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 II Cas9-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 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 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 second nucleic acid target sequence (“first 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.
2. 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 wherein the CRISPR Class 2 cross-type-NATNA/Cpf1 protein complex is capable of binding the second nucleic acid target sequence.
3. The engineered CRISPR Class 2 cross-type-NATNA of claim 2 , wherein the first Cas9-NATNA and the second Cas9-NATNA are capable of forming a complex with a CRISPR Type II Cas9 protein (“CRISPR Class 2 cross-type-NATNA/Cpf1 protein/Cas9 protein complex”), and wherein the CRISPR Class 2 cross-type-NATNA/Cpf1 protein/Cas9 protein complex is capable of binding the first nucleic acid target sequence and the second nucleic acid target sequence.
4. The engineered CRISPR Class 2 cross-type-NATNA of claim 1 , wherein at least one of the first Cpf1-NATNA, the first Cas9-NATNA, or the second Cas9-NATNA comprises RNA.
5. The engineered CRISPR Class 2 cross-type-NATNA of claim 1 , wherein at least one of the first Cpf1-NATNA, the first Cas9-NATNA, or the second Cas9-NATNA comprises DNA.
6. 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.
7. The nucleic acid/protein composition of claim 6 , 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.
8. The nucleic acid/protein composition of claim 7 , 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.
9. The nucleic acid/protein composition of claim 8 , further comprising one or more donor polynucleotides.