CRISPR/CAS9-BASED FUSION PROTEINS FOR MODULATING GENE EXPRESSION AND METHODS OF USE
The present disclosure generally relates to methods and compositions used for modulating or controlling gene expression involving sequence targeting, genome perturbation or gene-editing, that relate to Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and components thereof. In some embodiments, compositions comprising a catalytically inactive Cas9 (dCas9) fusion protein and methods for modulating expression of a gene of interest are disclosed.
1 . A fusion protein, comprising:
a dCas9 protein; and
two or more repressor domains selected from the group consisting of:
(a) a Krüppel-associated box domain of ZIM3 gene (ZIM3 KRAB domain);
(b) a transcription repression domain of methyl-CpG binding protein 2 (MeCP2 domain); and
(c) a transcription repression domain of Friend of GATA1 (FOG1 domain).
2 . The fusion protein according to claim 1 , further comprising one or more nuclear localization sequences (NLSs).
3 . The fusion protein according to claim 1 , further comprising a fluorescent marker (FM).
4 . The fusion protein according to claim 1 , wherein the fusion protein is a ZIM3 KRAB-MeCP2-NLS-dCas9 fusion protein comprising from the N-terminus to the C-terminus: ZIM3 KRAB domain, MeCP2 domain, a NLS, and a dCas9 protein.
5 . The fusion protein according to claim 1 , wherein the fusion protein is a ZIM3 KRAB-NLS-dCas9-FOG1 fusion protein comprising from the N-terminus to the C-terminus: ZIM3 KRAB domain, a NLS, a dCas9 protein, and FOG1 domain, or wherein the fusion protein is a ZIM3 KRAB-NLS-dCas9-NLS-FOG1 fusion protein comprising from the N-terminus to the C-terminus: ZIM3 KRAB domain, a NLS, a dCas9 protein, a NLS, and FOG1 domain.
6 . The fusion protein according to claim 1 , wherein the fusion protein is a MeCP2-NLS-dCas9-FOG1 fusion protein comprising from the N-terminus to the C-terminus: MeCP2 domain, a NLS, a dCas9 protein, and FOG1 domain, or wherein the fusion protein is a MeCP2-NLS-dCas9-NLS-FOG1 fusion protein comprising from the N-terminus to the C-terminus: MeCP2 domain, a NLS, a dCas9 protein, a NLS, and FOG1 domain.
7 . The fusion protein according to claim 1 , wherein the fusion protein is a ZIM3 KRAB-MeCP2-NLS-dCas9-NLS-FOG1 fusion protein comprising from the N-terminus to the C-terminus: ZIM3 KRAB domain, MeCP2 domain, a NLS, a dCas9 protein, a NLS, and FOG1 domain.
8 . The fusion protein according to claim 3 , wherein the fusion protein is a ZIM3 KRAB-MeCP2-NLS-dCas9-NLS-FM-NLS-FOG1 fusion protein comprising from the N-terminus to the C-terminus: ZIM3 KRAB domain, MeCP2 domain, a NLS, a dCas9 protein, a NLS, a FM, a NLS, and FOG1 domain, optionally wherein the FM comprises mTagBFP.
9 . The fusion protein according to claim 1 , further comprising one or more linkers.
10 . The fusion protein according to claim 1 , wherein the dCas9 protein comprises at least one domain selected from the group consisting of: a Rec1 domain, a bridge helix domain, and a protospacer adjacent motif interacting domain.
11 . The fusion protein according to claim 1 , wherein the dCas9 protein comprises a D10A mutation in a RuvC1 domain and a H840A mutation in a HNH domain.
12 . The fusion protein according to claim 1 , wherein the dCas9 protein comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:16; and/or wherein ZIM3 KRAB domain comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:14; and/or wherein MeCP2 domain comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:15; and/or wherein FOG1 domain comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:17; and/or wherein mTagBFP comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:18; and/or wherein the NLS comprises at least one sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 19 and/or SEQ ID NO:20.
13 . The fusion protein according to claim 1 , wherein the fusion protein comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:13, or an amino acid sequence having one, two, three, four, five or more amino acid substitutions, insertions, or deletions relative to SEQ ID NO:13.
14 . A polynucleotide encoding the fusion protein according to claim 1 .
15 . The polynucleotide according to claim 14 , wherein the polynucleotide comprises a sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 7, or a sequence having one, two, three, four, five or more substitutions, insertions, or deletions relative to SEQ ID NO:7; and/or wherein the polynucleotide encoding the dCas9 protein comprises a sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:10; and/or wherein the polynucleotide encoding ZIM3 KRAB domain comprises a sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:8; and/or wherein the polynucleotide encoding MeCP2 domain comprises a sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:9; and/or wherein the polynucleotide encoding FOG1 domain comprises a sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:11; and/or wherein the polynucleotide encoding mTagBFP comprises a sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 12.
16 . A method of repressing expression of a gene in a cell, comprising contacting the cell with an effective amount of:
(a) the fusion protein according to claim 1 or the polynucleotide according to claim 14 ; and
(b) one or more gRNAs that bind the dCas 9 protein according to claim 1 .
17 . The method according to claim 16 , wherein the one or more gRNAs comprises a sequence having sufficient complementarity with a target polynucleotide sequence within the gene, and/or wherein the one or more gRNAs are capable of hybridizing with the target polynucleotide sequence.
18 - 21 . (canceled)
22 . A viral vector, comprising the polynucleotide according to claim 1 , and optionally further comprising one or more gRNAs that bind the dCas9 protein according claim 1 .
23 . The viral vector according to claim 22 , wherein the one or more gRNAs comprises a sequence having sufficient complementarity with a target polynucleotide sequence within the gene, and/or wherein the one or more gRNAs are capable of hybridizing with the target polynucleotide sequence.
24 . The viral vector according to claim 22 , wherein the viral vector comprises a lentiviral vector.