Retrons for gene targeting

Abstract

The invention provides methods and nucleic acid constructs that may be used to modify a nucleic acid of interest at a target locus within the genome of a host. In some aspects, the invention contemplates producing in vivo a gene targeting substrate (GTS), which may be comprised of both DNA and RNA components. The gene targeting substrate may comprise a gene targeting nucleotide sequence (GTNS), which is homologous to the target locus, but comprises a sequence modification compared to the target locus. The gene targeting substrate may be produced by reverse transcription of a gene targeting message RNA (gtmRNA). The gene targeting message RNA may be folded for self-priming for reverse transcription by a reverse transcriptase. The gene targeting message RNA may in turn be the product of transcription of a gene targeting construct (GTC) encoding the gene targeting message RNA. The gene targeting construct may for example be a DNA sequence integrated into the genome of the host, or integrated into an extrachromosomal element. Following expression of the gene targeting systems of the invention, hosts may for example be selected having genomic modifications at a target locus that correspond to the sequence modification present on the gene targeting nucleotide sequence. In some embodiments, the structure of retrons may be adapted for use in the gene targeting systems of the invention.

1 ) A method of modifying a target nucleic acid of interest at a target locus within a genome of a host comprising: 
 a) introducing into the host a gene targeting construct (GTC) and culturing the host so as to: 
 i) express the gene targeting construct encoding a gene targeting RNA, to produce the gene targeting message RNA capable of priming reverse transcription by a reverse transcriptase (RT);  
 ii) reverse transcribe at least a portion of the gene targeting message RNA to produce an in vivo gene targeting substrate (GTS) having a gene targeting nucleotide sequence (GTNS), wherein the GTNS is homologous to the target locus and comprises a sequence modification compared to the target nucleic acid; and,  
   b) selecting a host having the sequence modification at the target locus.    
     
     
         2 ) The method of  claim 1 , wherein the host is capable of expressing the RT prior to transforming the host with the gene targeting conduct.  
     
     
         3 ) The method of  claim 1 , wherein the host is modified to be capable of expressing the RT at the same time as, or after, transforming the host with the gene targeting conduct.  
     
     
         4 ) The method of  claim 1 , wherein the GTC is introduced into the host by transformation, by cross breeding or by cell fusion.  
     
     
         5 ) A gene targeting construct comprised of recombinant nucleic acid sequence in a host having a host genome, wherein the gene targeting construct encodes and is capable of expressing a gene targeting message RNA, wherein the gene targeting message RNA is capable of self-priming reverse transcription by a reverse transcriptase in the host to produce a gene targeting substrate having a gene targeting nucleotide sequence, wherein the gene targeting nucleotide sequence is homologous to a target locus in the host genome and comprises a sequence modification compared to the target locus, wherein expression of the gene targeting construct in the host introduces the sequence modification as a heritable genetic change in the target sequence in the genome of the host.  
     
     
         6 ) The gene targeting construct of  claim 5 , wherein the gene targeting construct comprises an msr coding region and an msd coding region.  
     
     
         7 ) A recombinant reverse transcriptase coding sequence encoding a reverse transcriptase having a nuclear localization signal sequence.  
     
     
         8 ) The gene targeting construct of  claim 6 , wherein the msr and msd coding regions are in operative association with a first regulatory region, and the construct further comprises a nucleotide sequence encoding a reverse transcriptase.  
     
     
         9 ) The gene targeting construct of  claim 8 , wherein the nucleotide sequence encoding the reverse transcriptase is in operative association with the first regulatory region or with a second regulatory region.  
     
     
         10 ) The gene targeting construct of  claim 8 , wherein the reverse transcriptase comprises a nuclear localization signal sequence.  
     
     
         11 ) The gene targeting construct of  claim 8  wherein the regulatory region is operatively active in an S phase, a G1/S boundary of a cell cycle, or during meiosis.  
     
     
         12 ) The gene targeting construct of  claim 11 , wherein the regulatory region is selected from the group consisting of a histone promoter, a cyclin promoter, a promoter associated with a gene involved in DNA replication, a promoter associated with a gene involved in DNA repair and a promoter associated with a gene involved in DNA homologous recombination.  
     
     
         13 ) The gene targeting construct of  claim 8 , further comprising a nucleotide sequence encoding a selectable marker.  
     
     
         14 ) A vector comprising the gene targeting construct of  claim 8 .  
     
     
         15 ) An host comprising the vector of  claim 14 .  
     
     
         16 ) The host of  claim 15 , selected from the group consisting of a plant cell, an animal cell, a yeast cell, and an insect cell.  
     
     
         17 ) The host of  claim 16 , wherein the host is a plant cell.  
     
     
         18 ) A method of modifying a target locus in a host comprising transforming the host with the gene targeting construct of  claim 8 .  
     
     
         19 ) A method of modifying a target locus in a host comprising transforming the host with the vector of  claim 14 .  
     
     
         20 ) The method of  claim 19  wherein the host is a eukaryotic organism.  
     
     
         21 ) The method of  claim 20 , wherein the host is selected from the group consisting of a plant cell, an animal cell, a yeast cell, and an insect cell.  
     
     
         22 ) The gene targeting construct of  claim 1 , wherein the gene targeting nucleotide sequence comprises one, or more than one, region of 15 to about 500 nucleotides, exhibiting about 70% to about 99% sequence similarity with the target locus sequence, as determined using the following conditions: Program: blastp; Database: nr; Expect 10; filter: default; Alignment: pairwise; Query genetic Codes: Standard (1).  
     
     
         23 ) The gene targeting construct of  claim 22 , wherein the one or more than one region is of less than 300 nucleotides in length.