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Methods and Compositions for T-Rna

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Methods and Compositions for T-Rna (19) *EP003390631B1* (11) EP 3 390 631 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C12N 15/11 (2006.01) C12N 15/113 (2010.01) (2006.01) 08.04.2020 Bulletin 2020/15 C12N 15/81 (21) Application number: 16822827.8 (86) International application number: PCT/US2016/065537 (22) Date of filing: 08.12.2016 (87) International publication number: WO 2017/105991 (22.06.2017 Gazette 2017/25) (54) METHODS AND COMPOSITIONS FOR T-RNA BASED GUIDE RNA EXPRESSION VERFAHREN UND ZUSAMMENSETZUNGEN ZUR T-RNA-BASIERTEN GUIDE-RNA-EXPRESSION PROCÉDÉS ET COMPOSITIONS POUR L’EXPRESSION D’ARN DE GUIDAGE À BASE D’ARNT (84) Designated Contracting States: (56) References cited: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB WO-A1-2015/138855 WO-A2-2015/126927 GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR • ANDREW A. HORWITZ ET AL: "Efficient Multiplexed Integration of Synergistic Alleles and (30) Priority: 18.12.2015 US 201562269121 P Metabolic Pathways in Yeasts via CRISPR-Cas", CELL SYSTEMS, vol. 1, no. 1, 12 March 2015 (43) Date of publication of application: (2015-03-12), pages 88-96, XP055230552, ISSN: 24.10.2018 Bulletin 2018/43 2405-4712, DOI: 10.1016/j.cels.2015.02.001 • KABIN XIE ET AL: "Boosting CRISPR/Cas9 (60) Divisional application: multiplex editing capability with the endogenous 20155737.8 tRNA-processing system", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, vol. (73) Proprietor: Danisco US Inc. 112, no. 11, 2 March 2015 (2015-03-02), pages Palo Alto, California 94304 (US) 3570-3575, XP055196411, ISSN: 0027-8424, DOI: 10.1073/pnas.1420294112 (72) Inventors: • ADAM L. MEFFERD ET AL: "Expression of • BENDEZU, Felipe Oseas CRISPR/Cas single guide RNAs using small tRNA Palo Alto promoters", RNA, vol. 21, no. 9, 17 July 2015 California 94304 (US) (2015-07-17), pages 1683-1689, XP055348964, US • FAN, Xiaochun ISSN: 1355-8382, DOI: 10.1261/rna.051631.115 Palo Alto • OWEN W RYAN ET AL: "Selection of California 94304 (US) chromosomal DNA libraries using a multiplex • FRISCH, Ryan L. CRISPR system", ELIFE, vol. 3, 19 August 2014 Palo Alto (2014-08-19), XP055175718, DOI: California 94304 (US) 10.7554/eLife.03703 • HONG, Seung-Pyo • CORY M. SCHWARTZ ET AL: "Synthetic RNA Palo Alto Polymerase III Promoters Facilitate California 94304 (US) High-Efficiency CRISPR-Cas9-Mediated Genome Editing in Yarrowia lipolytica", ACS SYNTHETIC (74) Representative: Mewburn Ellis LLP BIOLOGY, vol. 5, no. 4, 29 December 2015 City Tower (2015-12-29), pages 356-359, XP055343808, USA 40 Basinghall Street ISSN: 2161-5063, DOI: London EC2V 5DE (GB) 10.1021/acssynbio.5b00162 Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 3 390 631 B1 Printed by Jouve, 75001 PARIS (FR) EP 3 390 631 B1 Description FIELD 5 [0001] The disclosure relates to the field of molecular biology, in particular, to methods for producing guide RNAs and methods for altering the genome of a cell. BACKGROUND 10 [0002] Recombinant DNA technology has made it possible to insert DNA sequences at targeted genomic locations and/or modify (edit) specific endogenous chromosomal sequences, thus altering the organism’s phenotype. Site-specific integration techniques, which employ site-specific recombination systems, as well as other types of recombination technologies, have been used to generate targeted insertions of genes of interest in a variety of organism. Genome- editing techniques such as designer zinc finger nucleases (ZFNs), transcription activator-like effector nucleases 15 (TALENs), homing meganucleases, engineered nucleases are available for producing targeted genome perturbations, but these systems tend to have a low specificity and employ designed nucleases that need to be redesigned for each target site, which renders them costly and time-consuming to prepare. CRISPR-associated (Cas) RNA-guided endonu- clease systems have been developed as a means for introducing site-specific DNA strand breaks at specific target sites. These nuclease based systems can create a single strand or double strand break (DSB) in a target nucleotide, which 20 can increase the frequency of homologous recombination at the target locus. [0003] WO2015/138855 and Ryan et al. ("Selection of chromosomal DNA libraries using a multiplex CRISPR system", ELIFE, vol. 3, 19 August 2014) describe an expression construct for Cas9 sgRNA in yeast comprising a tRNA promoter operably linked to a polynucleotide encoding a ribozyme and the sgRNA. [0004] Inhibition of gene expression can be accomplished, for example, by interrupting or deleting the DNA sequence 25 of the gene, resulting in "knock-out" of the gene. Gene knock-outs mostly have been carried out through homologous recombination (HR), a technique applicable across a wide array of organisms from bacteria to mammals. Another tool for