Using nucleosome interacting protein domains to enhance targeted genome modification

Patent metadata

Field	Value
Publication number	US-10604752-B2
Application number	US-201816031819-A
Country	US
Kind code	B2
Filing date	Jul 10, 2018
Priority date	Jul 11, 2017
Publication date	Mar 31, 2020
Grant date	Mar 31, 2020

Abstract

Compositions and methods for using nucleosome interacting protein domains to increase accessibility of programmable DNA modification proteins to target chromosomal sequences, thereby increasing efficiency of targeted genome/epigenetic modification in eukaryotic cells.

First claim

What is claimed is:

1. A fusion protein comprising a clustered regularly interspersed short palindromic repeats (CRISPR) protein linked to at least one nucleosome interacting protein domain, wherein the CRISPR protein is a type II CRISPR/Cas9 nuclease from Streptococcus pyogenes Cas9 (SpCas9), Streptococcus thermophilus Cas9 (StCas9), Streptococcus pasteurianus (SpaCas9), Campylobacter jejuni Cas9 (CjCas9), Francisella novicida Cas9 (FnCas9), or Neisseria cinerea Cas9 (NcCas9), and the at least one nucleosome interacting protein domain 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:45, or a combination thereof, and wherein the at least one nucleosome interacting protein domain is linked to the CRISPR protein at its N-terminus, C-terminus, or a combination thereof.

2. The fusion protein of claim 1 , wherein the at least one nucleosome interacting protein domain is HMGB1 box A domain, HMGN1 protein, HMGN2 protein, HMGN3a protein, HMGN3b protein, a central globular domain from a histone H1 variant comprising SEQ ID NO: 45, or a combination thereof.

3. The fusion protein of claim 1 , wherein the at least one nucleosome interacting protein domain is linked to the CRISPR protein directly via a chemical bond, indirectly via a linker, or a combination thereof.

4. The fusion protein of claim 1 , further comprising at least one nuclear localization signal, at least one cell-penetrating domain, at least one marker domain, or a combination thereof.

5. The fusion protein of claim 1 , wherein the fusion protein comprises the amino acid sequence as set forth in SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:72, or SEQ ID NO:73.

6. The fusion protein of claim 1 , wherein the fusion protein comprises the amino acid sequence as set forth in SEQ ID NO:78 or SEQ ID NO:79.

7. The fusion protein of claim 1 , wherein the type II CRISPR/Cas9 nuclease is from Streptococcus pyogenes Cas9 (SpCas9).

8. The fusion protein of claim 1 , wherein the type II CRISPR/Cas9 nuclease is from Streptococcus thermophilus Cas9 (StCas9).

9. The fusion protein of claim 1 , wherein the type II CRISPR/Cas9 nuclease is from Streptococcus pasteurianus (SpaCas9).

10. The fusion protein of claim 1 , wherein the type II CRISPR/Cas9 nuclease is from Campylobacter jejuni Cas9 (CjCas9).

11. The fusion protein of claim 1 , wherein the type II CRISPR/Cas9 nuclease is from Francisella novicida Cas9 (FnCas9).

12. The fusion protein of claim 1 , wherein the type II CRISPR/Cas9 nuclease is from Neisseria cinerea Cas9 (NcCas9).

13. A protein-RNA complex comprising at least one fusion protein of claim 1 and at least one guide RNA.

14. A nucleic acid encoding a fusion protein comprising a clustered regularly interspersed short palindromic repeats (CRISPR) protein linked to at least one nucleosome interacting protein domain, wherein the CRISPR protein is a type II CRISPR/Cas9 nuclease from Streptococcus pyogenes Cas9 (SpCas9), Streptococcus thermophilus Cas9 (StCas9), Streptococcus pasteurianus (SpaCas9), Campylobacter jejuni Cas9 (CjCas9), Francisella novicida Cas9 (FnCas9), or Neisseria cinerea Cas9 (NcCas9), and the at least one nucleosome interacting protein domain 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: 45, or a combination thereof, and wherein the at least one nucleosome interacting protein domain is linked to the CRISPR protein at its N-terminus, C-terminus, or a combination thereof.

15. The nucleic acid of claim 14 , which is codon optimized for translation in a eukaryotic cell.

16. The nucleic acid of claim 15 , which is part of a viral vector, a plasmid vector, or a self-replicating RNA.

17. A fusion protein comprising a clustered regularly interspersed short palindromic repeats (CRISPR) protein linked to at least one nucleosome interacting protein domain, wherein the CRISPR protein is a type II CRISPR/Cas9 protein from Streptococcus pyogenes Cas9 (SpCas9), Streptococcus thermophilus Cas9 (StCas9), Streptococcus pasteurianus (SpaCas9), Campylobacter jejuni Cas9 (CjCas9), Francisella novicida Cas9 (FnCas9), or Neisseria cinerea Cas9 (NcCas9), modified to lack all nuclease activity and linked to a non-nuclease domain and the at least one nucleosome interacting protein domain 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: 45, or a combination thereof, and wherein the at least one nucleosome interacting protein domain is linked to the CRISPR protein at its N-terminus, C-terminus, or a combination thereof.

18. The fusion protein of claim 17 , wherein the non-nuclease domain has cytosine deaminase activity, histone acetyltransferase activity, transcriptional activation activity, or transcriptional repressor activity.

19. A protein-RNA complex comprising at least one fusion protein of claim 17 , and at least one guide RNA.

20. A nucleic acid encoding a fusion protein comprising a clustered regularly interspersed short palindromic repeats (CRISPR) protein linked to at least one nucleosome interacting protein domain, wherein the CRISPR protein is a type II CRISPR/Cas9 protein from Streptococcus pyogenes Cas9 (SpCas9), Streptococcus thermophilus Cas9 (StCas9), Streptococcus pasteurianus (SpaCas9), Campylobacter jejuni Cas9 (CjCas9), Francisella novicida Cas9 (FnCas9), or Neisseria cinerea Cas9 (NcCas9), modified to lack all nuclease activity and linked to a non-nuclease domain and the at least one nucleosome interacting protein domain 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: 45, or a combination thereof, and wherein the at least one nucleosome interacting protein domain is linked to the CRISPR protein at its N-terminus, C-terminus, or a combination thereof.

21. The nucleic acid of claim 20 , which is codon optimized for translation in a eukaryotic cell.

22. The nucleic acid of claim 21 , which is part of a viral vector, a plasmid vector, or a self-replicating RNA.

Assignees

• Sigma Aldrich Co Llc

Inventors

• Chen Fuqiang
• Ding Xiao
• Feng Yongmei
• Davis Gregory D

Classifications

• C07K2319/80

  containing a DNA binding domain, e.g. Lacl or Tet-repressor · CPC title

• C07K2319/10

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

• C12N9/22 Primary

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title

• C12N15/907

  in mammalian cells · CPC title

• C12N15/85

  for animal cells · CPC title