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. A method of binding a first nucleic acid target sequence, a second nucleic acid target sequence, and a third nucleic acid target sequence, comprising:
contacting the first nucleic acid target sequence, the second nucleic acid target sequence, and the third nucleic acid target sequence with a nucleic acid/protein composition comprising
a catalytically active or catalytically inactive CRISPR Type II Cas9 protein;
a first catalytically active or catalytically inactive CRISPR Type V Cpf1 protein;
a second catalytically active or catalytically inactive CRISPR Type V Cpf1 protein; and
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 first spacer element complementary to the 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 second spacer element complementary to the 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 third spacer element complementary to the 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;
thereby facilitating binding of the nucleic acid/protein composition to the first nucleic acid target sequence, the second nucleic acid target sequence, and the third nucleic acid target sequence; and wherein the method is carried out in vitro, in a cultured cell, or in a non-human subject.
2. The method of claim 1 , wherein at least one of the Cpf1-NATNA, the first Cas9-NATNA, or the second Cas9-NATNA comprises RNA.
3. The method of claim 2 , wherein at least one of the Cpf1-NATNA, the first Cas9-NATNA, or the second Cas9-NATNA comprises DNA.
4. The method of claim 1 , wherein
the first CRISPR Type V Cpf1 protein and the second CRISPR Type V Cpf1 protein are both catalytically active and the CRISPR Type II Cas9 protein is catalytically inactive, and the binding of the nucleic acid/protein composition to the first nucleic acid target sequence, the second nucleic acid target sequence, and the third nucleic acid target sequence results in cutting of the first nucleic acid target sequence and the second nucleic acid target sequence by the first and second catalytically active CRISPR Type V Cpf1 proteins, respectively; or
the first CRISPR Type V Cpf1 protein and the second CRISPR Type V Cpf1 protein are both catalytically inactive and the CRISPR Type II Cas9 protein is catalytically active, and the binding of the nucleic acid/protein composition to the first nucleic acid target sequence, the second nucleic acid target sequence, and the third nucleic acid target sequence results in cutting of the third nucleic acid target sequence by the catalytically active CRISPR Type II Cas9 protein; or
the first CRISPR Type V Cpf1 protein is catalytically active, the second CRISPR Type V Cpf1 protein is catalytically inactive, and the CRISPR Type II Cas9 protein is catalytically inactive, and the binding of the nucleic acid/protein composition to the first nucleic acid target sequence, the second nucleic acid target sequence, and the third nucleic acid target sequence results in cutting of the first nucleic acid target sequence by the first catalytically active CRISPR Type V Cpf1 protein; or
the first CRISPR Type V Cpf1 protein and the second CRISPR Type V Cpf1 protein are each catalytically active, and the CRISPR Type II Cas9 protein is catalytically active, and the binding of the nucleic acid/protein composition to the first nucleic acid target sequence, the second nucleic acid target sequence, and the third nucleic acid target sequence results in cutting of the first nucleic acid target sequence, the second nucleic acid target sequence, and the third nucleic acid target sequence by the first and second catalytically active CRISPR Type V Cpf1 proteins and the catalytically active CRISPR Type II Cas9 protein, respectively.
5. The method of claim 1 , wherein the nucleic acid/protein composition further comprises at least one donor polynucleotide.
6. The method of claim 5 , wherein the donor polynucleotide comprises single-stranded DNA.
7. The method of claim 1 , wherein the first nucleic acid target sequence, the second nucleic acid target sequence, and the third nucleic acid target sequence are in a single polynucleotide.
8. The method of claim 7 , wherein the single polynucleotide comprises genomic DNA.
9. The method of claim 1 , wherein a first polynucleotide comprises the first nucleic acid target sequence, a second polynucleotide comprises the second nucleic acid target sequence, and a third polynucleotide comprises the third nucleic acid target sequence.
