Sequence-specific in vivo cell targeting
Enhanced, specific nucleic acid targeting complexes comprising endo and exonuclease activity, and related methods that allow both targeted degradation of specific and/or non-specific nucleic acids in vivo and specific temporal regulation of nuclease activity to prevent off-target activity are disclosed herein. Through practice of the disclosure, nucleic acids, and cells harboring them, such as cancer cells or pathogens, are selectively degraded in vivo.
1. A method of inducing cell death of a cancer cell in a tissue of a subject in need thereof, the method comprising:
a) administering to the subject a chimeric polypeptide comprising a first domain comprising a Cas9 domain with endonuclease activity and a second domain comprising RecJ domain with exonuclease activity, and a guide nucleic acid comprising a sequence complementary to a target nucleic acid in the cancer cell, wherein the target nucleic acid is an oncogene; and
b) cleaving the target nucleic acid, thereby inducing cell death of the cancer cell.
2. The method of claim 1 , wherein the cancer cell comprises a lung cancer cell, a pancreatic cancer cell, a breast cancer cell, an ovarian cancer cell, a colon cancer cell, or a cervical cancer cellk.
3. The method of claim 1 , wherein the target nucleic acid comprises a cancer-specific sequence.
4. The method of claim 3 , wherein the cancer-specific sequence comprises a single nucleotide polymorphism specific to a cancer, a translocation, a chromosomal abnormality, or a sequence associated with cancer progression.
5. The method of claim 4 , wherein the chromosomal abnormality is selected from the group consisting of: a translocation, a deletion, a duplication, an inversion, an insertion, a ring, copy number variations, an indel, and an isochromosome.
6. The method of claim 1 , wherein the tissue comprises a healthy cell.
7. The method of claim 6 , wherein after the administering, the healthy cell proliferates.
8. The method of claim 1 , wherein the cleaving comprises cleaving at at least 2 cleavage sites in the cancer cell.
9. The method of claim 1 , wherein the second domain comprises an enzyme having cleaved end resection activity.
10. The method of claim 1 , wherein the Cas9 domain comprises a sequence having at least 90% identity the full length of to SEQ ID NO: 68.