Compact promoters for gene editing
AssigneeHunterian Medicine LLC
InventorVinod Jaskula-Ranga
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Abstract
The invention relates generally to compact promoters and their use in gene editing e.g., for treating disease. The disclosure is based, in part, upon the discovery of compact, bidirectional promoters that can be used to express both a nuclease (e.g., a Cas9 nuclease) and a guide RNA (gRNA). For example, in certain embodiments disclosed herein, a compact, bidirectional promoter can comprise at least one regulatory element that directs expression of a gRNA in one direction and at least one regulatory element that directs expression of a nuclease in the other direction. Accordingly, the promoters disclosed herein use less space than prior art promoters, allowing both a nuclease and a gRNA to be packaged in a single vector (e.g., a plasmid or an AAV).
Claims90
A non-naturally occurring nuclease system comprising a vector comprising a compact bidirectional promoter, wherein the compact bidirectional promoter comprises:
- a) at least one regulatory element that provides for transcription in one direction of at least one nucleotide sequence encoding a guide RNA (gRNA), wherein the gRNA hybridizes with a target sequence of a nucleic acid;
- and b) at least one regulatory element that provides for transcription in the opposite direction of a nucleotide sequence encoding a nuclease, wherein the gRNA targets and hybridizes with the target sequence and directs the nuclease to the nucleic acid, wherein the bidirectional promoter is not one or more of SEQ ID NO:
- 70-SEQ ID NO:
- 106 and SEQ ID NO:
- 241-SEQ ID NO:
- 255.
The system of any preceding claim, wherein the bidirectional promoter comprises a nucleic acid sequence selected from SEQ ID NOs:
- 107-255 or the portion of any one of SEQ ID NOs:
- 25-106, 469-476, 559-564, 609-614, 673-678, 681, 692-697, 706-711, 719-724, 729-734, 748-753, 784-789, 904-909, 920-925, 936-1303, or any sequence in FIGS. 3-19 that corresponds to an H1 promoter (e.g., from about nucleotide 20 to about nucleotide 490 as numbered in FIG. 3), or a nucleic acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or at least about 99.5% identity thereto.
The system of any preceding claim, wherein the compact bidirectional promoter comprises an H1 promoter.
The system of any preceding claim, wherein the compact promoter comprises at least 95%, 98%, 99%, 99.5% or 100% identity to a naturally-occurring mammalian promoter.
The system of any preceding claim, wherein the target sequence comprises the nucleotide sequence AN19NGG, GN19NGG, CN19NGG, or TN19NGG.
The system of any preceding claim, wherein the nuclease is a nuclease-dead nuclease.
The system of any preceding claim, wherein the nuclease is an RNA-directed nuclease.
The system of any preceding claim, wherein the system is packaged into a single vector.
An expression construct comprising the system of any preceding claim.
A method, the method comprising introducing into a cell a non-naturally occurring nuclease system comprising a vector comprising a compact bidirectional promoter, wherein the compact bidirectional promoter comprises:
- a) at least one regulatory element that provides for transcription in one direction of at least one nucleotide sequence encoding a guide RNA (gRNA), wherein the gRNA hybridizes with a target sequence of a nucleic acid molecule;
- and b) at least one regulatory element that provides for transcription in the opposite direction of a nucleotide sequence encoding a nuclease, wherein the gRNA targets and hybridizes with the target sequence and directs the nuclease to the nucleic acid molecule, wherein the bidirectional promoter is not one or more of SEQ ID NO:
- 70-SEQ ID NO:
- 106 and SEQ ID NO:
- 241-SEQ ID NO:
- 255.
A non-naturally occurring nuclease system comprising a vector comprising a compact bidirectional promoter, wherein the compact bidirectional promoter comprises both RNA pol II and RNA pol III activity, wherein a) the promoter provides for transcription of at least one nucleotide sequence encoding a guide RNA (gRNA), wherein the gRNA hybridizes with a target sequence of a nucleic acid;
- and b) the promoter provides for transcription of a nucleotide sequence encoding a nuclease, wherein the gRNA targets and hybridizes with the target sequence and directs the nuclease to the nucleic acid, wherein the bidirectional promoter is not one or more of SEQ ID NO:
- 70-SEQ ID NO:
- 106 and SEQ ID NO:
- 241-SEQ ID NO:
- 255.
The system of any preceding claim, wherein the bidirectional promoter comprises a nucleic acid sequence selected from SEQ ID NOs:
- 107-255 or the portion of any one of SEQ ID NOs:
- 25-106, 469-476, 559-564, 609-614, 673-678, 681, 692-697, 706-711, 719-724, 729-734, 748-753, 784-789, 904-909, 920-925, 936-1303, or any sequence in FIGS. 3-19 that corresponds to an H1 promoter (e.g., from about nucleotide 20 to about nucleotide 490 as numbered in FIG. 3), or a nucleic acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or at least about 99.5% identity thereto.
The system of any preceding claim, wherein the compact bidirectional promoter comprises an H1 promoter.
A method, the method comprising introducing into a cell a non-naturally occurring nuclease system comprising a vector comprising a compact bidirectional promoter, wherein the compact bidirectional promoter comprises both RNA pol II and RNA pol III activity, wherein a) the promoter provides for transcription of at least one nucleotide sequence encoding a guide RNA (gRNA), wherein the gRNA hybridizes with a target sequence of a nucleic acid;
- and b) the promoter provides for transcription of a nucleotide sequence encoding a nuclease, wherein the gRNA targets and hybridizes with the target sequence and directs the nuclease to the nucleic acid, wherein the bidirectional promoter is not one or more of SEQ ID NO:
- 70-SEQ ID NO:
- 106 and SEQ ID NO:
- 241-SEQ ID NO:
- 255.
Dependencies are inferred from the claim text.