10. A method of binding a first nucleic acid target sequence and a second nucleic acid target sequence, comprising:
contacting the first nucleic acid target sequence and the second nucleic acid target sequence with a nucleic acid/protein composition comprising
a catalytically active or catalytically inactive CRISPR Type II Cas9 protein;
a catalytically active or catalytically inactive CRISPR Type V Cpf1 protein; and
an engineered CRISPR Class 2 cross-type-nucleic-acid targeting nucleic acid (“CRISPR Class 2 cross-type-NATNA”), comprising
a first CRISPR Type II Cas9-associated nucleic-acid targeting nucleic acid, having a 5′ end and a 3′ end, comprising a first spacer element complementary to the first nucleic acid target sequence (“first Cas9-NATNA”);
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”) and a first linker element, having a 5′ end and a 3′ end, wherein the 3′ end of the first linker element is covalently connected at the 5′ end of the second Cas9-NATNA;
a CRISPR Type V Cpf1-associated nucleic-acid targeting nucleic acid, having a 5′ end and a 3′ end, comprising a second spacer element complementary to the second nucleic acid target sequence (“Cpf1-NATNA”), wherein the 3′ end is covalently connected with the 5′ end of the first linker element; and
a second linker element, having a 5′ and a 3′ end, wherein the second linker element is covalently connected at the 5′ end of the first Cas9-NATNA or at the 3′ end of the first Cas9-NATNA or at the 3′ end of the second Cas9-NATNA;
thereby facilitating binding of the nucleic acid/protein composition to the first nucleic acid target sequence and the second nucleic acid target sequence; and wherein the method is carried out in vitro, in a cultured cell, or in a non-human subject.
11. The method of claim 10 , wherein at least one of the Cpf1-NATNA, the first Cas9-NATNA, or the second Cas9-NATNA comprises RNA.
12. The method of claim 11 , wherein at least one of the Cpf1-NATNA, the first Cas9-NATNA, or the second Cas9-NATNA comprises DNA.
13. The method of claim 10 , wherein
the CRISPR Type V Cpf1 protein is catalytically inactive and the CRISPR Type II Cas9 protein is catalytically active, and the binding of the nucleic acid/protein composition to the first nucleic acid target sequence and the second nucleic acid target sequence results in cutting of the first nucleic acid target sequence by the catalytically active CRISPR Type II Cas9 protein; or
the CRISPR Type V Cpf1 protein is catalytically active and the CRISPR Type II Cas9 protein is catalytically inactive, and the binding of the nucleic acid/protein composition to the first nucleic acid target sequence and the second nucleic acid target sequence results in cutting of the second nucleic acid target sequence by the catalytically active CRISPR Type V Cpf1 protein; or
the CRISPR Type V Cpf1 protein is catalytically active and the CRISPR Type II Cas9 protein is catalytically active, and the binding of the nucleic acid/protein composition to the first nucleic acid target sequence and the second nucleic acid target sequence results in cutting of the first nucleic acid target sequence and the second nucleic acid target sequence by the catalytically active CRISPR Type II Cas9 protein and the catalytically active CRISPR Type V Cpf1 protein, respectively.
14. The method of claim 10 , wherein the nucleic acid/protein composition further comprises a donor polynucleotide, the donor polynucleotide comprising:
a nucleotide sequence complementary to the second spacer element of the Cpf1-NATNA if the CRISPR Type V Cpf1 protein is catalytically inactive, or
a nucleotide sequence complementary to the first spacer element of the first Cas9-NATNA if the CRISPR Type II Cas9 protein is catalytically inactive; and
the donor polynucleotide is capable of associating with the first or second spacer element through hydrogen bonding.
15. The method of claim 14 , wherein the donor polynucleotide comprises single-stranded DNA.
16. The method of claim 10 , wherein the first nucleic acid target sequence and the second nucleic acid target sequence are in a single polynucleotide.
17. The method of claim 16 , wherein the single polynucleotide comprises genomic DNA.
18. The method of claim 10 , wherein a first polynucleotide comprises the first nucleic acid target sequence and a second polynucleotide comprises the second nucleic acid target sequence.