Methods and compositions for modifying a targeted locus

Patent metadata

Field	Value
Publication number	US-12060571-B2
Application number	US-201916369565-A
Country	US
Kind code	B2
Filing date	Mar 29, 2019
Priority date	Jun 6, 2014
Publication date	Aug 13, 2024
Grant date	Aug 13, 2024

Abstract

Methods and compositions are provided for modifying one or more target loci in a cell. Such methods comprise providing a cell comprising a first polynucleotide encoding a first selection marker operably linked to a first promoter active in the cell, wherein the first polynucleotide further comprises a first recognition site for a first nuclease agent. A first nuclease agent is introduced into a cell, wherein the first nuclease agent induces a nick or double-strand break at the first recognition site. Further introduced into the cell is a first targeting vector comprising a first insert polynucleotide flanked by a first and a second homology arm that correspond to a first and a second target site located in sufficient proximity to the first recognition site. At least one cell is then identified comprising in its genome the first insert polynucleotide integrated at the target locus.

First claim

That which is claimed:

1. A method for serial modification of a target locus in a cell, comprising:
(a) providing the cell comprising the target locus, wherein the target locus comprises a first selection cassette comprising: (1) a nucleic acid encoding a first selection marker; and (2) a first nuclease recognition site for a first nuclease agent, wherein the first nuclease recognition site is located in a coding region of the first selection marker or any non-protein-coding region of the first selection marker;
(b) introducing into the cell:
(i) the first nuclease agent, wherein the first nuclease agent induces a nick or a double-strand break at the first nuclease recognition site; and
(ii) a first large targeting vector (LTVEC) that is at least 10 kb and comprises a first insert polynucleotide flanked by a first homology arm corresponding to a first target site located in the target locus and a second homology arm corresponding to a second target site located in the target locus, wherein the first insert polynucleotide comprises a second selection cassette comprising: (1) a nucleic acid encoding a second selection marker, wherein the first selection marker and the second selection marker are different; and (2) a second nuclease recognition site for a second nuclease agent, wherein the second nuclease recognition site is located in a coding region of the second selection marker or any non-protein-coding region of the second selection marker;

(c) identifying a modified cell comprising the first insert polynucleotide at the target locus, wherein the modified cell has the activity of the second selection marker but does not have the activity of the first selection marker;
(d) introducing into the modified cell:
(i) the second nuclease agent, wherein the second nuclease agent induces a nick or a double-strand break at the second nuclease recognition site; and
(ii) a second LTVEC that is at least 10 kb and comprises a second insert polynucleotide flanked by a third homology arm corresponding to a third target site located in the target locus and a fourth homology arm corresponding to a fourth target site located in the target locus, wherein the second insert polynucleotide comprises a third selection cassette comprising: (1) a nucleic acid encoding a third selection marker, wherein the first selection marker and the third selection marker are identical; and (2) a third nuclease recognition site for a third nuclease agent, wherein the third nuclease recognition site is identical to the first nuclease recognition site and different from the second nuclease recognition site, and the first nuclease agent and the third nuclease agent are identical to one another and are different from the second nuclease agent; and

(e) identifying at least one cell comprising the second insert polynucleotide integrated at the target locus, wherein the at least one cell has the activity of the third selection marker but does not have the activity of the second selection marker.

2. The method of claim 1 , wherein:
(I) the identifying step (c) comprises:
(i) culturing the cell under conditions that allow identification of cells that do not have the activity of the first selection marker; or
(ii) identifying at least one cell comprising the first insert polynucleotide integrated at the first and second target sites; and/or

(II) the identifying step (e) comprises:
(i) culturing the cell under conditions that allow identification of cells that do not have the activity of the second selection marker; or
(ii) identifying at least one cell comprising the second insert polynucleotide integrated at the third and fourth target sites.

3. The method of claim 1 , wherein the identifying step (c) is carried out via a modification of allele (MOA) assay, and/or the identifying step (e) is carried out via a MOA assay.

