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

Patent metadata

Field	Value
Publication number	US-10760043-B2
Application number	US-202016823269-A
Country	US
Kind code	B2
Filing date	Mar 18, 2020
Priority date	Aug 14, 2018
Publication date	Sep 1, 2020
Grant date	Sep 1, 2020

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.

First claim

We claim:

1. 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;
diluting the transformed cells to a cell concentration to substantially singulate the transformed cells on a first substrate;
growing the substantially singulated cells to form cell colonies on the first substrate under conditions that allow genome repair;
making a replica of the first substrate on a second substrate;
making a replica of the first substrate forming a third substrate;
growing and inducing the replicated cells on the second substrate under conditions that allow genome repair;
growing and inducing the replicated cells on the third substrate under conditions that do not allow genome repair;
comparing cell growth of the replicated cells on the second and third substrates; and
selecting cells from the cell colonies from the first substrate that grow on the second substrate but do not grow on the third substrate.

2. The method of claim 1 , wherein the promoter driving transcription of 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 cells grown on the first, second and third substrates are yeast cells.

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

10. The method of claim 1 , wherein the cells grown on the first, second and third substrates are mammalian cells.

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;
diluting the transformed cells to a cell concentration to substantially singulate the transformed cells on a first substrate;
growing the substantially singulated cells to form colonies on the first substrate under conditions that allow genome repair;
making a replica of the first substrate on a second substrate;
making a replica of the first substrate on a third substrate;
growing the replicated cells on the second substrate for two or more doublings;
inducing the replicated cells on the second substrate under conditions that allow genome repair;
growing the replicated cells on the third substrate for two or more doublings;
inducing the replicated cells on the third substrate under conditions that do not allow genome repair; and
selecting cells from the colonies on the first substrate that grew on the second substrate but do not grow on the third substrate.

12. The method of claim 11 , wherein the promoter driving transcription of 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.

Assignees

• Inscripta Inc

Inventors

• Garst Andrew
• Fox Richard
• Belgrader Phillip
• Masquelier Don

Classifications

• C12N2800/80

  Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites · CPC title

• C12N15/87

  Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation · CPC title

• C12N15/102

  Mutagenizing nucleic acids · CPC title

• C12N9/22 Primary

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

• C12M47/04 Primary

  Cell isolation or sorting (purging biological preparations of unwanted cells C12N5/0081, determining the presence or kind of microorganism C12Q1/04) · CPC title