Patent Issued for Methods and Means for Exact Replacement of Target DNA in Eukaryotic Organisms

By a News Reporter-Staff News Editor at Life Science Weekly — According to news reporting originating from Alexandria, Virginia, by NewsRx journalists, a patent by the inventors Rolland, Anne (Fontaine sur Saone, FR); Dubald, Manuel (Raleigh, NC); Van Lookeren Campagne, Michiel (Chapel Hill, NC); Ruiter, Rene (Heusden, BE), filed on June 3, 2008, was published online on July 1, 2014 (see also Bayer Cropscience, N.V.).
The assignee for this patent, patent number 8765448, is Bayer Cropscience, N.V. (Diegem, BE).
Reporters obtained the following quote from the background information supplied by the inventors: “Homologous recombination allows numerous targeted genetic modifications in prokaryotic and selected eukaryotic organisms including selected deletions, insertions or replacements.
“In higher eukaryotic organisms, homologous recombination may be stimulated through the induction of double stranded DNA breaks via rare-cutting endonucleases, such as e.g. I-SceI.
“WO2004/067753 describes the use of meganucleases for inducing homologous recombination ex vivo and in toto in vertebrate somatic tissues and the application thereof for genome engineering and gene therapy.
“WO2000/46386 describes methods of modifying, repairing, attenuating and inactivating a gene or other chromosomal DNA in a cell through I-SceI induced double stranded breaks. Also disclosed are methods of treating or phrophylaxis of a genetic disease in an individual in need thereof.
“In plants, induction of double stranded DNA breaks using I-SceI has been shown to increase the frequency of homologous recombination by at least two orders of magnitude using Agrobacteria to deliver the repair DNA to the plant cells (Puchta et al., 1996, Proc. Natl. Acad. Sci. U.S.A., 93, pp 5055-5060). Chilton and Que (2003, Plant Physiol. 133: pp 956-965) and Tzifira et al. (2003, Plant Physiol. 133: pp 1011-1023) report that T-DNA preferentially integrates in double stranded DNA breaks, artificially induced by the rare-cleaving enzymes I-SceI or I-CeuI. The reports also included donor T-DNA vectors which comprised a recognition site for the respective rare-cleaving enzyme.
“In addition, methods have been described which allow the design of rare cleaving endonucleases to alter substrate or sequence-specificity of the enzymes, thus allowing to induce a double stranded break at virtually any locus of interest without being dependent on the presence of a recognition site for any of the natural rare-cleaving endonucleases. Briefly, chimeric restriction enzymes can be prepared using hybrids between a zinc-finger domain designed to recognize a specific nucleotide sequence and the non-specific DNA-cleavage domain from a natural restriction enzyme, such as FokI. Such methods have been described e.g. in WO 03/080809, WO94/18313 or WO95/09233 and in Isalan et al., 2001, Nature Biotechnology 19, 656-660; Liu et al. 1997, Proc. Natl. Acad. Sci. USA 94, 5525-5530). Another way of producing custom-made meganucleases, by selection from a library of variants, is described in WO2004/067736. Custom made meganucleases with altered sequence specificity and DNA-binding affinity may also be obtained through rational design as described in WO2007/047859.
“WO2007/049095 describes ‘LADGLIDADG’ homing endonuclease variants having mutations in two separate subdomains, each binding a distinct part of a modified DNA target half site, such that the endonuclease variant is able to cleave a chimeric DNA target sequence comprising the nucleotides bound by each subdomain.
“WO2007/049156 and WO 2007/093836 describe I-CreI homing endonuclease variants having novel cleavage specificity and uses thereof.
“WO2007/047859 describes rationally designed meganucleases with altered sequence specificity and DNA binding affinity.
“WO2006/105946 described a method for the exact exchange in plant cells and plants of a target DNA sequence for a DNA sequence of interest through homologous recombination, whereby the selectable or screenable marker used during the homologous recombination phase for temporal selection of the gene replacement events can subsequently be removed without leaving a foot-print and without resorting to in vitro culture during the removal step, employing the therein described method for the removal of a selected DNA by microspore specific expression of a double stranded break inducing rare cleaving endonuclease.
“U.S. provisional patent application 60/828,042 and European patent application 06020370.0, and WO2008/037436 describe variants of the methods and means of WO2006/105946 wherein the removal step of a selected DNA fragment induced by a double stranded break inducing rare cleaving endonuclease is under control of a germline-specific promoter. Other embodiments of the method relied on non-homologous endjoining at one end of the repair DNA and homologous recombination at the other end.
“Some of the embodiments of the above identified methods and means for exact exchange of a target DNA fragment for a DNA fragment of interest require that the introduced repair DNA is introduced in the plant cell in the presence of the double stranded break inducing enzyme. The repair DNA normally also contains the preselected site recognized by a double stranded break inducing rare cleaving endonuclease and therefore the repair DNA is also prone to DNA cleavage. Accordingly, the efficiency of DNA insertion by homologous recombination may be lowered. To avoid this decrease in efficiency, the preselected site in the repair DNA may be altered in such a way that it is no longer recognized by the double stranded break inducing rare cleaving endonuclease. However, this entails the introduction of an extra change in the repair DNA compared to the target DNA in addition to the desired change.
