METHODS OF IN VIVO ENGINEERING OF LARGE SEQUENCES USING MULTIPLE CRISPR/CAS SELECTIONS OF RECOMBINEERING EVENTS

• US 20160024529A1
• Filed: 01/07/2014
• Published: 01/28/2016
• Est. Priority Date: 03/09/2013
• Status: Active Grant

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Carstens, Carsten-Peter
• Granted Patent: US 10,612,043 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12N 15/8509 for producing genetically modified animals, e.g. transgenic
• CPC Class Codes: C12N 15/902 using homologous recombination
• CPC Class Codes: C12N 2800/80 Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

Abstract

The present invention provides a method for making a large nucleic acid having a defined sequence in vivo. The method combines recombineering techniques with a CRISPR/Cas system to permit multiple insertions of defined sequences into a target nucleic acid at one time, double stranded cleavage of target nucleic acids in which the defined sequences were not successfully inserted, and selection of successful recombinant cells. The method further includes repeating the process one or more times, using a successful recombinant from one round as the host cell for the next round.

Claims

1. A method for engineering, in vivo, a nucleic acid having a desired sequence, said method comprising:

a) identifying or creating a double stranded DNA CRISPR/Cas cleavage sequence at a pre-determined site on a target nucleic acid in a host cell;

b) obtaining a recombineering segment for the pre-determined site, where the recombineering segment comprises a sequence that is desired to be inserted into the target nucleic acid and that, when inserted into the target nucleic acid by homologous recombination, eliminates the cleavage sequence;

c) introducing into the host cell the recombineering construct;

d) if not already present in the host cell, introducing into the host cell a nucleic acid encoding a CRISPR/Cas system that is specific for the cleavage sequence;

e) maintaining the cell in a viable state until recombineering insertion of the desired sequence and CRISPR/Cas cleavage of the cleavage site has occurred;

f) selecting for recombinant cells that survive the CRISPR/Cas cleavage event; and

g) repeating steps a)-f) one or more times using the recombinant cell produced in step f) as the host cell for step a) of the following round;

wherein multiple, site-specific insertions of recombineering segments into the target nucleic acid creates a nucleic acid having a desired sequence.

2. The method of claim 1, wherein, after performance of steps a)-f) one time, each successive performance of steps a)-f) uses recombineering segments that include sequences for homologous recombination that recombine with sequences present in a recombineering segment used in a prior performance of steps a)-f).

3. The method of claim 1, wherein, after performance of steps a)-f) one time, at least one successive performance of steps a)-f) uses at least one recombineering segment that includes a sequence for homologous recombination that recombines with at least one sequence present in a recombineering segment used in a prior performance of steps a)-f).

4. The method of claim 1, wherein a recombineering segment used in one round of performance of steps a)-f) comprise a cleavage sequence for a subsequent round of performance of steps a)-f).

5. The method of claim 1, wherein, for each round of performance of steps a)-f), the cleavage sequence is different than the cleavage sequence used for the immediate prior round.

6. A recombinant cell made by the method of claim 5.

7. A method for engineering, in vivo, a nucleic acid having a desired sequence, said method comprising:

a) obtaining a double stranded plasmid containing the sequence for a recombineering segment flanked on each end by a cleavage site for a pre-selected double stranded DNA cleaving CRISPR/Cas system, the orientation of one cleavage site on the plasmid being opposite to the orientation of the other cleavage site,

wherein the recombineering segment has sufficient identity with a pre-determined site on a target nucleic acid in a host cell to participate in homologous recombination with that site via recombineering, and

wherein the recombineering segment comprises a sequence that is desired to be inserted into the target nucleic acid and that, when inserted into the target nucleic acid by homologous recombination via recombineering, eliminates a cleavage sequence that is different than the cleavage sequence for excision of the recombineering segment from the double stranded plasmid;

b) introducing into host cells the recombineering plasmid of a);

c) if not already present in the host cells, introducing into the host cells a nucleic acid encoding a CRISPR/Cas system that is specific for the recombineering plasmid cleavage sequence;

d) if not already present in the host cells, introducing into the host cells a nucleic acid encoding a CRISPR/Cas system that is specific for the cleavage sequence to be excised by insertion of the desired sequence into the target nucleic acid;

e) expressing the CRISPR/Cas system that is specific for the recombineering cleavage sequence to effect release of the recombineering segment from the recombineering plasmid;

f) maintaining the cells under conditions that permit viable cells to continue to live until recombineering insertion of the recombineering segment has occurred;

g) expressing the CRISPR/Cas system that is specific for the cleavage sequence to be excised via recombineering;

h) maintaining the cells under conditions that permit viable cells to continue to live until cleavage of cleavage sequences on the target nucleic acid has occurred;

i) selecting for recombinant cells that survive the CRISPR/Cas cleavage event at the target nucleic acid sequence; and

j) repeating steps a)-i) one or more times using the recombinant cell produced in step i) as the host cell for step a) of the following round;

wherein multiple, site-specific insertions of recombineering segments into the target nucleic acid creates a nucleic acid having a desired sequence.

8. The method of claim 7, wherein expressing in steps e), f), or both comprises expressing the respective CRISPR/Cas system by inducing an inducible promoter or de-repressing a repressible promoter, wherein expression of each system is under the control of a different inducer or repressor.

9. The method of claim 7, wherein the host cells comprise a nucleic acid encoding a CRISPR/Cas system that is specific for the recombineering plasmid cleavage sequence and a nucleic acid encoding a CRISPR/Cas system that is specific for the cleavage sequence to be excised by insertion of the desired sequence into the target nucleic acid, and steps c) and d) are not practiced.

10. The method of claim 7, wherein each of the nucleic acids encoding a CRISPR/Cas system further comprises a coding sequence for a selectable marker, each selectable marker being different than the other, to allow for selection, prior to expression of the CRISPR/Cas systems, for host cells containing both nucleic acids.

11. The method of claim 7, wherein, after performance of steps a)-i) one time, each successive performance of steps a)-i) uses recombineering segments that include sequences for homologous recombination that recombine with sequences present in a recombineering segment used in a prior performance of steps a)-i).

12. The method of claim 7, wherein, after performance of steps a)-i) one time, at least one successive performance of steps a)-i) uses at least one recombineering segment that includes a sequence for homologous recombination that recombines with at least one sequence present in a recombineering segment used in a prior performance of steps a)-i).

13. The method of claim 7, wherein a recombineering segment used in one round of performance of steps a)-i) comprise a cleavage sequence for a subsequent round of performance of steps a)-i).

14. The method of claim 7, wherein, for each round of performance of steps a)-i), the cleavage sequence is different than the cleavage sequence used for the immediate prior round.

15. A recombinant cell made by the method of claim 7.