Engineered CAS9 systems for eukaryotic genome modification

Patent metadata

Field	Value
Publication number	US-12297449-B2
Application number	US-202016943813-A
Country	US
Kind code	B2
Filing date	Jul 30, 2020
Priority date	Feb 15, 2018
Publication date	May 13, 2025
Grant date	May 13, 2025

Abstract

Engineered Cas9 systems that utilize alternate protospacer adjacent motifs for target DNA binding, nucleic acids encoding the engineered Cas9 systems, and methods of using the engineered Cas9 systems for modifying target chromosomal sequences in eukaryotic cells.

First claim

What is claimed is:

1. A system for eukaryotic genome modification comprising (a) an engineered Lactobacillus rhamnosus Cas9 protein comprising a nuclear localization signal (NLS), and (b) an engineered guide RNA, wherein the engineered guide RNA is designed to complex with the engineered Lactobacillus rhamnosus Cas9 protein and the engineered guide RNA comprises a 5′ guide sequence designed to hybridize with a target sequence in a double-stranded sequence, and wherein the target sequence is 5′ to a protospacer adjacent motif (PAM) comprising the sequence 5′-NGAAA-3′, wherein N is A, C, G, or T, and
wherein said system comprises (1) (a) the modified engineered Cas9 protein Lactobacillus rhamnosus Cas9 (LrhCas9)/NLS protein comprising the amino acid sequence of SEQ ID NO: 4; (b) an engineered guide RNA encoded by the nucleotide sequence of SEQ ID NO: 32 designed to hybridize to the target sequence 5′ to the PAM sequence, or
(2)(a) the modified engineered Cas9 protein comprising the amino acid sequence of SEQ ID NO: 121 or SEQ ID NO: 122; and (b) an engineered guide RNA designed to hybridize to the target sequence 5′ to the PAM sequence.

2. The system of claim 1 , wherein the engineered Cas9 protein further comprises at least one heterologous domain and the at least one heterologous domain is a cell-penetrating domain, a marker domain, a chromatin modulating motif, an epigenetic modification domain, a transcriptional regulation domain, an RNA aptamer binding domain, or combination thereof.

3. The system of claim 1 , wherein the engineered Cas9 protein further comprises at least one modification within a RuvC domain, HNH domain, or combination thereof.

4. The system of claim 1 , wherein the engineered Cas9 protein further comprises a chromatin modulating motif.

5. The system of claim 1 , wherein the engineered Cas9 protein comprises at least one modification within a RuvC domain or an HNH domain, such that the engineered Cas9 protein functions as a nickase, and wherein the engineered Cas9 protein further comprises a cell-penetrating domain, a marker domain, an RNA aptamer binding domain, or combination thereof.

6. The system of claim 1 , wherein the engineered Cas9 protein comprises at least one modification within a RuvC domain and an HNH domain, such that the engineered Cas9 protein is catalytically inactive, and wherein the engineered Cas9 protein is modified to add a chromatin modulating motif, an epigenetic modification domain, a transcriptional regulation domain or combination thereof.

7. A plurality of nucleic acids encoding a system for eukaryotic genome modification comprising (a) an engineered Lactobacillus rhamnosus Cas9 protein comprising a nuclear localization signal (NLS); and (b) an engineered guide RNA, wherein the engineered guide RNA is designed to complex with the engineered Lactobacillus rhamnosus Cas9 protein and the engineered guide RNA comprises a 5′ guide sequence designed to hybridize with a target sequence in a double-stranded sequence, and wherein the target sequence is 5′ to a protospacer adjacent motif (PAM) comprising the sequence 5′-NGAAA-3′, wherein N is A, C, G, or T; the plurality of nucleic acids comprising at least one nucleic acid encoding the engineered Lactobacillus rhamnosus Cas9 protein, and at least one nucleic acid encoding the engineered guide RNA, wherein the engineered guide RNA comprises the nucleotide sequence of SEQ ID NO:32.

8. The plurality of nucleic acids of claim 7 , wherein the at least one nucleic acid encoding the engineered Lactobacillus rhamnosus Cas9 protein is RNA.

9. The plurality of nucleic acids of claim 7 , wherein the at least one nucleic acid encoding the engineered Lactobacillus rhamnosus Cas9 protein is DNA.

