	US 12,252,706 B2
	RNA-guided nucleases and active fragments and variants thereof and methods of use
Tyson D. Bowen, Morrisville, NC (US); Michael Coyle, Chapel Hill, NC (US); Alexandra Briner Crawley, Cary, NC (US); and Tedd D. Elich, Durham, NC (US)
Assigned to Life Edit Therapeutics, Inc., Durham, NC (US)
Filed by LifeEDIT Therapeutics, Inc., Durham, NC (US)
Filed on Apr. 29, 2024, as Appl. No. 18/649,317.
Application 18/649,317 is a continuation of application No. 17/929,165, filed on Sep. 1, 2022, granted, now 11,981,916.
Application 17/929,165 is a continuation of application No. 17/851,887, filed on Jun. 28, 2022.
Application 17/851,887 is a continuation of application No. PCT/US2021/028843, filed on Apr. 23, 2021.
Claims priority of provisional application 63/014,970, filed on Apr. 24, 2020.
Claims priority of provisional application 63/077,211, filed on Sep. 11, 2020.
Prior Publication US 2024/0294947 A1, Sep. 5, 2024
This patent is subject to a terminal disclaimer.
Int. Cl. C12N 15/90 (2006.01); C12N 9/22 (2006.01); C12N 15/113 (2010.01); C12N 15/64 (2006.01); C12N 15/66 (2006.01)

CPC C12N 15/902 (2013.01) [C12N 9/22 (2013.01); C12N 15/113 (2013.01); C12N 15/64 (2013.01); C12N 15/66 (2013.01); C12N 2310/20 (2017.05); C12Q 2521/301 (2013.01)]

28 Claims

1. A nucleic acid molecule comprising a polynucleotide encoding an RNA-guided nuclease (RGN) polypeptide, wherein said polynucleotide comprises a nucleotide sequence encoding an RGN polypeptide comprising an amino acid sequence having at least 92% sequence identity to any one of SEQ ID NOs: 63, 70, and 570-579;

wherein said polynucleotide encoding an RGN polypeptide is operably linked to a promoter heterologous to said polynucleotide.

12. An RNA-guided nuclease (RGN) polypeptide, wherein said RGN polypeptide comprises an amino acid sequence having at least 92% sequence identity to any one of SEQ ID NOs: 63, 70, and 570-579; and

wherein said RGN polypeptide is operably fused to an effector domain.

17. A system for binding a target DNA sequence of a DNA molecule, said system comprising:

a) one or more guide RNAs (gRNAs) or one or more polynucleotides comprising one or more nucleotide sequences encoding the one or more gRNAs, wherein said target DNA sequence is a eukaryotic target DNA sequence; and

b) an RNA-guided nuclease (RGN) polypeptide comprising an amino acid sequence having at least 92% sequence identity to any one of SEQ ID NOs: 63, 70, and 570-579, or a polynucleotide comprising a nucleotide sequence encoding the RGN polypeptide;

wherein the one or more gRNAs are capable of forming a complex with the RGN polypeptide.

23. A method for cleaving and/or modifying a target DNA sequence of a DNA molecule, comprising contacting the DNA molecule with:

a) an RNA-guided nuclease (RGN) polypeptide, or a polynucleotide comprising a nucleotide sequence encoding the RGN polypeptide, wherein said RGN comprises an amino acid sequence having at least 92% sequence identity to any one of SEQ ID NOs: 63, 70, and 570-579; and

b) one or more guide RNAs;

wherein the one or more guide RNAs hybridize to the target DNA sequence.