studying gene function can be through genetic "knock-in", which is also usually performed by HR. HR for purposes of gene targeting (knock-out or knock-in) can use the presence of an exogenously supplied DNA having homology with the target site. Although gene targeting by HR is a powerful tool, it can be a complex, labor-intensive procedure. Most 30 studies using HR have generally been limited to knock-out of a single gene rather than multiple genes in a pathway, since HR is generally difficult to scale-up in a cost-effective manner. This difficulty is exacerbated in organisms in which HR is not efficient. Such low efficiency typically forces practitioners to rely on selectable phenotypes or exogenous markers to help identify cells in which a desired HR event occurred. [0005] Thus there remains a need for new and more efficient genome engineering technologies that are affordable, 35 easy to set up, scalable, and amenable to targeting multiple positions within the genome of an organism. BRIEF SUMMARY [0006] Compositions and methods are provided for editing nucleotides and/or altering target sites in the genome of a 40 cell. The methods and compositions employ a recombinant DNA construct comprising a tRNA promoter operably linked to a polynucleotide encoding a single guide RNA, wherein said recombinant DNA construct does not comprise a nucleotide sequence encoding a ribozyme, wherein said guide RNA is capable of forming a guide RNA/Cas endonuclease complex, wherein said complex can bind to and cleave a target site sequence in the genome of a non-conventional yeast. [0007] In one embodiment of the disclosure, the disclosure comprises a recombinant DNA construct comprising a 45 tRNA promoter operably linked to a polynucleotide encoding a single guide RNA, wherein said recombinant DNA construct does not comprise a nucleotide sequence encoding a ribozyme, wherein said guide RNA is capable of forming a guide RNA/Cas endonuclease complex, wherein said complex can bind to and cleave a target site sequence in the genome of a non-conventional yeast. The tRNA promoter is selected from the group consisting of a tRNA or a tRNA fragment capable of functioning as a promoter sequence. The tRNA can be selected from the group consisting of a tRNA-Lys, 50 tRNA-Val, tRNA-Glu, tRNA Leu, tRNA-ile, tRNA-trp, tRNA-tyr, tRNA-his, or any one combination thereof. The tRNA fragment can be selected from the group consisting of a polynucleotide comprising the S-, D-, A-, V-, and T- domains of a tRNA, a polynucleotide comprising the S-, D-, V-, and T-domains of the tRNA, a polynucleotide comprising the S-, D-, and T- domains of the tRNA, and a polynucleotide comprising the S-, and T- domains of the tRNA. [0008] Also provided is a non-conventional yeast comprising any one of the recombinant DNA constructs described 55 herein. The non-conventional yeast can be a member of a genus selected from the group consisting of Yarrowia, Pichia, Schwanniomyces, Kluyveromyces, Arxula, Trichosporon, Candida, Ustilago, Torulopsis, Zygosaccharomyces, Trigo- nopsis, Cryptococcus, Rhodotorula, Phaffia, Sporobolomyces, and Pachysolen [0009] In one embodiment of the disclosure, the method comprises a method for modifying a target site on a chromo- 2 EP 3 390 631 B1 some or episome in a non-conventional yeast, the method comprising providing to a non-conventional yeast at least a first recombinant DNA construct as described herein and a second recombinant DNA construct encoding a Cas endo- nuclease, wherein the Cas endonuclease introduces a single or double-strand break at said target site. The method can further comprise comprising identifying at least one non-conventional yeast cell that has a modification at said target 5 site, wherein the modification includes at least one deletion, addition or substitution of one or more nucleotides in said target site. [0010] These methods can further comprise identifying the mutation efficiency in said non-conventional yeasts. In one embodiment, the mutation efficiency can be least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20 fold higher compared to a method for modifying a target site in said non-conventional yeast utilizing a ribozyme linked single guide RNA. 10 [0011] In one embodiment of the disclosure, the method comprises a method for editing a nucleotide sequence on a chromosome or episome in a non-conventional yeast, the method comprising providing to a non-conventional yeast a polynucleotide modification template DNA, a first recombinant DNA construct comprising a DNA sequence encoding a Cas endonuclease, and a second recombinant DNA construct described herein, wherein the Cas9 endonuclease intro- duces a single or double-strand break at a target site in the chromosome or episome of said yeast, wherein said 15 polynucleotide modification template DNA comprises at least one nucleotide modification of said nucleotide sequence.
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