Engineered nucleic-acid targeting nucleic acids
The present disclosure provides engineered cross-type-nucleic-acid targeting nucleic acids and compositions thereof. Nucleic acid sequences encoding the engineered cross-type-nucleic-acid targeting nucleic acids, as well as expression cassettes, vectors and cells comprising such nucleic acid sequences, are described. Also, methods are disclosed for making and using the engineered cross-type-nucleic-acid targeting nucleic acids and compositions thereof.
1. An engineered CRISPR Class 2 cross-type-nucleic-acid targeting nucleic acid (“CRISPR Class 2 cross-type-NATNA”), comprising:
a first CRISPR Type V Cpf1-associated nucleic-acid targeting nucleic acid, having a 5′ end and a 3′ end, comprising a spacer element complementary to a first nucleic acid target sequence (“first Cpf1-NATNA”);
a second CRISPR Type V Cpf1-associated nucleic-acid targeting nucleic acid, having a 5′ end and a 3′ end, comprising a spacer element complementary to a second nucleic acid target sequence (“second Cpf1-NATNA”);
a first CRISPR Type II Cas9-associated nucleic-acid targeting nucleic acid, having a 5′ end and a 3′ end, comprising a spacer element complementary to a third nucleic acid target sequence (“first Cas9-NATNA”); and
a second CRISPR Type II Cas9-associated nucleic-acid targeting nucleic acid, having a 5′ end and a 3′ end, comprising a tracr element (“second Cas9-NATNA”);
wherein the 3′ end of the first Cas9-NATNA is covalently connected through a loop element with the 5′ end of the second Cas9-NATNA, resulting in a single-Cas9-associated nucleic-acid targeting nucleic acid (“single-Cas9-NATNA”), having a 5′ end and a 3′ end; and
wherein the single-Cas9-NATNA is connected with the first Cpf1-NATNA and the second Cpf1-NATNA.
2. The engineered CRISPR Class 2 cross-type-NATNA of claim 1 , wherein the single-Cas9-NATNA further comprises a linker element nucleotide sequence covalently connected with the 5′ end, the 3′ end, or the 5′ end and the 3′ end of the single-Cas9-NATNA, and wherein, if the linker element nucleotide sequence is covalently connected with the 5′ end of the single-Cas9-NATNA, the linker element nucleotide sequence is located 5′ of the single-Cas9-NATNA spacer element.
3. The engineered CRISPR Class 2 cross-type-NATNA of claim 2 , wherein, if a first linker element nucleotide sequence is covalently connected to the 5′ end of the single-Cas9-NATNA and a second linker element nucleotide sequence is covalently connected to the 3′ end of the single-Cas9-NATNA,
the first Cpf1-NATNA further comprises a linker element nucleotide sequence covalently connected with the 5′ end or the 3′ end of the first Cpf1-NATNA, and the single-Cas9-NATNA is non-covalently connected with the first Cpf1-NATNA through hydrogen base-pair bonding of the first linker element nucleotide sequence of the single-Cas9-NATNA and the first Cpf1-NATNA linker element nucleotide sequence, and
the second Cpf1-NATNA further comprises a linker element nucleotide sequence covalently connected with the 5′ end or the 3′ end of the second Cpf1-NATNA, and the single-Cas9-NATNA is non-covalently connected with the second Cpf1-NATNA through hydrogen base-pair bonding of the second linker element nucleotide sequence of the single-Cas9-NATNA and the second Cpf1-NATNA linker element nucleotide sequence; and
wherein, if the linker element nucleotide sequence is covalently connected with the 3′ end of the first Cpf1-NATNA, the linker element nucleotide sequence is located 3′ of the first Cpf1-NATNA spacer element, and
wherein, if the linker element nucleotide sequence is covalently connected with the 3′ end of the second Cpf1-NATNA, the linker element nucleotide sequence is located 3′ of the second Cpf1-NATNA spacer element.
4. The engineered CRISPR Class 2 cross-type-NATNA of claim 2 , wherein, if a linker element nucleotide sequence is covalently connected to the 5′ end of the single-Cas9-NATNA,
the first Cpf1-NATNA further comprises a linker element nucleotide sequence covalently connected with the 5′ end or the 3′ end of the first Cpf1-NATNA, and the single-Cas9-NATNA is non-covalently connected with the first Cpf1-NATNA through hydrogen base-pair bonding of the linker element nucleotide sequence of the single-Cas9-NATNA and the first Cpf1-NATNA linker element nucleotide sequence, and
the 5′ end of the second Cpf1-NATNA is covalently connected to the 3′ end of the single-Cas9-NATNA; and
wherein, if the linker element nucleotide sequence is covalently connected with the 3′ end of the first Cpf1-NATNA, the linker element nucleotide sequence is located 3′ of the first Cpf1-NATNA spacer element.
