# US 9,040,677 B2 · Transcription activator-like effector assembly

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- Number: US 9,040,677 B2
- Document ID: US9040677B2
- Status: Granted
- Kind: B2
- Application number: 13/965,469
- Filed: 2013-08-13
- Published: 2014-03-13
- Granted: 2015-05-26
- Earliest claimed: 2012-09-12
- Assignees: SIDANSAI Biotechnology CO., LTD
- Applicants: SIDANSAI Biotechnology CO., LTD
- Inventors: Jinlong Zhao; Zhao Wu
- Web page: https://patentgrep.com/patent/US9040677B2

## CPC

- C12P 19/34: Polynucleotides, e.g. nucleic acids, oligoribonucleotides
- C07H 21/00: Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 15/10: Processes for the isolation, preparation or purification of DNA or RNA (chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34)
- C12N 15/102: {Mutagenizing nucleic acids}
- C12N 15/1027: {by DNA shuffling, e.g. RSR, STEP, RPR}
- C12N 15/1031: {mutagenesis by gene assembly, e.g. assembly by oligonucleotide extension PCR}
- C12N 15/64: General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
- C12N 15/66: General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
- C12Q 1/68: involving nucleic acids
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

Described herein are techniques for assembling a polynucleotide encoding a transcription activator-like effector nucleases (TALEN). The techniques ligate and digest necessary modules for a TALEN assembly in one reactor or system. Methods and Kits for generating a TALEN are also described.

## Claims

### Claim 1

A method for assembling a polynucleotide encoding a transcription activator-like effector (TALE) polypeptide, the method comprising:
generating multiple Deoxyribonucleic acid (DNA) segments, an individual DNA segment of the multiple DNA segments corresponding to a repeat sequence of the TALE polypeptide, the repeat sequence identifying a single nucleotide of a particular target polynucleotide or two contiguous nucleotides of the particular target polynucleotide;
mixing the multiple DNA segments with restriction enzymes, DNA ligases, and a TALE backbone vector to generate the polynucleotide encoding the TALE polypeptide, a number of multiple DNA segments being greater than 14; and
purifying the polynucleotide using a plasmid-safe Deoxyribonuclease (DNase).

### Claim 2 (depends on 1)

The method of claim 1, wherein the multiple DNA segments are polymerase chain reaction (PCR) amplification products or a plasmid.

### Claim 3 (depends on 1)

The method of claim 1, wherein the number of multiple DNA segments is greater than 18.

### Claim 4 (depends on 1)

The method of claim 1, wherein the mixing the multiple DNA segments with the restriction enzymes, the DNA ligases, and the TALE backbone vector comprises mixing the multiple DNA segments with the restriction enzymes, the DNA ligases, and the TALE backbone vector in a reaction system.

### Claim 5 (depends on 1)

The method of claim 1, wherein the mixing the multiple DNA segments with the restriction enzymes, the DNA ligases, and the TALE backbone vector comprises mixing the multiple DNA segments with the restriction enzymes, the DNA ligases, and the TALE backbone vectors under a condition including multiple temperature cyclings.

### Claim 6 (depends on 5)

The method of claim 5, wherein the multiple temperature cyclings is followed by inactivation of the restriction enzymes.

### Claim 7 (depends on 1)

The method of claim 1, wherein the mixing the multiple DNA segments with the restriction enzymes, the DNA ligases, and the TALE backbone vector comprises mixing the multiple DNA segments with the restriction enzymes that belongs to a type of type II restriction enzymes, the DNA ligases, and the TALE backbone vectors without other types of the type II restriction enzymes.

### Claim 8 (depends on 1)

The method of claim 1, wherein the individual DNA segment of the multiple DNA segments has two cleavage sites that are generated using type II restriction enzymes, and wherein the restriction enzymes are the type II restriction enzymes.

### Claim 9

A kit for generating polynucleotides encoding a transcription activator-like effector (TALE) polypeptide, the kit comprising:
multiple sets of dimer DNA segments, an individual set of dimer DNA segments including multiple dimer DNA segments, an individual dimer DNA segment encoding two recognition domains of a designed TALE that identify two contiguous nucleotides of a particular target polynucleotide;
multiple sets of monomer DNA segments, an individual set of monomer DNA segments including multiple monomer DNA modules, an individual monomer DNA module encoding a recognition domain of the designed TALE that identifies a single nucleotide of the particular target polynucleotide, wherein the individual dimer DNA segments and the individual monomer DNA segments including a first overhang and a second overhang that are generated using a type II restriction endonuclease;
restriction enzymes;
DNA ligases; and
TALE backbone vectors.

### Claim 10 (depends on 9)

The Kit of claim 9, wherein a sequence of a first overhang of a dimer DNA segment of multiple dimer DNA segments is complementary to a sequence of a second overhang of a particular dimer NDA segment that is ligated to the dimer DNA segment.

### Claim 11 (depends on 9)

The Kit of claim 9, wherein a sequence of a first overhang of a monomer DNA segment of multiple monomer DNA segments is complementary to a sequence of a second overhang of a particular monomer DNA segment that is ligated to the monomer DNA segment.

### Claim 12 (depends on 9)

The Kit of claim 9, wherein the type II restriction endonuclease is Bsa I.

### Claim 13 (depends on 9)

The Kit of claim 9, wherein a number of the multiple sets of dimer DNA segments is from 8 to 10, and a number of the multiple sets of monomer DNA segments is from 6 to 8.

### Claim 14 (depends on 9)

The Kit of claim 9, wherein an individual set of dimer DNA segments includes 15 to 17 dimer DNA segments, and an individual set of monomer DNA segments include 3 to 5 monomer DNA segments.

### Claim 15 (depends on 9)

The Kit of claim 9, wherein the multiple dimer DNA segments and the multiple monomer DNA segments include an overlap extension polymerase chain reaction (PCR) product or a plasmid.

### Claim 16 (depends on 9)

The Kit of claim 9, wherein the multiple dimer DNA segments and the multiple monomer DNA segments include pMD18-T.