High-throughput precision genome editing

Abstract

The invention provides compositions and methods for high-efficiency genome editing. In some aspects, the invention provides retron-guide RNA cassettes and vectors comprising the cassettes. Also provided are host cells that have been transformed with the vectors. In other aspects, the invention provides retron donor DNA-guide molecules. In some other aspects, methods for genome editing and the screening of genetic loci are provided. In further aspects, methods and compositions are provided for the prevention or treatment of genetic diseases. Kits for genome editing and screening are also provided.

1 . A retron-guide RNA cassette comprising:
 (a) a retron comprising:
 (i) an msr locus; 
 (ii) a first inverted repeat sequence coding region; 
 (iii) an msd locus; 
 (iv) a donor DNA sequence located within the msd locus; and 
 (v) a second inverted repeat sequence coding region; and 
   (b) a guide RNA (gRNA) coding region,
 wherein the donor DNA sequence is at least about 500 to 10000 nucleotides in length. 
   
     
     
         2 . The cassette of  claim 1 , wherein the first inverted repeat sequence coding region is located within a 5′ end of the msr locus. 
     
     
         3 . The cassette of  claim 1 , wherein the second inverted repeat sequence coding region is located 3′ of the msd locus. 
     
     
         4 . The cassette of  claim 1 , wherein the retron encodes an RNA molecule that is capable of self-priming reverse transcription by a reverse transcriptase (RT). 
     
     
         5 . The cassette of  claim 4 , wherein reverse transcription of the RNA molecule results in a multicopy single-stranded DNA (msDNA) molecule that comprises RNA and DNA. 
     
     
         6 . The cassette of  claim 1 , wherein transcription products of the retron and the gRNA coding region are physically coupled. 
     
     
         7 . The cassette of  claim 1 , wherein transcription products of the retron and the gRNA coding region are not physically coupled. 
     
     
         8 . The cassette of  claim 1 , wherein the gRNA coding region is 3′ of the retron. 
     
     
         9 . The cassette of  claim 1 , wherein the gRNA coding region is 5′ of the retron. 
     
     
         10 . The cassette of  claim 1 , further comprising a ribozyme sequence. 
     
     
         11 . The cassette of  claim 10 , wherein the ribozyme sequence encodes a hepatitis delta virus ribozyme. 
     
     
         12 . The cassette of  claim 1 , wherein the donor DNA sequence is at least about 1000 nucleotides in length. 
     
     
         13 . The cassette of  claim 1 , wherein the donor DNA sequence comprises two homology arms, wherein each homology arm has at least about 70% to about 99% similarity to a portion of the sequence of a genetic locus of interest on either side of a nuclease cleavage she. 
     
     
         14 . A vector comprising the cassette of  claim 1 . 
     
     
         15 . The vector of  claim 14 , further comprising a promoter that is operably linked to the cassette. 
     
     
         16 . The vector of  claim 15 , wherein the promoter is inducible. 
     
     
         17 . The vector of  claim 15 , wherein the promoter is selected from the group consisting of an RNA polymerase II promoter, an RNA polymerase III promoter, and a combination thereof. 
     
     
         18 . The vector of  claim 14 , further comprising a reverse transcriptase (RT) coding sequence. 
     
     
         19 . The vector of  claim 18 , further comprising a nuclear localizing sequence located 5′ of the RT coding sequence. 
     
     
         20 . The vector of  claim 14 , further comprising a nuclease coding sequence. 
     
     
         21 . The vector of  claim 20 , wherein the nuclease encoded by the nuclease coding sequence is selected from the group consisting of Cas9, Cpf1, and a combination thereof. 
     
     
         22 . A retron donor DNA-guide molecule comprising:
 (a) a retron transcript comprising:
 (i) an msr region; 
 (ii) a first inverted repeat sequence; 
 (iii) an msd region; 
 (iv) a donor DNA sequence coding region located within the msd region; and 
 (v) a second inverted repeat sequence; and 
   (b) a guide RNA (gRNA) molecule,
 wherein the donor DNA sequence is at least about 500 to 10000 nucleotides in length. 
   
     
     
         23 . The retron donor DNA-guide molecule of  claim 22 , wherein the first inverted repeat sequence is located within a 5′ end of the msr region. 
     
     
         24 . The retron donor DNA-guide molecule of  claim 22 , wherein the second inverted repeat sequence is located 3′ of the msd region. 
     
     
         25 . The retron donor DNA-guide molecule of  claim 22 , wherein the retron transcript is capable of self-priming reverse transcription by a reverse transcriptase (RT). 
     
     
         26 . The retron donor DNA-guide molecule of  claim 22 , wherein the retron transcript and gRNA molecule are physically coupled. 
     
     
         27 . The retron donor DNA-guide molecule of  claim 26 , wherein the gRNA molecule is 3′ of the retron transcript. 
     
     
         28 . The retron donor DNA-guide molecule of  claim 26 , wherein the gRNA molecule is 5′ of the retron transcript. 
     
     
         29 . The retron donor DNA-guide molecule of  claim 22 , further comprising a ribozyme. 
     
     
         30 . The retron donor DNA-guide molecule of  claim 29 , wherein the ribozyme is a hepatitis delta virus ribozyme. 
     
     
         31 . The retron donor DNA-guide molecule of  claim 22 , wherein the retron transcript and gRNA molecule are physically uncoupled after transcription. 
     
     
         32 . The retron donor DNA-guide molecule of  claim 22 , wherein reverse transcription of the retron transcript results in a multicopy single-stranded DNA (msDNA) molecule that comprises RNA and DNA. 
     
     
         33 . The retron donor DNA-guide molecule of  claim 32 , wherein at least some of the RNA content of the msDNA molecule is degraded. 
     
     
         34 . The retron donor DNA-guide molecule of  claim 22 , wherein the donor DNA sequence coding region comprises sequences encoding two homology arms, wherein each homology arm has at least about 70% to about 99% similarity to a portion of the sequence of a genetic locus of interest on either side of a nuclease cleavage site. 
     
     
         35 - 63 . (canceled) 
     
     
         64 . A host cell that has been transformed by a vector of  claim 14 . 
     
     
         65 . A pharmaceutical composition comprising:
 (a) the retron-guide RNA cassette of  claim 1 ; and   (b) a pharmaceutically acceptable carrier.   
     
     
         66 - 79 . (canceled) 
     
     
         80 . The vector of  claim 20 , wherein the nuclease encoded by the nuclease coding sequence is a CRISPR-associated protein (Cas) nuclease. 
     
     
         81 . The retron donor DNA-guide molecule of  claim 22 , wherein the donor DNA sequence is at least about 1000 nucleotides in length.