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 Type V nucleic-acid targeting nucleic acid (“CRISPR Type V NATNA”), having a 5′ end and a 3′ end, comprising:
(a) a stem-loop element, having a 5′ end and a 3′ end; and
(b) a spacer element complementary to a nucleic acid target sequence, having a 5′ end and a 3′ end,
wherein the 3′ end of the stem-loop element is covalently connected with the 5′ end of the spacer element; and the stem-loop element has a stem of increased length compared to the stem of the polynucleotide of SEQ ID NO: 28 formed between nucleotides 6-10 and 15-19 of the polynucleotide of SEQ ID NO:28, and wherein the CRISPR Class 2 Type V NATNA enables cleavage with a Cpf1 protein having an active Ruv-C-like nuclease domain.
2. The engineered CRISPR Class 2 Type V NATNA of claim 1 , wherein said NATNA comprises an additional secondary structural element.
3. The engineered CRISPR Class 2 Type V NATNA of claim 2 , wherein the additional secondary structural element is covalently connected to the 5′ end of the stem-loop element or the 3′ end of the spacer element.
4. The engineered CRISPR Class 2 Type V NATNA of claim 2 , wherein said NATNA further comprises an additional linker element, wherein the additional linker element is covalently connected to the 5′ end or the 3′ end of the additional secondary structural element.
5. The engineered CRISPR Type V NATNA of claim 2 , wherein the additional secondary structural element is selected from the group consisting of a hairpin, a pseudo-knot, and a ribozyme.
6. The engineered CRISPR Type V NATNA of claim 1 , wherein the engineered CRISPR Type V NATNA comprises DNA.
7. The engineered CRISPR Type V NATNA of claim 1 , wherein the engineered CRISPR Type V NATNA comprises DNA and RNA.
8. A composition comprising the engineered CRISPR Class 2 Type V NATNA of claim 1 and a Cpf1 protein having an active Ruv-C-like nuclease domain.
9. The composition of claim 8 , wherein the Cpf1 protein is an Acidaminococcus sp. Cpf1 protein.
10. A cell comprising the composition of claim 8 .
11. The cell of claim 10 , wherein the cell is a eukaryotic cell.
12. The cell of claim 10 , wherein the cell is a stem cell or a progenitor cell.
13. The cell of claim 11 , wherein the eukaryotic cell is a human cell.
14. The cell of claim 11 further comprising a donor polynucleotide.