Retrons for gene targeting

摘要

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.

主权项

1. A method of modifying a target nucleic acid of interest at a target locus within a genome, comprising:
a) introducing directly into a eukaryotic host cell a gene targeting construct (GTC) by transformation of the host cell with a DNA molecule comprising the GTC, and culturing the host cell or transformed progeny of the host cell so as to:
i) express a gene targeting RNA from the GTC, wherein the RNA self-primes reverse transcription by a reverse transcriptase (RT) expressed by the host cell or the transformed progeny of the host cell, wherein the RT has a nuclear localization sequence;ii) wherein at least a portion of the gene targeting RNA is reverse transcribed in the nucleus to produce a 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;iii) wherein the GTNS recombines with the target nucleic acid to insert, delete, or substitute one or more bases of the sequence of the target nucleic acid to produce a sequence modification at the target locus within the genome;iv) wherein the GTC comprises an msr coding region encoding an RNA component of the GTS called an msr element and an msd coding region encoding a DNA component of the GTS called an msd element, wherein the msr and msd elements comprise inverted repeat sequences b 1 and b2, and are further flanked by inverted repeat sequences a1′ and a2′, and wherein inverted repeat sequences a1′ and a2′ are configured to form double-stranded regions in the gene targeting RNA by base-pairing; and, b) selecting a host cell or transformed progeny of the host cell having the sequence modification at the target locus.