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.

Description

FIELD OF THE INVENTION [0001] The present invention relates to nucleic acid constucts and manipulations that may be used for in vivo gene modification. More specifically the present invention relates to systems for producing gene targeting substrates using reverse transcriptase, as well as methods for promoting in vivo gene modification using such gene targeting substrates. BACKGROUND OF THE INVENTION [0002] Gene targeting generally refers to the directed alteration of a specific DNA sequence in its genomic locus in vivo. This may involve the transfer of genetic information from a nucleic acid molecule, which may be referred to as a gene targeting substrate, to a specific target locus in the host cell genome. In current methods, the gene targeting substrate usually exists as an extrachromosomal nucleic acid molecule. The target locus may be present in the host cell's nuclear chromosomes or organellar chromosomes (e.g. mitochondria or plastids) or a cellular episome. The gene targeti...

AI Technical Summary

Benefits of technology

[0040] In alternative embodiments, a gene targeting construct may be excised from the genome of a host. For example, the gene targeting construct may be flanked on each side by a recognition sequence for a site-specific recombinase such as, for example, FLP protein of the 2 micron element. Such embodiments may be adapted so that by the action of the recombinase on its respective recognition sequence the gene targeting construct is excised, typically as as a circular dsDNA molecule (having been excised from a chromosomal locus or an extrachromosomal locus on a vector where it is integrated). This may for example be useful for producing subsequent generations of hosts in which the heritable genetic change mediated by the gene targeting construct is present, while the construct itself is absent from such hosts. Accordingly, in one aspect the invention provides hosts having a heritable genetic change mediated by the methods and constructs of the invention, in which the heterologous constructs used to mediate the genetic change are not present.

Problems solved by technology

In this manner, the novel sequence of the gene targeting substrate is transferred into the host genome at the targeted locus, which may result in loss of the wild-type genetic information at this locus.

Thus transgenes which integrate at random sites in the genome may not be expressed in the correct fashion to accurately reflect the biological effect of the gene under basic study, or provide the desired phenotype in a biotechnology application.

In contrast, placement of a transgene encoding a modified version of an endogenous gene at random sites in the genome may not enable accurate analysis of the effect of this transgene because the endogenous gene copy is still functioning.

However, the frequency of these events is low.

Application of gene targeting methods, especially in plants and mammals, may be inhibited by several limitations in conventional technology, which may be technically demanding, rely on tedious and expensive in vitro procedures, or be successful only in specialized cell lines.

These limitations may be compounded by a low frequency of gene targeting events which may not be easily identifiable.

Conventional gene targeting strategies may rely on incorporation of a selectable marker at the target locus resulting in insertional-inactivation mutants by interruption of the target gene with the selectable marker, an approach that may not enable more subtle modifications such as single base-pair changes.

Current selection and enrichment procedures may also be ineffective if they select false-positives with high frequency.

Such methods require extensive screening since the frequency of modifying the target locus is low, and background levels of insertion at non-target loci is high.

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