5. The engineered CRISPR Class 2 cross-type-NATNA of claim 2 , wherein, if a linker element nucleotide sequence is covalently connected to the 3′ end of the single-Cas9-NATNA,
the first Cpf1-NATNA further comprises a linker element nucleotide sequence covalently connected with the 5′ end or the 3′ end of the first Cpf1-NATNA, and the single-Cas9-NATNA is non-covalently connected with the first Cpf1-NATNA through hydrogen base-pair bonding of the linker element nucleotide sequence of the single-Cas9-NATNA and the first Cpf1-NATNA linker element nucleotide sequence, and
the 3′ end of the second Cpf1-NATNA is covalently connected to the 5′ end of the single-Cas9-NATNA; and
wherein, if the linker element nucleotide sequence is covalently connected with the 3′ end of the first Cpf1-NATNA, the linker element nucleotide sequence is located 3′ of the first Cpf1-NATNA spacer element.
6. The engineered CRISPR Class 2 cross-type-NATNA of claim 1 , wherein the 5′ end of the single-Cas9-NATNA is covalently connected with the first Cpf1-NATNA, and the 3′ end of the single-Cas9-NATNA is covalently connected with the second Cpf1-NATNA.
7. The engineered CRISPR Class 2 cross-type-NATNA of claim 1 , wherein the first Cpf1-NATNA is capable of forming a complex with a CRISPR Type V Cpf1 protein (“CRISPR Class 2 cross-type-NATNA/Cpf1 protein complex”), and the CRISPR Class 2 cross-type-NATNA/Cpf1 protein complex is capable of binding the first nucleic acid target sequence.
8. The engineered CRISPR Class 2 cross-type-NATNA of claim 7 , wherein the single-Cas9-NATNA is capable of forming a complex with a CRISPR Type II Cas9 protein (“CRISPR Class 2 cross-type-NATNA/Cpf1 protein/Cas9 protein complex”), and the CRISPR Class 2 cross-type-NATNA/Cpf1 protein/Cas9 protein complex is capable of binding the first nucleic acid target sequence and the third nucleic acid target sequence.
9. The engineered CRISPR Class 2 cross-type-NATNA of claim 1 , wherein the second Cpf1-NATNA is capable of forming a complex with a CRISPR Type V Cpf1 protein (“CRISPR Class 2 cross-type-NATNA/Cpf1 protein complex”), and the CRISPR Class 2 cross-type-NATNA/Cpf1 protein complex is capable of binding the second nucleic acid target sequence.
10. The engineered CRISPR Class 2 cross-type-NATNA of claim 9 , wherein the single-Cas9-NATNA is capable of forming a complex with a CRISPR Type II Cas9 protein (“CRISPR Class 2 cross-type-NATNA/Cpf1 protein/Cas9 protein complex”), and the CRISPR Class 2 cross-type-NATNA/Cpf1 protein/Cas9 protein complex is capable of binding the second nucleic acid target sequence and the third nucleic acid target sequence.
11. The engineered CRISPR Class 2 cross-type-NATNA of claim 1 , wherein the single-Cas9-NATNA is capable of forming a complex with a CRISPR Type II Cas9 protein (“CRISPR Class 2 cross-type-NATNA/Cas9 protein complex”), and the CRISPR Class 2 cross-type-NATNA/Cas9 protein complex is capable of binding the third nucleic acid target sequence.
12. The engineered CRISPR Class 2 cross-type-NATNA of claim 1 , wherein at least one of the first Cpf1-NATNA, the second Cpf1-NATNA, or the single-Cas9-NATNA comprises RNA.
13. The engineered CRISPR Class 2 cross-type-NATNA of claim 1 , wherein at least one of the first Cpf1-NATNA, the second Cpf1-NATNA, or the single-Cas9-NATNA comprises DNA.
14. A nucleic acid/protein composition, comprising:
the engineered CRISPR Class 2 cross-type-NATNA of claim 1 ;
a catalytically active or catalytically inactive CRISPR Type II Cas9 protein; and a catalytically active or catalytically inactive CRISPR Type V Cpf1 protein.
15. The nucleic acid/protein composition of claim 14 , wherein the engineered CRISPR Class 2 cross-type-NATNA is in a complex with the CRISPR Type II Cas9 protein and the CRISPR Type V Cpf1 protein.
16. The nucleic acid/protein composition of claim 15 , wherein the CRISPR Type V Cpf1 protein is catalytically inactive, the CRISPR Type II Cas9 protein is catalytic ally inactive, or the CRISPR Type V Cpf1 protein and CRISPR Type II Cas9 protein are catalytically inactive.
17. A cell comprising the nucleic acid/protein composition of claim 14 .
18. The cell of claim 17 , wherein the cell is a eukaryotic cell.
19. The cell of claim 18 , wherein the eukaryotic cell is selected from the group consisting of a single-cell eukaryotic organism, a protozoa cell, a cell from a plant, an algal cell, a fungal cell, a cell from an invertebrate animal, a cell from a vertebrate animal, and a cell from a mammal.
20. The cell of claim 17 , wherein the cell is a bacterial cell or an archaeal cell.