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US 2024/0247262 A1Published application
Crispr/cas system and method for genome editing and modulating transcription
AssigneeThe Board of Trustees of the University of Illinois
InventorsRyan E. Clarke, Bradley J. Merrill, Matthew S. MacDougall, Hannah M. Pennington, Brian R. Shy
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Abstract
A CRISPR/Cas system and method for editing or regulating transcription of a genome of a cell are provided, wherein the system includes a Cas endonuclease fused with one or more degron sequences and at least one activatable cognate single guide RNA harboring an inactivation sequence in a non-essential region of the cognate sgRNA, wherein said inactivation sequence comprises one or more endonuclease recognition sites of, e.g., a ribozyme.
Claims19
1:
- A CRISPR/Cas system comprising a Cas endonuclease and a cognate single guide RNA (sgRNA), wherein:
- (a) the Cas endonuclease is fused with one or more degron sequences, wherein said degron has:
- (i) a non-canonical Cdc20 or Chd1 recognition motif, or
- (ii) a sequence targeted by a ligase selected from the group of EMI1, TRP1, CBL-PTK, CBL-MET, COP1, CRL4-CDT2, Kelch KEAP1, Kelch KLHL3, MDM2-SWIB, ODPH-VHL, SCF-SKP2, SCF-SKP2-CKS1, SCF-CULLIN, SCF-FBW7, SCF-FBX05, SCF-TRCP1, SCF-CUL4, E6-AP, SIAH, HECT domain family, RING finger family, U box family, and combinations thereof;
- (b) the cognate sgRNA is an activatable sgRNA harboring an inactivation sequence in a non-essential region of the activatable sgRNA, wherein said inactivation sequence comprises one or more endonuclease recognition sites;
- or
- (c) a combination of (a) and (b).
4 more elements
2:
- The system of claim 1, wherein the activatable sgRNA targets a transcribed strand of a nucleic acid molecule.
3:
- The system of claim 1, wherein the inactivation sequence is a cis-acting ribozyme encoded by a nucleic acid molecule of SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4.
4:
- The system of claim 1, wherein the activatable sgRNA is encoded by a nucleic acid molecule of SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21 or SEQ ID NO:22.
5:
- The system of claim 1, further comprising an array of activatable sgRNAs, where the cognate sgRNA targets at least one activatable sgRNA in the array of activatable sgRNAs.
6:
- A fusion protein comprising a Cas endonuclease fused with one or more degron sequences selected from the group of:
- (i) a non-canonical Cdc20 or Chd1 recognition motif, or
- (ii) a sequence targeted by a ligase selected from the group of EMI1, TRP1, CBL-PTK, CBL-MET, COP1, CRL4-CDT2, Kelch KEAP1, Kelch KLHL3, MDM2-SWIB, ODPH-VHL, SCF-SKP2, SCF-SKP2-CKS1, SCF-CULLIN, SCF-FBW7, SCF-FBX05, SCF-TRCP1, SCF-CUL4, E6-AP, SIAH, HECT domain family, RING finger family, U box family, and combinations thereof.
7:
- The fusion protein of claim 6, wherein the one or more degron sequences are fused to the Cas endonuclease with one or more linkers disposed therebetween.
8:
- The fusion protein of claim 6, wherein the one or more degron sequences are fused to the Cas endonuclease at the N-terminus, C-terminus, or N-terminus and C-terminus of the Cas endonuclease.
9:
- An activatable single guide RNA (sgRNA) harboring an inactivation sequence in a non-essential region of the sgRNA, wherein said inactivation sequence comprises one or more endonuclease recognition sites.
10:
- The activatable sgRNA of claim 9, wherein the activatable sgRNA binds a transcribed strand of a target nucleic acid molecule.
11:
- The activatable sgRNA of claim 9, wherein the inactivation sequence is a cis-acting ribozyme encoded by a nucleic acid molecule of SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4.
12:
- The activatable sgRNA of claim 9, wherein the activatable sgRNA is encoded by a nucleic acid molecule of SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21 or SEQ ID NO:22.
13:
- A method of editing or modulating transcription of a genome in a cell comprising introducing into the cell at least one Cas endonuclease and at least one cognate single guide RNA (sgRNA) that targets a nucleic acid sequence of the genome, wherein:
- (a) the Cas endonuclease is fused with one or more degron sequences selected from the group of:
- (i) a non-canonical Cdc20 or Chd1 recognition motif, or
- (ii) a sequence targeted by a ligase selected from the group of EMI1, TRP1, CBL-PTK, CBL-MET, COP1, CRL4-CDT2, Kelch KEAP1, Kelch KLHL3, MDM2-SWIB, ODPH-VHL, SCF-SKP2, SCF-SKP2-CKS1, SCF-CULLIN, SCF-FBW7, SCF-FBX05, SCF-TRCP1, SCF-CUL4, E6-AP, SIAH, HECT domain family, RING finger family, U box family, and combinations thereof;
- (b) the cognate sgRNA is an activatable sgRNA of claim 9;
- or
- (c) a combination of (a) and (b),
- thereby editing or modulating transcription of the genome of the cell.
5 more elements
14:
- The method of claim 13, wherein expression of the Cas endonuclease, activatable sgRNA or Cas endonuclease and activatable sgRNA are controlled by one or more regulatable promoters.
15:
- The method of claim 13, wherein at least two cognate sgRNAs that target two or more sequences in the genome are introduced into the cell.
16:
- The method of claim 15, wherein the at least two cognate sgRNAs are activatable sgRNA, which are sequentially activated to introduce two or more edits in the genome.
17:
- The method of claim 15, wherein the at least two cognate sgRNAs are activatable sgRNA, which are sequentially activated and comprise:
- (i) a first activatable sgRNA which targets nucleic acids encoding a second activatable sgRNA, and
- (ii) the second activatable sgRNA which targets a promoter in the genome.
18:
- The method of claim 13, wherein the at least one cognate sgRNA is inserted into nucleic acids encoding a 5′- or 3′-untranslated region of a gene.
19:
- A nucleic acid comprising a coding sequence, a 5′-untranslated region and a 3′-untranslated region, wherein said nucleic acid has inserted in the 5′-untranslated region or 3′-untranslated region an activatable single guide RNA of claim 9.