10. The plurality of nucleic acids of claim 7 , wherein the at least one nucleic acid encoding the engineered Lactobacillus rhamnosus Cas9 protein is codon optimized for expression in a eukaryotic cell.

11. The plurality of nucleic acids of claim 7 , wherein the codon optimized for expression in a eukaryotic cell is a human cell, a non-human mammalian cell, a non-mammalian vertebrate cell, an invertebrate cell, a plant cell, or a single cell eukaryotic organism.

12. The plurality of nucleic acids of claim 7 , wherein the at least one nucleic acid encoding the engineered guide RNA is DNA.

13. The plurality of nucleic acids of claim 7 , wherein the at least one nucleic acid encoding the engineered Lactobacillus rhamnosus Cas9 protein is operably linked to a phage promoter sequence for in vitro RNA synthesis or protein expression in a bacterial cell, and the at least one nucleic acid encoding the engineered guide RNA is operably linked to a phage promoter sequence for in vitro RNA synthesis.

14. The plurality of nucleic acids of claim 7 , wherein the at least one nucleic acid encoding the engineered Lactobacillus rhamnosus Cas9 protein is operably linked to a eukaryotic promoter sequence for expression in a eukaryotic cell, and the at least one nucleic acid encoding the engineered guide RNA is operably linked to a eukaryotic promoter sequence for expression in a eukaryotic cell.

15. At least one vector comprising the plurality of nucleic acids of claim 7 , wherein the at least one vector is a plasmid vector, a viral vector, or a self-replicating viral RNA replicon.

16. A eukaryotic cell comprising at least one system comprising (a) an engineered Lactobacillus rhamnosus Cas9 protein comprising a nuclear localization signal (NLS); and (b) an engineered guide RNA, wherein the engineered guide RNA is designed to complex with the engineered Lactobacillus rhamnosus Cas9 protein and the engineered guide RNA comprises a 5′ guide sequence designed to hybridize with a target sequence in a double-stranded sequence, and wherein the target sequence is 5′ to a protospacer adjacent motif (PAM) comprising the sequence 5′-NGAAA-3′, wherein N is A, C, G, or T, wherein the eukaryotic cell is a non-human mammalian cell, a plant cell, a nonmammalian vertebrate cell, an invertebrate cell, or a single cell eukaryotic organism and wherein the eukaryotic cell is in vivo, ex vivo, or in vitro, and, wherein the engineered guide RNA comprises the nucleotide sequence of SEQ ID NO:32.

17. A human eukaryotic cell comprising at least one system comprising (a) an engineered Lactobacillus rhamnosus Cas9 protein comprising a nuclear localization signal (NLS); and (b) an engineered guide RNA, wherein the engineered guide RNA is designed to complex with the engineered Lactobacillus rhamnosus Cas9 protein and the engineered guide RNA comprises a 5′ guide sequence designed to hybridize with a target sequence in a double-stranded sequence, and wherein the target sequence is 5′ to a protospacer adjacent motif (PAM) comprising the sequence 5′-NGAAA-3′, wherein the human eukaryotic cell is ex vivo, or in vitro, and, wherein the engineered guide RNA comprises the nucleotide sequence of SEQ ID NO:32.

18. A fusion protein comprising an engineered Lactobacillus rhamnosus Cas9 protein linked to at least one chromatin modulating motif,
wherein the chromatin modulating motif is a high mobility group (HMG) box (HMGB) DNA binding domain, a HMG nucleosome-binding (HMGN) protein, a central globular domain from a histone H1 variant comprising SEQ ID NO: 74, or a combination thereof,
wherein the at least one chromatin modulating motif is linked to the engineered Lactobacillus rhamnosus Cas9 protein protein at its N-terminus, C-terminus, or a combination thereof,
wherein, together with an engineered guide RNA designed to complex with t

Assignees

• Sigma Aldrich Co Llc

Inventors

• Seebeck Timothy
• Chen Fuqiang
• Davis Gregory D

Classifications

• C07K2319/09

  containing a nuclear localisation signal · CPC title

• C07K2319/85

  containing an RNA binding domain · CPC title

• C12N15/63

  Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression · CPC title

• C07K2319/10

  containing a tag for extracellular membrane crossing, e.g. TAT or VP22 · CPC title

• C07K2319/00

  Fusion polypeptide · CPC title