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 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 (“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 second 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 first Cas9-NATNA or the second Cas9-NATNA is connected with the Cpf1-NATNA.
2. The engineered CRISPR Class 2 cross-type-NATNA of claim 1 , wherein the first Cas9-NATNA or the second Cas9-NATNA is covalently connected with the Cpf1-NATNA; and wherein the Cpf1-NATNA is covalently connected with the 5′ end or the 3′ end of the first Cas9-NATNA or to the 5′ end or the 3′ end of the second Cas9-NATNA.
3. The engineered CRISPR Class 2 cross-type-NATNA of claim 2 , wherein the Cpf1-NATNA is capable of forming a first complex with a CRISPR Type V Cpf1 protein (“Cas9-Cpf1-NATNA/Cpf1 protein complex”); and wherein the first Cas9-NATNA and the second Cas9-NATNA are capable of forming a second complex with a CRISPR Type II Cas9 protein (“Cas9-Cpf1-NATNA/Cas9 protein complex”).
4. The engineered CRISPR Class 2 cross-type-NATNA of claim 3 , wherein if the Cpf1-NATNA forms the first complex with the CRISPR Type V Cpf1 protein, the first complex is capable of binding a first double-stranded nucleic acid target sequence complementary to the Cpf1 spacer element; and wherein if the first Cas9-NATNA and the second Cas9-NATNA form the second complex with the CRISPR Type II Cas9 protein, the second complex is capable of binding a second double-stranded nucleic acid target sequence complementary to the first Cas9-NATNA spacer element.
5. The engineered CRISPR Class 2 cross-type-NATNA of claim 3 , wherein if the first complex is formed and the second complex is formed, resulting in a Cas9-Cpf1-NATNA/Cas9&Cpf1 protein complex, the Cas9-Cpf1-NATNA/Cas9&Cpf1 protein complex is capable of binding a first double-stranded nucleic acid target sequence complementary to the Cpf1 spacer element and a second double-stranded nucleic acid target sequence complementary to the first Cas9-NATNA spacer element.
6. The engineered CRISPR Class 2 cross-type-NATNA of claim 1 , wherein the Cpf1-NATNA further comprises a linker element nucleotide sequence covalently connected with the 5′ end or the 3′ end of the Cpf1-NATNA; and wherein if the linker element nucleotide sequence is covalently connected with the 3′ end of the Cpf1-NATNA, the linker element nucleotide sequence is located 3′ of the Cpf1-NATNA spacer element.
7. The engineered CRISPR Class 2 cross-type-NATNA of claim 6 , wherein the first Cas9-NATNA further comprises a linker element nucleotide sequence covalently connected with the 5′ end or the 3′ end of the first Cas9-NATNA, and if the linker element nucleotide sequence is covalently connected with the 5′ end of the first Cas9-NATNA, the linker element nucleotide sequence is located 5′ of the first Cas9-NATNA spacer element, or the second Cas9-NATNA further comprises a linker element nucleotide sequence covalently connected with the 5′ end or the 3′ end of the second Cas9-NATNA.
8. The engineered CRISPR Class 2 cross-type-NATNA of claim 7 , wherein the first Cas9-NATNA or the second Cas9-NATNA is non-covalently connected with the Cpf1-NATNA through hydrogen base-pair bonding of the linker element nucleotide sequence of the first Cas9-NATNA or of the linker element nucleotide sequence of the second Cas9-NATNA with the Cpf1-NATNA linker element nucleotide sequence.
9. The engineered CRISPR Class 2 cross-type-NATNA of claim 1 , 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 comprises the first Cas9-NATNA and the second Cas9-NATNA.
10. The engineered CRISPR Class 2 cross-type-NATNA of claim 9 , wherein (a) the single-Cas9-NATNA further comprises a linker element nucleotide sequence covalently connected with the 5′ end or the 3′ end of the single-Cas9-NATNA; 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, (b) the Cpf1-NATNA further comprises a linker element nucleotide sequence covalently connected with the 5′ end or the 3′ end of the Cpf1-NATNA; and wherein if the linker element nucleotide sequence is covalently connected with the 3′ end of the Cpf1-NATNA, the linker element nucleotide sequence is located 3′ of the Cpf1-NATNA spacer element, and (c) the single-Cas9-NATNA is non-covalently connected with the Cpf1-NATNA through hydrogen base-pair bonding of the single-Cas9-NATNA linker element nucleotide sequence with the Cpf1-NATNA linker element nucleotide sequence.
11. The engineered CRISPR Class 2 cross-type-NATNA of claim 9 , wherein the single-Cas9-NATNA is covalently connected with the Cpf1-NATNA; and wherein the Cpf1-NATNA is covalently connected with the 5′ end or the 3′ end of the single-Cas9-NATNA.
12. The engineered CRISPR Class 2 cross-type-NATNA of claim 9 , wherein the single-Cas9-NATNA comprises a linker element nucleotide sequence covalently connected with the 5′ end or 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.
13. The engineered CRISPR Class 2 cross-type-NATNA of claim 9 , wherein the Cpf1-NATNA comprises a linker element nucleotide sequence covalently connected with the 5′ end or the 3′ end of the Cpf1-NATNA; and wherein if the linker element nucleotide sequence is covalently connected with the 3′ end of the Cpf1-NATNA, the linker element nucleotide sequence is located 3′ of the Cpf1-NATNA spacer element.
14. The engineered CRISPR Class 2 cross-type-NATNA of claim 1 , wherein at least one of the Cpf1-NATNA, the first Cas9-NATNA, or the second Cas9-NATNA comprises RNA.
15. The engineered CRISPR Class 2 cross-type-NATNA of claim 1 , wherein at least one of the Cpf1-NATNA, the first Cas9-NATNA, or the second Cas9-NATNA comprises DNA.
16. 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.
17. The nucleic acid/protein composition of claim 16 , 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.
18. The nucleic acid/protein composition of claim 17 , wherein the CRISPR Type V Cpf1 protein, the CRISPR Type II Cas9 protein, or the CRISPR Type V Cpf1 protein and CRISPR Type II Cas9 protein are catalytically inactive.
19. A kit, comprising:
the engineered CRISPR Class 2 cross-type-NATNA of claim 1 and a buffer.
20. The kit of claim 19 , further comprising a CRISPR Type II Cas9 protein, a CRISPR Type V Cpf1 protein, or both a CRISPR Type II Cas9 and a CRISPR Type V Cpf1 protein.