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 Cpf1 nucleic acid target sequence that is in proximity to a Cpf1 PAM (“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 Cas9 nucleic acid target sequence that is in proximity to a Cas9 PAM (“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 is covalently connected with the Cpf1-NATNA.
2. The engineered CRISPR Class 2 cross-type-NATNA of claim 1 , wherein the Cpf1-NATNA is covalently connected with the 5′ end of the first 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 Cas9-Cpf1-NATNA/Cpf1 protein complex with the CRISPR Type V Cpf1 protein, the Cas9-Cpf1-NATNA/Cpf1 protein 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 Cas9-Cpf1-NATNA/Cas9 protein complex with the CRISPR Type II Cas9 protein, the Cas9-Cpf1-NATNA/Cas9 protein 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 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.
6. The engineered CRISPR Class 2 cross-type-NATNA of claim 5 , 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 wherein, 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.
7. 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.
8. The engineered CRISPR Class 2 cross-type-NATNA of claim 7 , wherein the single-Cas9-NATNA is covalently connected with the Cpf1-NATNA; and wherein the Cpf1-NATNA is covalently connected with the 5′ end of the single-Cas9-NATNA.
9. The engineered CRISPR Class 2 cross-type-NATNA of claim 7 , 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.
10. The engineered CRISPR Class 2 cross-type-NATNA of claim 7 , 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.
11. The engineered CRISPR Class 2 cross-type-NATNA of claim 1 , wherein the Cpf1 target sequence is in proximity to the Cas9 PAM.
12. The engineered CRISPR Class 2 cross-type-NATNA of claim 11 , wherein the Cas9 target sequence is in proximity to the Cpf1 PAM.
13. The engineered CRISPR Class 2 cross-type-NATNA of claim 1 , wherein the Cas9 target sequence is in proximity to the Cpf1 PAM.
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 ; and
a catalytically active or a catalytically inactive CRISPR Type II Cas9 protein, and/or
a catalytically active or a 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 catalytically active CRISPR Type II Cas9 protein or the catalytically inactive CRISPR Type II Cas9 protein.
18. The nucleic acid/protein composition of claim 16 , wherein the engineered CRISPR Class 2 cross-type-NATNA is in a complex with the catalytically active CRISPR Type V Cpf1 protein or the catalytically inactive CRISPR Type V Cpf1 protein.
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 protein and a CRISPR Type V Cpf1 protein.