US-20260125711-A1 - RNA-GUIDED NUCLEASES AND ACTIVE FRAGMENTS AND VARIANTS THEREOF AND METHODS OF USE

Abstract

Compositions and methods for binding to a target sequence of interest are provided. The compositions find use in cleaving or modifying a target sequence of interest, visualization of a target sequence of interest, and modifying the expression of a sequence of interest. Compositions comprise RNA-guided nuclease (RGN) polypeptides, CRISPR RNAs, trans-activating CRISPR RNAs, guide RNAs, and nucleic acid molecules encoding the same. Vectors and host cells comprising the nucleic acid molecules are also provided. Further provided are RGN systems for binding a target sequence of interest, wherein the RGN system comprises an RNA-guided nuclease polypeptide and one or more guide RNAs.

Inventors

• Alexandra Briner Crawley
• Michael Coyle
• Gunjan H. Arya
• Lisle MOSE

Assignees

• LIFE EDIT THERAPEUTICS, INC.

Dates

Publication Date

20260507

Application Date

20230812

Claims (20)

1. 1 . A nucleic acid molecule comprising a polynucleotide encoding an RNA-guided nuclease (RGN) polypeptide comprising an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 19, 13, 3, 7, 16, 1, 2, 4-6, 8-12, 14, 15, 17, 18, and 20.
2. 2 . (canceled)
3. 3 . The nucleic acid molecule of claim 1 , wherein said polynucleotide encoding an RGN polypeptide is: a) operably linked to a promoter heterologous to said polynucleotide; and/or b) codon optimized for expression in a eukaryotic cell.
4. 4 . The nucleic acid molecule of claim 1 , wherein said RGN polypeptide comprises the amino acid sequence set forth as any one of SEQ ID NOs: 19, 13, 3, 7, 16, 1, 2, 4-6, 8-12, 14, 15, 17, 18, and 20.
5. 5 .- 7 . (canceled)
6. 8 . The nucleic acid molecule of claim 1 , wherein said RGN polypeptide: a) is nuclease inactive or is a nickase; and/or b) comprises one or more nuclear localization signals.
7. 9 . The nucleic acid molecule of claim 1 , wherein the RGN polypeptide is operably fused to a base-editing polypeptide.
8. 10 . The nucleic acid molecule of claim 9 , wherein the base-editing polypeptide is a deaminase.
9. 11 .- 13 . (canceled)
10. 14 . A vector comprising the nucleic acid molecule of claim 1 .
11. 15 . The vector of claim 14 , further comprising at least one nucleotide sequence encoding a gRNA capable of hybridizing to the non-target strand of said target sequence.
12. 16 .- 19 . (canceled)
13. 20 . A cell comprising the nucleic acid molecule of claim 1 .
14. 21 .- 29 . (canceled)
15. 30 . An RNA-guided nuclease (RGN) polypeptide, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 19, 13, 3, 7, 16, 1, 2, 4-6, 8-12, 14, 15, 17, 18, and 20.
16. 31 . (canceled)
17. 32 . The RGN polypeptide of claim 30 , wherein said RGN polypeptide comprises the amino acid sequence set forth as any one of SEQ ID NOs: 19, 13, 3, 7, 16, 1, 2, 4-6, 8-12, 14, 15, 17, 18, and 20.
18. 33 .- 35 . (canceled)
19. 36 . The RGN polypeptide of claim 30 , wherein said RGN polypeptide: a) is nuclease inactive or a nickase; and/or b) comprises one or more nuclear localization signals.
20. 37 . The RGN polypeptide of claim 30 , wherein the RGN polypeptide is operably fused to a base-editing polypeptide.