“The current invention provides an alternative solution to this problem, which does not require the modification of the preselected site in the repair DNA and consequently allows the exchange of the target DNA with only the desired nucleotide change, without modification of the preselected site. These and other problems are solved as described hereinafter in the different detailed embodiments of the invention, as well as in the claims.”
In addition to obtaining background information on this patent, NewsRx editors also obtained the inventors’ summary information for this patent: “In one embodiment of the invention, a method is provided for exchanging a target DNA sequence in the genome a eukaryotic cell or eukaryotic organism for a DNA sequence of interest comprising the following steps: a. Inducing a first double stranded DNA break at a preselected site in the genome of a cell of the eukaryotic organism, the preselected site being located within the target DNA sequence or in the vicinity of the target DNA sequence and the preselected site being recognized by a first double-stranded break inducing (DSBI) enzyme; b. Introducing a repair DNA molecule into the eukaryotic cell, the repair DNA molecule comprising i. a DNA sequence of interest located between two flanking DNA regions having at least 80\% sequence homology to a DNA region flanking the target DNA sequence, and preferably flanking the preselected site in the genome of the eukaryotic cell; ii. A selectable or screenable marker gene located between the flanking DNA regions, the selectable or screenable marker gene further being located between a first repeat sequence consisting of the 5′-terminal part of the preselected site and a second sequence consisting of the 3′ terminal part of the preselected site, whereby the sequences common between the first and second repeat sequences are in direct repeat; and iii. At least one recognition site for a second DSBI enzyme located between the one of the flanking DNA regions and the first and second repeat sequence, preferably between the first and sequence direct repeat sequence; c. Selecting a population of cells comprising the selectable or screenable marker; d. Selecting a cell wherein the selectable or screenable marker has been introduced by homologous recombination through the flanking DNA regions; e. Introducing a double stranded break at the recognition site for the second DSBI enzyme in the cell; f. Selecting a progeny cell wherein the selectable or screenable marker gene is deleted by homologous recombination between the direct repeats thereby recreating the preselected site.
“In another embodiment of the invention, a method is provided for exchanging a target DNA sequence in the genome, particularly the nuclear genome, of a plant for a DNA sequence of interest comprising the following steps: a) Inducing a first double stranded DNA break at a preselected site in the genome of a cell of the plant, the preselected site being located within the target DNA sequence or in the vicinity of the target DNA sequence and the preselected site being recognized by a first double-stranded break inducing (DSBI) enzyme b) Introducing a repair DNA molecule into the eukaryotic cell, the repair DNA molecule comprising i) The DNA sequence of interest located between two flanking DNA regions having at least 80\% sequence homology to a DNA region flanking the target DNA sequence, and preferably flanking the preselected site in the genome of the eukaryotic cell; ii) A selectable or screenable marker gene located between the flanking DNA regions, the selectable or screenable marker gene further being located between a first repeat sequence consisting of the 5′-terminal part of the preselected site and a second sequence consisting of the 3′ terminal part of the preselected site, whereby the sequences common between the first and second repeat sequences are in direct repeat; and iii) At least one recognition site for a second DSBI enzyme located between the one of the flanking DNA regions and the first and second repeat sequence; c) Selecting a population of plant cells comprising the selectable or screenable marker; d) Selecting a plant cell wherein the DNA sequence of interest (and the selectable or screenable marker) has been introduced by homologous recombination through the flanking DNA regions, and regenerating a plant from the plant cell; e) Crossing the regenerated plant or a progeny plant thereof comprising the selectable marker gene with a plant comprising a rare cleaving double stranded break inducing (‘DSBI’) enzyme encoding chimeric gene, the chimeric gene comprising the following operably linked DNA segments: i. a germline specific promoter; ii. a DNA region encoding a double stranded DNA break inducing enzyme recognizing the recognition site located in the DNA of interest (i.e. the second double stranded DNA break inducing enzyme); iii. a transcription termination and polyadenylation region; f) Selecting a progeny plant (F1-plant) comprising the selectable or screenable marker gene and the DSBI enzyme encoding chimeric gene; g) Crossing the progeny plant with another plant whereby the progeny plant is used as pollen donor; h) Selecting a population of progeny plants (F2-population) which comprises the DSBI enzyme encoding chimeric gene; and i) Selecting a progeny plant wherein the selectable or screenable marker gene is deleted by homologous recombination between the first and second direct repeat sequence. The invention relates to the eukaryotic cells and plants obtainable by the above described methods.”
For more information, see this patent: Rolland, Anne; Dubald, Manuel; Van Lookeren Campagne, Michiel; Ruiter, Rene. Methods and Means for Exact Replacement of Target DNA in Eukaryotic Organisms. U.S. Patent Number 8765448, filed June 3, 2008, and published online on July 1, 2014. Patent URL: http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO1&Sect2=HITOFF&d=PALL&p=1&u=\%2Fnetahtml\%2FPTO\%2Fsrchnum.htm&r=1&f=G&l=50&s1=8765448.PN.&OS=PN/8765448RS=PN/8765448
Keywords for this news article include: Esterases, DNA Research, Endonucleases, Eukaryotic Cells, Enzymes and Coenzymes, Bayer Cropscience N.V..
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