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US-12624369-B2 - RNA-guided nucleases and active fragments and variants thereof and methods of use

US12624369B2US 12624369 B2US12624369 B2US 12624369B2US-12624369-B2

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

  • Tyson D. Bowen
  • Michael Coyle
  • Alexandra Briner Crawley
  • Tedd D. Elich

Assignees

  • LIFE EDIT THERAPEUTICS, INC.

Dates

Publication Date
20260512
Application Date
20220628

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 SEQ ID NO: 117, and wherein the RGN polypeptide is operably fused to a base-editing polypeptide.
  2. 2 . The nucleic acid molecule of claim 1 , wherein the RGN polypeptide is operably fused to one or more nuclear localization signals.
  3. 3 . The nucleic acid molecule of claim 1 , wherein the RGN polypeptide is codon optimized for expression in a eukaryotic cell.
  4. 4 . A cell comprising the nucleic acid molecule of claim 1 .
  5. 5 . The nucleic acid molecule of claim 1 , wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 117.
  6. 6 . The nucleic acid molecule of claim 1 , wherein the RGN polypeptide comprises the amino acid sequence set forth as SEQ ID NO: 117.
  7. 7 . The nucleic acid molecule of claim 1 , wherein said RGN polypeptide is nuclease inactive or is a nickase.
  8. 8 . The nucleic acid molecule of claim 1 , wherein said polynucleotide encoding an RGN polypeptide is operably linked to a promoter heterologous to said polynucleotide.
  9. 9 . A vector comprising 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 SEQ ID NO: 117, wherein said polynucleotide encoding an RGN polypeptide is operably linked to a promoter heterologous to said polynucleotide, wherein the vector further comprises at least one nucleotide sequence encoding a guide RNA capable of hybridizing to a target DNA sequence, wherein the guide RNA comprises: i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 118; and ii) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 119.
  10. 10 . The vector of claim 9 , wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 117.
  11. 11 . The vector of claim 9 , wherein the RGN polypeptide comprises the amino acid sequence set forth as SEQ ID NO: 117.
  12. 12 . The vector of claim 9 , wherein the CRISPR repeat sequence has at least 95% sequence identity to SEQ ID NO: 118.
  13. 13 . The vector of claim 9 , wherein the CRISPR repeat sequence is set forth as SEQ ID NO: 118.
  14. 14 . The vector of claim 9 , wherein the tracrRNA has at least 95% sequence identity to SEQ ID NO: 119.
  15. 15 . The vector of claim 9 , wherein the tracrRNA has the nucleotide sequence set forth as SEQ ID NO: 119.
  16. 16 . 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 SEQ ID NO: 117, wherein the RGN polypeptide is operably fused to a deaminase, and wherein the RGN polypeptide is capable of recognizing a protospacer adjacent motif (PAM) comprising NGG (SEQ ID NO: 116).
  17. 17 . The nucleic acid molecule of claim 16 , wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 117.
  18. 18 . The nucleic acid molecule of claim 16 , wherein the RGN polypeptide comprises the amino acid sequence set forth as SEQ ID NO: 117.
  19. 19 . The nucleic acid molecule of claim 16 , wherein said RGN polypeptide is nuclease inactive or is a nickase.
  20. 20 . The nucleic acid molecule of claim 16 , wherein said polynucleotide encoding an RGN polypeptide is operably linked to a promoter heterologous to said polynucleotide.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of International Application No. PCT/US2021/028843, filed Apr. 23, 2021, which claims priority to U.S. Provisional Application No. 63/014,970, filed Apr. 24, 2020, and U.S. Provisional Application No. 63/077,211, filed Sep. 11, 2020, each of which application is incorporated by reference herein in its entirety. STATEMENT REGARDING THE SEQUENCE LISTING The Sequence Listing associated with this application is provided in ASCII format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The ASCII copy named L103438_1200WO_0084_1_SL is 878,362 bytes in size, was created on Apr. 21, 2021, and is being submitted electronically via EFS-Web. 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 are provided. The compositions find use in cleaving or modifying a target sequence of interest, detection of a target sequence of interest, and modifying the expression of a sequence of interest. Compositions comprise RNA-guided nuclease (RGN) polypeptides, CRISPR RNAs (crRNAs), trans-activating CRISPR RNAs (tracrRNAs), guide RNAs (gRNAs), nucleic acid molecules encoding the same, and vectors and host cells comprising the nucleic acid molecules. Also 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. Thus, methods disclosed herein are drawn to binding a target sequence of interest, and in some embodiments, cleaving or modifying the target sequence of interest. The target sequence 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. DETAILED DESCRIPTION Many modifications and other embodiments of the inventions set forth herein will come to mind to one skilled in the art to which these inventions pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the inventions are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended embodiments. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation. I. Overview RNA-guided nucleases (RGNs) allow for the targeted manipulation of specific site(s) within a genome and are useful in the context of gene targeting for therapeutic and research applications. In a variety of organisms, including mammals, RNA-guided nucleases have been used for genome engineering by stimulating non-homologous end joining and homologous recombination, for example. The compositions and methods described herein are useful for creating single- or double-stranded breaks in polynucleotides, modifying polynucleotides, detecting a particular site within a polynucleotide, or modifying the expression of a particular gene. The RNA-guided nucleases disclosed herein can alter gene expression by modifying a target sequence. In specific embodiments, the RNA-guided nucleases are directed to the target sequence by a guide RNA (gRNA) as part of a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) RNA-guided nuclease system. The RGNs are considered “RNA-guided” because guide RNAs form a complex with the RNA-guided nucleases to direct the RNA-guided nuclease to bind to a target seque