	US 12,351,839 B2
	Rationally-designed single-chain meganucleases with non-palindromic recognition sequences
James Jefferson Smith, Morrisville, NC (US); and Derek Jantz, Durham, NC (US)
Assigned to Precision BioSciences, Inc., Durham, NC (US)
Filed by Precision Biosciences, Inc., Durham, NC (US)
Filed on Mar. 17, 2023, as Appl. No. 18/185,726.
Application 18/185,726 is a continuation of application No. 17/079,377, filed on Oct. 23, 2020, abandoned.
Application 17/079,377 is a continuation of application No. 16/025,747, filed on Jul. 2, 2018, abandoned.
Application 16/025,747 is a continuation of application No. 15/132,941, filed on Apr. 19, 2016, granted, now 10,041,053, issued on Aug. 7, 2018.
Application 15/132,941 is a continuation of application No. 14/858,986, filed on Sep. 18, 2015, granted, now 9,340,777.
Application 14/858,986 is a continuation of application No. 14/723,840, filed on May 28, 2015, abandoned.
Application 14/723,840 is a continuation of application No. 13/897,923, filed on May 20, 2013, abandoned.
Application 13/897,923 is a continuation of application No. 12/771,163, filed on Apr. 30, 2010, granted, now 8,445,251, issued on May 1, 2013.
Application 12/771,163 is a continuation of application No. PCT/US2008/082072, filed on Oct. 31, 2008.
Claims priority of provisional application 61/001,247, filed on Oct. 31, 2007.
Prior Publication US 2023/0416711 A1, Dec. 28, 2023
This patent is subject to a terminal disclaimer.
Int. Cl. C12N 9/22 (2006.01); A61P 31/00 (2006.01); A61P 31/04 (2006.01); A61P 31/12 (2006.01); A61P 33/02 (2006.01); A61P 43/00 (2006.01); C12N 9/16 (2006.01); C12N 15/85 (2006.01); C12N 15/90 (2006.01)

CPC C12N 9/22 (2013.01) [C12N 9/16 (2013.01); C12N 15/8509 (2013.01); C12N 15/902 (2013.01); C12N 15/907 (2013.01); C12N 2800/80 (2013.01); C12Y 301/00 (2013.01); C12Y 301/04 (2013.01)]

11 Claims

1. A recombinant single-chain meganuclease comprising:

a first LAGLIDADG (SEQ ID NO: 55) subunit comprising a polypeptide sequence having at least 85% sequence identity to residues 9-151 of a wild-type I-CreI meganuclease of SEQ ID NO: 1, said first LAGLIDADG (SEQ ID NO: 55) subunit having a first recognition half-site;

a second LAGLIDADG (SEQ ID NO: 55) subunit comprising a polypeptide sequence having at least 85% sequence identity to residues 9-151 of a wild-type I-CreI meganuclease of SEQ ID NO: 1, said second LAGLIDADG (SEQ ID NO: 55) subunit having a second recognition half-site;

wherein said first and second LAGLIDADG (SEQ ID NO: 55) subunits are covalently joined by a polypeptide linker such that said first LAGLIDADG (SEQ ID NO: 55) domain is N-terminal to said linker and said second LAGLIDADG (SEQ ID NO: 55) domain is C-terminal to said linker; wherein said polypeptide linker comprises 15-56 residues, and

wherein said first and second LAGLIDADG (SEQ ID NO: 55) subunits are capable of functioning together to recognize and cleave a non-palindromic DNA sequence which is a hybrid of said first recognition half-site and said second recognition half-site.