Instruments, modules, and methods for improved detection of edited sequences in live cells

• US 10,633,627 B2
• Filed: 10/09/2019
• Issued: 04/28/2020
• Est. Priority Date: 08/14/2018
• Status: Active Grant

• Current Assignee: Inscripta, Inc.
• Original Assignee: Inscripta, Inc.
• Inventors: Garst, Andrew, Fox, Richard, Belgrader, Phillip, Masquelier, Don
• Primary Examiner(s): Leith, Nancy J
• Application Number:
  US16/597,831
• Publication Number:
  US 20200095533A1
• Time in Patent Office: 202 Days
• Field of Search: None
• CPC Class Codes: B01D 2201/0407 Perforated supports on both sides of the filtering element
• CPC Class Codes: B01D 2313/50 Specific extra tanks
• CPC Class Codes: B01D 2315/10 Cross-flow filtration
• CPC Class Codes: B01D 63/082 comprising a stack of flat membranes
• CPC Class Codes: B01D 69/06 Flat membranes
• CPC Class Codes: B01D 71/0213 Silicon
• CPC Class Codes: C12M 23/44 Multiple separable units; Modules
• CPC Class Codes: C12M 29/04 Filters; Permeable or porous membranes or plates, e.g. dialysis
• CPC Class Codes: C12M 33/00 Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus chemical or physical laboratory apparatus in general B01L, devices for taking cell samples A61B10/0045, withdrawing or distributing predetermined quantities of fluid B01L99/00
• CPC Class Codes: C12M 47/02 Separating microorganisms from the culture medium; Concentration of biomass separating microorganisms from their culture media C12N1/02
• CPC Class Codes: C12M 47/04 Cell isolation or sorting purging biological preparations of unwanted cells C12N5/0081, determining the presence or kind of microorganism C12Q1/04
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 15/11 DNA or RNA fragments; Modified forms thereof DNA or RNA not used in recombinant technology, C07H21/00; Non-coding nucleic acids having a biological activity
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 15/68 Stabilisation of the vector
• CPC Class Codes: C12N 15/70 Vectors or expression systems specially adapted for E. coli
• CPC Class Codes: C12N 15/81 for yeasts
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 15/87 Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
• CPC Class Codes: C12N 15/90 Stable introduction of foreign DNA into chromosome
• CPC Class Codes: C12N 15/907 : in mammalian cells
• CPC Class Codes: C12N 2310/20 : involving clustered regularly interspaced short palindromic repeats [CRISPRs]
• CPC Class Codes: C12N 2800/80 : Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
• CPC Class Codes: C12N 2830/001 : controllable enhancer/promoter combination
• CPC Class Codes: C12N 2830/002 : inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
• CPC Class Codes: C12N 9/22 : Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

Abstract

The present disclosure provides instruments, modules and methods for improved detection of edited cells following nucleic acid-guided nuclease genome editing. The disclosure provides improved automated instruments that perform methods—including high throughput methods—for screening cells that have been subjected to editing and identifying cells that have been properly edited.

Claims

1. A method for enriching edited cells during CRISPR editing comprising:

transforming cells with one or more vectors comprising a promoter driving transcription of a coding sequence for a CRISPR nuclease, an inducible promoter driving transcription of a guide nucleic acid sequence covalently-linked to a DNA donor sequence and wherein each of the one or more vectors comprises a gene for a selectable marker;

diluting the transformed cells to a cell concentration to substantially singulate the transformed cells on a substrate;

growing the substantially singulated transformed cells on the substrate for at least 5 doublings;

initiating editing by inducing the inducible promoter driving transcription of the guide nucleic acid; and

growing the edited cells to form colonies of terminal size.

2. The method of claim 1, wherein the inducible promoter is a pL promoter.

3. The method of claim 1, wherein all of the coding sequence for the CRISPR nuclease, guide nucleic acid sequence covalently-linked DNA donor sequence are on the same vector.

4. The method of claim 1, wherein the coding sequence for the CRISPR nuclease is on a first vector and the guide nucleic acid sequence and DNA donor sequence are on a second vector.

5. The method of claim 1, wherein the DNA donor sequence further comprises a PAM-altering sequence.

6. The method of claim 1, wherein the cells are mammalian cells.

7. The method of claim 1, further comprising adding selective agents to the substrate to select for the selectable marker(s) on the one or more vectors.

8. The method of claim 1, wherein the substantially singulated transformed cells are grown for at least 20 doublings.

9. The method of claim 1, wherein the one or more vectors further comprises a recombineering system.

10. The method of claim 1, wherein the substrate is selected from an agar plate and a device with solid walls.

11. The method of claim 10, wherein the device with solid walls is a solid wall isolation, induction, and normalization (SWIIN) device.

12. A method for enriching edited cells during CRISPR nuclease editing comprising:

transforming cells with one or more vectors comprising a promoter driving transcription of a coding sequence for a CRISPR nuclease, an inducible promoter driving transcription of a guide nucleic acid sequence covalently-linked to a DNA donor sequence, and a gene for a selectable marker;

diluting the transformed cells to a cell concentration to substantially singulate the transformed cells on a solid wall device;

growing the substantially singulated transformed cells on the substrate for at least 5 doublings;

initiating editing by inducing the inducible promoter; and

growing the edited cells to form colonies of terminal size.

13. The method of claim 12, wherein the promoter driving transcription of the guide nucleic acid is a pL promoter.

14. The method of claim 12, wherein the solid wall device is a solid wall isolation, induction, and normalization (SWIIN) device.

15. The method of claim 12, further comprising adding selective agents to the substrate to select for the one or more vectors.

16. The method of claim 12, wherein the CRISPR nuclease is a MADzyme nuclease.

17. The method of claim 12, further comprising pooling the terminal-size colonies.

18. The method of claim 17, further comprising subjecting the pooled terminal-size colonies to another round of transformation, dilution, growth, editing, and growth to terminal-size colonies.

19. The method of claim 12, wherein the substantially singulated transformed cells are grown on the substrate for at least 10 doublings.

20. The method of claim 12, wherein the transformed cells are bacterial cells and the one or more vectors further comprise a recombineering system.