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

• US 10,633,626 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:
  US16/597,826
• Publication Number:
  US 20200063088A1
• 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 nucleic acid-guided nuclease editing comprising:

transforming cells with one or more vectors comprising a promoter driving expression of a CRISPR nuclease, an inducible promoter driving transcription of a guide nucleic acid 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 first substrate;

growing the cells to form colonies on the first substrate under conditions that allow genome repair;

initiating editing in the cells growing on the first substrate by inducing the inducible promoter driving transcription of the guide nucleic acid to produce induced cells;

growing the induced cells into terminal-sized colonies;

making a replica of the first substrate forming a second substrate;

growing and inducing cells on the second substrate under conditions that do not allow genome repair;

comparing cell growth on the first and second substrates; and

selecting cells from the first substrate that grow on the first substrate but do not grow on the second substrate.

2. The method of claim 1, wherein the promoter driving transcription the guide nucleic acid and donor DNA is a pL promoter.

3. The method of claim 1, wherein the promoter driving expression of the CRISPR nuclease is an inducible promoter.

4. The method of claim 3, wherein the inducible promoter driving expression of each of the guide nucleic acid and the CRISPR nuclease is the same inducible promoter.

5. The method of claim 4, wherein the inducible promoter driving expression of the guide nucleic acid and driving transcription of the guide nucleic acid is a pL promoter.

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

7. The method of claim 1, further comprising adding selective agents to medium of the first substrate to select for the one or more vectors.

8. The method of claim 1, wherein the guide nucleic acid is a guide RNA.

9. The method of claim 1, wherein the cells grown on the first and second substrates are bacteria cells and the engine vector further comprises a recombineering system.

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

11. A method for enriching edited cells during nucleic acid-guided CRISPR nuclease editing comprising:

transforming cells with one or more vectors comprising a promoter driving expression of a CRISPR nuclease, an inducible promoter driving transcription of a guide nucleic acid 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 first substrate;

growing the cells to form colonies on the first substrate under conditions that allow genome repair;

initiating editing in the cells growing on the first substrate by inducing the inducible promoter driving transcription of the guide nucleic acid to produce induced cells;

selecting cells from the substantially singulated induced cells from the first substrate and arraying the selected cells on a second substrate;

making a replica of the second substrate forming a third substrate;

growing and inducing cells on the second substrate under conditions that allow genome repair;

growing and inducing cells on the third substrate under conditions that do not allow genome repair;

comparing cell growth on the second and third substrates; and

selecting cells from the second substrate that grow on the second substrate but do not grow on the third substrate.

12. The method of claim 11, wherein the promoter driving transcription the guide nucleic acid and donor DNA is a pL promoter.

13. The method of claim 11, wherein the promoter driving expression of the CRISPR nuclease is an inducible promoter.

14. The method of claim 13, wherein the inducible promoter driving expression of each of the guide nucleic acid and the CRISPR nuclease is the same inducible promoter.

15. The method of claim 14, wherein the inducible promoter driving expression of the guide nucleic acid and driving transcription of the guide nucleic acid is a pL promoter.

16. The method of claim 11, wherein the DNA donor sequence further comprises a PAM-altering sequence.

17. The method of claim 11, further comprising adding selective agents to medium of the first substrate to select for the one or more vectors.

18. The method of claim 11, wherein the cells grown on the first, second and third substrates are yeast cells.

19. The method of claim 11, wherein the cells grown on the first, second and third substrates are bacteria cells and the engine vector further comprises a recombineering system.

20. The method of claim 11, wherein the cells grown on the first, second and third substrates are mammalian cells.