# US 11,111,504 B2 · Methods for scarless introduction of targeted modifications into targeting vectors

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- Number: US 11,111,504 B2
- Document ID: US11111504B2
- Status: Granted
- Kind: B2
- Application number: 16/838,621
- Filed: 2020-04-02
- Published: 2020-10-08
- Granted: 2021-09-07
- Earliest claimed: 2019-04-04
- Assignees: Regeneron Pharmaceuticals, Inc.
- Applicants: Regeneron Pharmaceuticals, Inc.
- Inventors: Susannah Brydges; Jose F. Rojas; Gregg S. Warshaw; Chia-Jen Siao
- Web page: https://patentgrep.com/patent/US11111504B2

## CPC

- C12N 15/85: for animal cells
- C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein}
- C12N 15/102: {Mutagenizing nucleic acids}
- C12N 15/64: General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Methods for introducing a scarless targeted genetic modification into a preexisting targeting vector are provided. The methods can use combinations of bacterial homologous recombination (BHR) and in vitro assembly to introduce such targeted genetic modifications into a preexisting targeting vector in a scarless manner.

## Claims

### Claim 1

A method for introducing a scarless targeted genetic modification in a preexisting targeting vector, comprising:
(a) performing bacterial homologous recombination between the preexisting targeting vector and a modification cassette in a population of bacterial cells,
wherein the modification cassette comprises the targeted genetic modification and comprises an insert nucleic acid flanked by a 5′ homology arm corresponding to a 5′ target sequence in the preexisting targeting vector and a 3′ homology arm corresponding to a 3′ target sequence in the preexisting targeting vector, wherein the insert nucleic acid comprises from 5′ to 3′:
(i) a first repeat sequence;
(ii) a first target site for a first nuclease agent;
(iii) a selection cassette;
(iv) a second target site for a second nuclease agent; and
(v) a second repeat sequence identical to the first repeat sequence,
wherein the repeat sequence is at least about 20 nucleotides in length;
(b) selecting bacterial cells comprising a modified targeting vector comprising the selection cassette;
(c) cleaving the first target site in the modified targeting vector with the first nuclease agent and cleaving the second target site in the modified targeting vector with the second nuclease agent to remove the selection cassette and expose the first repeat sequence and the second repeat sequence in the modified targeting vector,
wherein step (c) occurs in vitro; and
(d) assembling the exposed first repeat sequence with the exposed second repeat sequence in an intramolecular in vitro assembly reaction to generate the targeting vector comprising the scarless targeted genetic modification,
wherein neither the first target site for the first nuclease agent nor the second target site for the second nuclease agent are present and only a single copy of the repeat sequence is present in the targeting vector comprising the scarless targeted genetic modification.

### Claim 2 (depends on 1)

The method of claim 1, wherein the repeat sequence is identical to a sequence in the preexisting targeting vector.

### Claim 3 (depends on 1)

The method of claim 1, wherein the targeted genetic modification comprises an insertion, and the repeat sequence is identical to the 5′ end or the 3′ end of the insertion.

### Claim 4 (depends on 1)

The method of claim 1, wherein the repeat sequence is between about 20 nucleotides and about 100 nucleotides in length.

### Claim 5 (depends on 1)

The method of claim 1, wherein the modification cassette is a linear, double-stranded nucleic acid.

### Claim 6 (depends on 1)

The method of claim 1, wherein the modification cassette is from about 1 kb to about 15 kb in length.

### Claim 7 (depends on 1)

The method of claim 1, wherein the 5′ homology arm and the 3′ homology arm are each at least about 35 nucleotides in length.

### Claim 8 (depends on 7)

The method of claim 7, wherein the 5′ homology arm and the 3′ homology arm are each between about 35 nucleotides and about 500 nucleotides in length.

### Claim 9 (depends on 1)

The method of claim 1, wherein the first nuclease agent and/or the second nuclease agent is a rare-cutting nuclease agent.

