Sequence-specific in vivo cell targeting

Abstract

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.

What is claimed is: 
     
         1 . A method of treating cancer of a subject in need thereof, the method comprising:
 administering to the subject a chimeric polypeptide comprising a first domain comprising a sequence-specific programmable endonuclease and a second domain; and a guide nucleic acid comprising a sequence complementary to a target nucleic acid in a cell of the subject, wherein, upon contacting of the target nucleic acid, the chimeric polypeptide cleaves the target nucleic acid, whereby treating the cancer.   
     
     
         2 . The method of  claim 1 , wherein the target nucleic acid comprises a cancer-specific sequence. 
     
     
         3 . The method of  claim 2 , 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. 
     
     
         4 . The method of  claim 3 , 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. 
     
     
         5 . The method of  claim 1 , wherein the target nucleic acid is an oncogene. 
     
     
         6 . The method of  claim 1 , wherein the target nucleic acid comprises DNA. 
     
     
         7 . The method of  claim 1 , wherein the target nucleic acid comprises RNA. 
     
     
         8 . The method of  claim 1 , wherein the target nucleic acid is absent in the healthy cell of the subject. 
     
     
         9 . The method of  claim 1 , wherein the target nucleic acid comprises a portion of a gene selected from the group consisting of BRCA-1, BRAF, BCR-ABL, HER2, KIF5A, IRX1, ADAMTS16, GNPDA2, KCNE2, SLC15A5, SMIM11, DACH2, HERC2P2, CD68SHBG, ERBB2, KRT16, LINC00536, TRPS1, CDK8, TRAPPC9, HERC2P2, SIRPB1, MRC1, ATP 11 A, POTEB, HERC2P2, PRDM9, CDKN2B, HPV, LINE2 (MT2), CCR5, and HPRT1. 
     
     
         10 . The method of  claim 1 , wherein the chimeric polypeptide cleaves the target nucleic acid at one or more cleavage sites. 
     
     
         11 . The method of  claim 1 , wherein the cell is a cancer cell. 
     
     
         12 . The method of  claim 1 , wherein the cell is a healthy cell. 
     
     
         13 . The method of  claim 1 , wherein the cancer comprises lung cancer, pancreatic cancer, breast cancer, ovarian cancer, colon cancer, or cervical cancer. 
     
     
         14 . The method of  claim 1 , wherein the chimeric polypeptide comprises inducible non-specific nuclease activity. 
     
     
         15 . The method of  claim 14 , wherein the inducible non-specific nuclease activity is activated by site-specific cleavage of the target nucleic acid by the chimeric polypeptide. 
     
     
         16 . The method of  claim 1 , wherein the first domain comprises a Cas12a (or Cpf1), a Cas12a, a Cas12b, a Cas12c, a Cas12d, a Cas12e, a Cas12f, a Cas12g, a Cas12h, a Cas12i, a Cas13a, a Cas13b, a Cas14, or a Cas9. 
     
     
         17 . The method of  claim 16 , wherein the first domain comprises the Cas9. 
     
     
         18 . The method of  claim 1 , wherein the second domain comprises a RecE, a RecJ, a T5, a Lambda Exonuclease, a RecBCD, a DNA pol I small fragment, an Exo1, an ExoII, an ExoVII, an Exo T, a Trex1, a mung bean nuclease, or a Trex2. 
     
     
         19 . The method of  claim 18 , wherein the second domain comprises the RecJ. 
     
     
         20 . The method of  claim 1 , wherein the second domain comprises an enzyme having cleaved end resection activity.