editing cells to the editing module all without human intervention.

2. The method of nucleic acid-guided nuclease editing of claim 1 , wherein the stand-alone automated multi-module cell processing instrument is controlled by a processor.

3. The method of nucleic acid-guided nuclease editing of claim 2 , wherein the processor is configured to operate the instrument based on user input.

4. The method of nucleic acid-guided nuclease editing of claim 1 , wherein the synthetic chromosomes are produced by the top down method.

5. The method of nucleic acid-guided nuclease editing of claim 1 , wherein the synthetic chromosomes are produced by the bottom up method.

6. The method of nucleic acid-guided nuclease editing of claim 1 , wherein the synthetic chromosomes are produced by the engineering of minichromosomes method.

7. The method of nucleic acid-guided nuclease editing of claim 1 , wherein the synthetic chromosomes are produced by the induced de novo chromosome generation method.

8. The method of nucleic acid-guided nuclease editing of claim 1 , wherein one or more of the five hundred or more exogenous target polynucleotides are selected from a full-length gene; an open reading frame coding sequence; or a genomic locus of size 1000-10,000 nucleotides, 50-500 nucleotides, 10-100 nucleotides, or 10,000-100,000 nucleotides.

9. The method of nucleic acid-guided nuclease editing of claim 8 , wherein one or more of the five hundred or more exogenous target polynucleotides are single open reading frame coding sequences.

10. The method of nucleic acid-guided nuclease editing of claim 8 , wherein one or more of the five hundred or more exogenous target polynucleotides are genomic loci of size 1000-10,000 nucleotides.

11. The method of nucleic acid-guided nuclease editing of claim 1 , wherein the heterologous editing cells are selected from bacterial cells, fungal cells, plant cells, mammalian cells and human cells.

12. The method of nucleic acid-guided nuclease editing of claim 11 , wherein the heterologous editing cells are bacterial cells.

13. The method of nucleic acid-guided nuclease editing of claim 11 , wherein the heterologous editing cells are fungal cells.

14. The method of nucleic acid-guided nuclease editing of claim 11 , wherein the heterologous editing cells are plant cells.

15. The method of nucleic acid-guided nuclease editing of claim 11 , wherein the heterologous editing cells are mammalian cells.

16. The method of nucleic acid-guided nuclease editing of claim 1 , wherein the heterologous editing cells are human cells.

17. The method of nucleic acid-guided nuclease editing of claim 1 , wherein the editing vectors confer edits comprising saturation mutagenesis edits, knockout edits, loss of function edits, gain of function edits, coding sequence edits and noncoding sequence edits.

18. The method of nucleic acid-guided nuclease editing of claim 17 , wherein the editing vectors confer edits comprising saturation mutagenesis edits.

19. The method of nucleic acid-guided nuclease editing of claim 17 , wherein the editing vectors confer knockout edits.

20. The method of nucleic acid-guided nuclease editing of claim 17 , wherein the editing vectors confer loss of function edits.

21. The method of nucleic acid-guided nuclease editing of claim 17 , wherein the editing vectors confer coding sequence edits.

22. The method of nucleic acid-guided nuclease editing of claim 17 , wherein the editing vectors confer noncoding sequence edits.

23. The method of nucleic acid-guided nuclease editing of claim 1 , wherein the stand-alone automated multi-module cell processing instrument further comprises a cell concentration module.

24. The method of nucleic acid-guided nuclease editing of claim 23 , wherein the cell concentration module performs tangential flow filtration.

25. The method of nucleic acid-guided nuclease editing of claim 1 , wherein the stand-alone automated multi-module cell processing instrument further comprises a singulation module for singulating the transformed heterologous editing cells.

26. The method of nucleic acid-guided nuclease editing of claim 1 , wherein the stand-alone automated multi-module cell processing instrument further comprises a recovery module.

27. The method of nucleic acid-guided nuclease editing of claim 1 , wherein the stand-alone automated multi-module cell processing instrument further comprises a growth module.

28. The method of nucleic acid-guided nuclease editing of claim 1 , wherein one or more of the five hundred or more different target polynucleotides are selected from single open reading frame coding sequences, a gene sequence from 5′UTR to 3′UTR, and a genetic locus comprising at least 100 kb.

29. The method of nucleic acid-guided nuclease editing of claim 8 , wherein one or more of the five hundred or more exogenous target polynucleotides are a genomic locus of size 1000-10,000 nucleotides.

30. The method of nucleic acid-guided nuclease editing of claim 8 , wherein one or more of the five hundred or more exogenous target polynucleotides are a genomic locus of size 10,000-100,000 nucleotides.

Assignees

• Inscripta Inc

Inventors

• Fox Richard
• Held Daniel

Classifications

• C12N2310/20

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

• C40B20/04

  Identifying library members by means of a tag, label, or other readable or detectable entity associated with the library members, e.g. decoding processes · CPC title

• C12N15/905

  in yeast · CPC title

• C12N15/1037

  Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display · CPC title

• C07K16/2809

  against the T-cell receptor (TcR)-CD3 complex · CPC title