Viral delivery of rna utilizing self-cleaving ribozymes and crispr-based applications thereof

Abstract

The present disclosure relates to viral delivery of RNA utilizing self-cleaving ribozymes and applications of such, including but not limited to CRISPR-Cas related applications.

1 . A viral particle comprising a nucleic acid comprising a genome sequence of a single-stranded RNA (ssRNA) virus or antigenome sequence that is complementary to the genome sequence, the antigenome sequence comprising a first region comprising
 (i) a target segment; and   (ii) a first segment encoding a first self-cleaving ribozyme,   wherein the target segment is adjacent to the first segment.   
     
     
         2 . The viral particle of  claim 1 , wherein the first region further comprises (iii) a second segment encoding a second self-cleaving ribozyme, wherein the target segment is flanked by the first segment and the second segment. 
     
     
         3 . The viral particle of  claim 1 , wherein the first self-cleaving ribozyme is a 3′ self-cleaving ribozyme or a 5′ self-cleaving ribozyme. 
     
     
         4 . The viral particle of  claim 3 , wherein the first self-cleaving ribozyme comprises one of a hammerhead ribozyme and a hepatitis delta virus (HDV) ribozyme. 
     
     
         5 . The viral particle of  claim 1 , wherein the antigenome sequence further comprises a second region comprising a third segment encoding a nuclease. 
     
     
         6 . The viral particle of  claim 5 , wherein the second region further comprises a fourth segment encoding a reporter molecule. 
     
     
         7 . The viral particle of  claim 5 , wherein the nuclease comprises Cas9 or Cpf1. 
     
     
         8 . The viral particle of  claim 1 , wherein the target segment comprises guide RNA (gRNA), wherein the gRNA has a scaffold sequence and a targeting sequence. 
     
     
         9 . The viral particle of  claim 1 , wherein the first self-cleaving ribozyme comprises a hammerhead ribozyme. 
     
     
         10 . The viral particle of  claim 1 , further comprising a third region comprising a fifth segment, wherein the fifth segment comprises a mutant P gene. 
     
     
         11 . The viral particle of  claim 1 , further comprising a fourth region comprising a fourth region comprising a sixth segment, wherein the sixth segment comprises a mutant L gene. 
     
     
         12 . The viral particle of  claim 1 , wherein the viral particle is within the order mononegavirales. 
     
     
         13 . The viral particle of  claim 1 , wherein the viral particle is within the family paramyxoviridae. 
     
     
         14 . The viral particle of  claim 1 , wherein the viral particle is a Sendai virus (SeV) or a Newcastle disease virus (NDV). 
     
     
         15 . The viral particle of  claim 1 , wherein the viral particle is a temperature sensitive mutant. 
     
     
         16 . A method of introducing into a host cell a target RNA, comprising
 (i) contacting the viral particle of  claim 1  with said host cell; and   (ii) culturing the host cell under conditions allowing
 (a) producing a target RNA; and 
 (b) liberating the target RNA, wherein the first self-cleaving ribozyme liberates the target RNA from the transcribed first region. 
   
     
     
         17 . The method of  claim 16 , wherein the host cell is selected from the group consisting of an archaea cell, bacterial cell, and a eukaryotic cell. 
     
     
         18 . The method of  claim 17 , wherein the host cell is a stem cell. 
     
     
         19 . A method of introducing a site-specific modification to target DNA in a host cell comprising
 (i) contacting the viral particle of  claim 1  with said host cell;   (ii) culturing the host cell under conditions allowing
 (a) producing the gRNA flanked by the 5′ self-cleaving ribozyme and the nuclease; 
 (b) liberating the gRNA, wherein the 5′ self-cleaving ribozyme liberates the gRNA; 
 (c) expressing the nuclease; 
 (d) forming a complex between the nuclease and the gRNA, wherein the scaffold sequence of the gRNA is bound to the nuclease; and 
 (e) contacting the target DNA with the complex, wherein the targeting sequence of the gRNA binds to a sequence on the target DNA adjacent to a protospacer adjacent motif (PAM); and 
   (iii) introducing the site-specific modification to the target DNA.   
     
     
         20 . The method of  claim 19 , wherein the site-specific modification is one of an insertion, a deletion, a frameshift, and a point mutation. 
     
     
         21 . The method of  claim 19 , wherein the DNA is genomic. 
     
     
         22 . The method of  claim 19 , wherein the host cell is selected from the group consisting of an archaea cell, bacterial cell, and a eukaryotic cell.