IP Library › Granted Patent US 12,275,965
Granted Patent B2
US 12,275,965 · App. 17/271,481 · Granted Apr 15, 2025

Nuclease domain and use thereof

Inventors: Takashi Yamamoto (Higashi-Hiroshima, JP); Tetsushi Sakuma (Higashi-Hiroshima, JP); Masakazu Saito (Higashi-Hiroshima, JP)
Assignee: HIROSHIMA UNIVERSITY
C12N9/22C12N15/63C12N15/907C12N2800/80
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Quick Facts
Patent No.
US 12,275,965
App. No.
17/271,481
Granted
Apr 15, 2025
Kind
B2
Abstract

The present invention provides an artificial nucleic acid-cleaving enzyme comprising: a nuclease domain which is a polypeptide containing an amino acid sequence set forth at positions 391 to 585 of SEQ ID NO: 1 or positions 389 to 579 of SEQ ID NO: 3, or a mutant polypeptide thereof; and a nucleic acid-binding domain.

Claims (13)

1. A combination of two artificial nucleic acid-cleaving enzymes comprising:

a first artificial nucleic acid-cleaving enzyme comprising a nuclease domain which is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO 69 and a nucleic acid-binding domain, and

a second artificial nucleic acid-cleaving enzyme comprising a nuclease domain which is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO 70 and a nucleic acid-binding domain,

wherein the nucleic acid-binding domain of the first artificial nucleic acid-cleaving enzyme and of the second artificial nucleic acid-cleaving enzyme is a zinc finger.

2. The combination of two artificial nucleic acid-cleaving enzymes according to claim 1 , wherein each of the first and the second artificial nucleic acid-cleaving enzymes further comprises a linker located between the nuclease domain and the nucleic acid-binding domain.

3. An isolated nucleic acid comprising a nucleic acid sequence encoding the combination of two artificial nucleic acid-cleaving enzymes according to claim 1 .

4. A vector comprising: the nucleic acid according to claim 3 .

5. A method for modifying a target nucleic acid, comprising: introducing into a cell the combination of artificial nucleic acid-cleaving enzymes according to claim 1 .

6. A kit for modifying a target nucleic acid, comprising: the combination of two artificial nucleic acid-cleaving enzymes according to claim 1 .

7. A method for modifying a target nucleic acid, comprising: introducing into a cell the nucleic acid according to claim 3 .

8. A method for modifying a target nucleic acid, comprising: introducing into a cell the vector according to claim 4 .

9. A kit for modifying a target nucleic acid, comprising: the nucleic acid according to claim 3 .

10. A kit for modifying a target nucleic acid, comprising: the vector according to claim 4 .

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Apr 2, 2021
From: YAMAMOTO, TAKASHI; SAKUMA, TETSUSHI; SAITO, MASAKAZU
To: HIROSHIMA UNIVERSITY
Reel/Frame 055808/0267 →
Priority Claims (1)
JP 2018-158710 · Aug 27, 2018 · national
Continuity (1)
Related Publication 20210332339A1 · Oct 28, 2021
References Cited (20)
US 9410134B2 · Kuhn · 2016 [cited by applicant]
US 10975393B2 · Miller · 2021 [cited by examiner]
US 20170152527A1 · Paschon et al. · 2017 [cited by applicant]
US 20180087072A1 · Miller · 2018 [cited by examiner]
US 20190169240A1 · Yamamoto et al. · 2019 [cited by applicant]
WO 2014175284A1 · 2014 [cited by applicant]
Bhattacharya et al., Impact of genetic variation on three dimensional structure and function of proteins, 2017, PLoS One, vol. 12, Issue 3, pp. 1-22 (Year: 2017). [cited by examiner]
Fenton et al., Rheostat positions: A new classification of protein positions relevant to pharmacogenomics, 2020, Medicinal Chemistry Research, vol. 29, pp. 1133-1146 (Year: 2020). [cited by examiner]
Guo et al., Protein tolerance to random amino acid change, 2004, PNAS, vol. 101, No. 25, pp. 9205-9210 (Year: 2004). [cited by examiner]
Handel et al., Expanding or Restricting the Target Site Repertoire of Zinc-finger Nucleases: The Inter-domain Linker as a Major Determinant of Target Site Selectivity, 2009, Molecular Therapy, vol. 17, No. 1, pp. 104-11… [cited by examiner]
Sakuma et al., Repeating pattern of non-RVD variations in DNA-binding modules enhances TALEN activity, 2013, Scientific Reports, vol. 3, Issue 3379, pp. 1-8 (Year: 2013). [cited by examiner]
Extended European Search Report, dated May 20, 2022, issued by the European Patent Office in European Application No. 19853370.5. [cited by applicant]
Database Protein [online], “hypothetical protein [Clostridium botulinum]”, WP_05049162 8, 579 aa, linear BCT Aug. 7, 2015 (1 page total). [cited by applicant]
Database UniProt [online], “SubName: Full=Uncharacterized protein {ECO : 0000313|EMBL:KSU87972.1}”, AOAV8JLM2, Mar. 16, 2016, 585 AA (1 page total). [cited by applicant]
Communication, dated Mar. 11, 2021, issued by the International Bureau in International application No. PCT/JP2019/033045. [cited by applicant]
Eva-Maria Handel et al., “Expanding or Restricting the Target Site Repertoire of Zinc-finger Nucleases: The Inter-domain Linker as a Major Determinant of Target Site Selectivity”, [cited by applicant]
S. G. Dastager et al., “hypothetical protein AS180_10220 [ [cited by applicant]
S. G. Dastager et al., “ [cited by applicant]
A. Poehlein e al., “[Clostridium] thermoalcaliphilum strain DSM 7309 CLOTH_contig000003, whole genome shotgun sequence”, Database GenBank [online], Accession No. MZGW01000003. Mar. 1, 2017, Genomic and Applied Microbiol… [cited by applicant]
International Search Report for PCT/JP2019/033045, dated Oct. 15, 2019. [cited by applicant]