Description

CROSS-REFERENCE TO RELATED APPLICATION This application claims priority to US Provisional Patent Application Nos. 63/371,230, filed Aug. 12, 2022, which is fully incorporated by reference herein. REFERENCE TO A SEQUENCE LISTING SUBMITTED ELECTRONICALLY AS AN XML FILE The instant application contains a Sequence Listing which has been submitted in xml format via USPTO Patent Center and is hereby incorporated by reference in its entirety. Said xml copy, created on Dec. 22, 2025, is named L103438_1310WO_Seq_List.xml, and is 1,573,029 bytes in size. FIELD OF THE INVENTION The present invention relates to the field of molecular biology and gene editing. BACKGROUND OF THE INVENTION Targeted genome editing or modification is rapidly becoming an important tool for basic and applied research. Initial methods involved engineering nucleases such as meganucleases, zinc finger fusion proteins or TALENs, requiring the generation of chimeric nucleases with engineered, programmable, sequence-specific DNA-binding domains specific for each particular target sequence. RNA-guided nucleases, such as the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated (Cas) proteins of the CRISPR-Cas bacterial system, allow for the targeting of specific sequences by complexing the nucleases with guide RNA that specifically hybridizes with a particular target sequence. Producing target-specific guide RNAs is less costly and more efficient than generating chimeric nucleases for each target sequence. Such RNA-guided nucleases can be used to edit genomes optionally through the introduction of a sequence-specific, double-stranded break that is repaired via error-prone non-homologous end-joining (NHEJ) to introduce a mutation at a specific genomic location. Alternatively, heterologous DNA may be introduced into the genomic site via homology-directed repair. RNA-guided nucleases (RGNs) can also be used for base editing when fused with a deaminase. BRIEF SUMMARY OF THE INVENTION Compositions and methods for binding a target sequence of interest in a target nucleic acid molecule are provided. The compositions find use in cleaving or modifying a target nucleic acid molecule of interest, detection of a target sequence of interest, and modifying the expression of a gene of interest comprising a target sequence. Compositions comprise RNA-guided nuclease (RGN) polypeptides, CRISPR RNAs (crRNAs), trans-activating CRISPR RNAs (tracrRNAs), guide RNAs (gRNAs) such as single guide RNAs (sgRNAs), nucleic acid molecules encoding the same, compositions comprising the same, and vectors and host cells comprising the nucleic acid molecules. Also provided are RGN systems and ribonucleoprotein complexes for binding a target sequence of interest, wherein the RGN system and ribonucleoprotein complex comprises an RNA-guided nuclease polypeptide and one or more guide RNAs. Thus, methods disclosed herein are drawn to binding a target sequence of interest in a target nucleic acid molecule, and in some embodiments, cleaving or modifying the target nucleic acid molecule of interest. The target nucleic acid molecule of interest can be modified, for example, as a result of non-homologous end joining, homology-directed repair with an introduced donor sequence, or base editing. In one aspect, the present disclosure provides a nucleic acid molecule comprising a polynucleotide encoding an RNA-guided nuclease (RGN) polypeptide, wherein the polynucleotide comprises a nucleotide sequence encoding an RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs:1-20. In some embodiments of the above aspect, the RGN polypeptide is capable of binding a target sequence in a target nucleic acid molecule, wherein the target sequence comprises a target strand and a non-target strand, in an RNA-guided sequence specific manner when bound to a guide RNA (gRNA) capable of hybridizing to the non-target strand of the target sequence. In some embodiments of the above aspect, the polynucleotide encoding an RGN polypeptide is operably linked to a promoter heterologous to the polynucleotide. In some embodiments of the above aspect, the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NOs:1-20. In some embodiments, the RGN polypeptide comprises an amino acid sequence having 100% sequence identity to any one of SEQ ID NOs: 1-20. In some embodiments of the above aspect, the RGN polypeptide is capable of cleaving the target nucleic acid molecule upon binding. In some embodiments, the RGN polypeptide is capable of generating a double-stranded break. In some embodiments, the RGN polypeptide is capable of generating a single-stranded break. In some embodiments of the above aspect, the RGN polypeptide is nuclease inactive or is a nickase. In some embodiments of the above aspect, the RGN polypeptide is operably fused to a base-editing polypeptide. In some embodiments, the base-