### Claim 10 (depends on 1)

The method of claim 1, wherein the first target site and/or the second target site is not present in the preexisting targeting vector.

### Claim 11 (depends on 1)

The method of claim 1, wherein the first target site is identical to the second target site, and the first nuclease agent is identical to the second nuclease agent.

### Claim 12 (depends on 1)

The method of claim 1, wherein the first nuclease agent and/or the second nuclease agent comprises a rare-cutting restriction enzyme.

### Claim 13 (depends on 12)

The method of claim 12, wherein the rare-cutting restriction enzyme is NotI, XmaIII, SstII, Sall, NruI, NheI, Nb.BbvCI, BbvCI, AscI, AsiSI, FseI, PacI, PmeI, SbfI, SgrAI, SwaI, BspQI, SapI, SfiI, CspCI, AbsI, CciNI, FspAI, MauBI, MreI, MssI, PalAI, RgaI, RigI, SdaI, SfaAI, SgfI, SgrDI, SgsI, SmiI, SrfI, Sse2321, Sse83871, LguI, PciSI, AarI, AjuI, AloI, BarI, PpiI, or PsrI.

### Claim 14 (depends on 1)

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

### Claim 15 (depends on 14)

The method of claim 14, wherein the first nuclease agent and/or the second nuclease agent is the Cas protein and the gRNA, wherein the Cas protein is Cas9, and wherein the gRNA comprises a CRLSPR RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA).

### Claim 16 (depends on 1)

The method of claim 1, wherein the targeted genetic modification comprises a modification in the 5′ homology arm or the 3′ homology arm.

### Claim 17 (depends on 1)

The method of claim 1, wherein the targeted genetic modification comprises a modification in the insert nucleic acid.

### Claim 18 (depends on 1)

The method of claim 1, wherein the targeted genetic modification comprises a point mutation, a deletion, an insertion, a replacement, or a combination thereof.

### Claim 19 (depends on 1)

The method of claim 1, wherein the selection cassette imparts resistance to an antibiotic.

### Claim 20 (depends on 19)

The method of claim 19, wherein the selection cassette imparts resistance to ampicillin, chloramphenicol, tetracycline, kanamycin, spectinomycin, streptomycin, carbenicillin, bleomycin, erythromycin, or polymyxin B.

### Claim 21 (depends on 1)

The method of claim 1, wherein the preexisting targeting vector is a large targeting vector at least about 10 kb in length.

### Claim 22 (depends on 21)

The method of claim 21, wherein the preexisting targeting vector is at least about 100 kb in length.

### Claim 23 (depends on 1)

The method of claim 1, wherein the preexisting targeting vector comprises a second selection cassette.

### Claim 24 (depends on 23)

The method of claim 23, wherein the second selection cassette imparts resistance to an antibiotic.

### Claim 25 (depends on 24)

The method of claim 24, wherein the selection cassette in the modification cassette and the second selection cassette in the preexisting targeting vector each impart resistance to a different antibiotic.

### Claim 26 (depends on 23)

The method of claim 23, wherein the second selection cassette allows for selection in both bacterial and mammalian cells.

### Claim 27 (depends on 1)

The method of claim 1, wherein step (d) comprises:
(i) contacting the modified targeting vector with an exonuclease to expose complementary sequences between the first repeat sequence and the second repeat sequence;
(ii) annealing the exposed complementary sequences;
(iii) extending the 3′ ends of the annealed complementary sequences; and
(iv) ligating the annealed complementary sequences.

### Claim 28 (depends on 27)

The method of claim 27, wherein step (d) comprises incubating the modified targeting vector with an exonuclease, a DNA polymerase, and a DNA ligase.

### Claim 29 (depends on 1)

The method of claim 1, further comprising:
(e) treating the targeting vector with the first nuclease agent and the second nuclease agent following the in vitro assembly in step (d) to verify that neither the first target site for the first nuclease agent nor the second target site for the second nuclease agent are present.