Rapid, Efficient In Vivo Site-Directed Mutagenesis Using Oligonucleotides
Description of Invention:
The rapid modification of genes provides opportunities to study gene function and evaluate drug responsiveness. Scientists at the National Institute for Environmental and Health Sciences have developed a new system in yeast, delitto perfetto, which provides for rapid, efficient and accurate in vivo genomic mutagenesis using oligonucleotides (IROs) and involves the complete removal of the heterologous sequence previously integrated at the target locus (Nature Biotechnol. (2001) 19, 773-776). They have demonstrated that synthetic oligonucleotides can target a desired mutation to almost any chromosomal locus where a marker cassette has been previously integrated. The oligonucleotides, which are designed with short sequence homology to sites up- and down-stream of the marker cassette, replace the marker cassette with the chosen mutation without leaving any heterologous sequence in the targeted locus. Since the system always provides selection for the clones containing the desired mutation, it can be used to generate any kind of modification: i.e., it is not constrained by the generation of mutations that provide a detectable phenotype. Additionally, induction of double-strand breaks (delitto perfetto-DSB) in vivo before standard transformation procedures provides 1,000 to 10,000 fold stimulation of oligonucleotide targeting, resulting in 5-20% of all cells in the population being efficiently targeted by small oligonucleotides (PNAS (2003), 100, 14994-14999). With such a high stimulation of targeting even gross rearrangements, like large DNA deletions, chromosome fusions, circularizations, reciprocal or non reciprocal translocations are obtained with high frequency and direct selection.
The core invention is a novel self-cloning system for simple and high-throughput in vivo site-directed mutagenesis applicable to all organisms capable of homologous recombination and developed in the non-pathogenic yeast Saccharomyces cerevisiae. Since changes are created through a self-cloning process, this system could represent a highly versatile tool to generate modifications of genes in yeasts for commercial application in the food and beverage industries (such as, baking, brewing, wine and sake) without the resulting organisms being classified as GMO (genetically modified organisms). This approach could also be well positioned within drug discovery protocols where the need to mutagenize particular target sequences forms an integral part of the drug development process.
Delitto perfetto-DSB is efficient for targeting homologous sequences that are close or distant to the DSB and in the presence of a competing homologous chromosome in diploid cells, and can strongly stimulate recombination with single-stranded DNA, without strand bias. The mechanism of DSB repair with oligonucleotides follows primarily a single-strand annealing pathway of recombinational repair. This novel system is also independent of restriction sites, requires minimal sequence analysis. This method has been used in S. cerevisiae for many yeast chromosomal genes and the human gene p53 and has obvious potential for use with YAC and TAR clones. Claims are directed to several methods for generating DNA nucleic acid mutations in vivo and are applicable to any organism that has a homologous recombination system, as well as to kits. This methodology is available for licensing and is a highly versatile tool of direct use to drug discovery, pharma and research reagent companies as well as to companies working with industrial yeast strains.
Inventors:
Francesca Storici (NIEHS) Michael A. Resnick (NIEHS) Lysle Kevin Lewis (NIEHS)
Patent Status:
DHHS Reference No. E-204-2001/0 --
PCT Application No. PCT/US02/23634 filed 26 July 2002, which published as International Publication No. WO03/012036 on 13 Feb 2003
National Stage Entry: EPC, CA, AU, US, JP
U.S. Patent Application No. 10/484,989 filed 26 Jan 2004
Related Technologies: Related technologies also available for licensing include: DHHS Ref. No. E-121-1996/0-US-06, Transformation-Associated Recombination Cloning (U.S. Patent No. 6,391,642 issued 21 May 2002); and DHHS Ref. No. E-262-1984/0-US-03, Process for Site Specific Mutagenesis Without Phenotypic Selection (U.S. Patent No. 4,873,192 issued 10 Oct 1989).
Licensing Status: In addition to licensing, the technology is available for further development through collaborative research with the inventors via a Cooperative Research and Development Agreement (CRADA).
Portfolios: Gene Based Therapies
Gene Based Therapies -Therapeutics
For Additional Information Please Contact: Jasbir (Jesse) S. Kindra J.D.
NIH Office of Technology Transfer
6011 Executive Blvd, Suite 325
Rockville, MD 20852-3804
Phone: (301) 435-5559
Email: kindraj@mail.nih.gov
Fax: (301) 402-0220