4. The method of claim 1 , wherein the first selection cassette in step (a) is flanked by the first target site and the second target site, and/or wherein the second selection cassette in the modified cell in step (c) is flanked by the third target site and the fourth target site.

5. The method of claim 1 , wherein the first selection marker or the second selection marker imparts resistance to an antibiotic.

6. The method of claim 5 , wherein the first and third selection markers impart resistance to neomycin and the second selection marker imparts resistance to hygromycin, or
wherein the first and third selection markers impart resistance to hygromycin and the second selection marker imparts resistance to neomycin.

7. The method of claim 1 , wherein the first selection marker or the second selection marker is operably linked to an inducible promoter, and expression of the inducible selection marker is toxic to the cell.

8. The method of claim 1 , wherein the combined use of the first LTVEC with the first nuclease agent increases targeting efficiency at least two-fold compared to the use of the first LTVEC alone.

9. The method of claim 1 , wherein the first nuclease agent or the second nuclease agent comprises an expression construct comprising a nucleic acid sequence encoding the nuclease agent, wherein the nucleic acid sequence encoding the nuclease agent is operably linked to a promoter active in the cell.

10. The method of claim 1 , wherein the first nuclease agent or the second nuclease agent is a zinc finger nuclease (ZFN), a Transcription Activator-Like Effector Nuclease (TALEN), a meganuclease, or a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated (Cas) protein and a guide RNA (gRNA).

11. The method of claim 10 , wherein the first nuclease agent or the second nuclease agent is the ZFN, and the first nuclease recognition site or the second nuclease recognition site comprises any one of SEQ ID NOS: 9-12.

12. The method of claim 10 , wherein the first nuclease agent or the second nuclease agent is the Cas protein and the gRNA, wherein the Cas protein is Cas9, wherein the gRNA comprises a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) RNA (crRNA) that targets the first nuclease recognition site or the second nuclease recognition site and a trans-activating CRISPR RNA (tracrRNA), and wherein the first nuclease recognition site or the second nuclease recognition site is immediately flanked by a Protospacer Adjacent Motif (PAM) sequence.

13. The method of claim 12 , wherein the gRNA comprises any one of SEQ ID NOS: 13-20.

14. The method of claim 12 , wherein the gRNA comprises SEQ ID NO: 2, 3, 4, 5, 6, 7, or 8.

15. The method of claim 14 , wherein the gRNA comprises SEQ ID NO: 3 or SEQ ID NO: 7.

16. The method of claim 1 , wherein the first nuclease recognition site is located in an intron, an exon, a promoter, a promoter regulatory region, or an enhancer region of the first selection marker, and the second nuclease recognition site is located in an intron, an exon, a promoter, a promoter regulatory region, or an enhancer region of the second selection marker.

17. The method of claim 1 , wherein:
(I) the nick or the double-strand break induced by the first nuclease agent disrupts the activity of the first selection marker, or wherein insertion of the first insert polynucleotide at the target locus disrupts the activity of the first selection marker; and/or
(II) the nick or the double-strand break induced by the second nuclease agent disrupts the activity of the second selection marker, or wherein insertion of the second insert polynucleotide at the target locus disrupts the activity of the second selection marker.

18. The m

Assignees

• Regeneron Pharma

Inventors

• Auerbach Wojtek
• Frendewey David
• Droguett Gustavo
• Gagliardi Anthony
• Kuno Junko
• Valenzuela David M

Classifications

• C12N2810/00

  Vectors comprising a targeting moiety · CPC title

• A01K2227/105

  Murine · CPC title

• C12N15/64

  General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host · CPC title

• A01K2207/15

  Humanized animals · CPC title

• A01K2217/15

  Animals comprising multiple alterations of the genome, by transgenesis or homologous recombination, e.g. obtained by cross-breeding · CPC title