Genome Editing by Guided Endonuclease and Single-stranded Oligonucleotide

15:

• A method for introducing one or more desired nucleotide modification(s) in at least one target sequence in the genome of a microorganism cell using a polynucleotide-guided endonuclease, said method comprising the steps of:
• a) providing a microorganism host cell comprising at least one genome target sequence to be modified located in the vicinity of a protospacer adjacent motif (PAM) sequence for the polynucleotide-guided endonuclease;
• b) transforming the microorganism host cell with:
• i) the polynucleotide-guided endonuclease and at least one suitable guide polynucleotide for the at least one target sequence to be modified, OR one or more polynucleotide encoding the polynucleotide-guided endonuclease and encoding at least one suitable guide polynucleotide for the at least one target sequence to be modified, and
• ii) at least one single-stranded oligonucleotide capable of hybridizing with the at least one genome target sequence, said oligonucleotide comprising the one or more desired nucleotide modification(s);
• wherein the at least one genome target sequence to be modified is located from 20 to 250 nucleotides away from the protospacer adjacent motif (PAM) sequence for the polynucleotide-guided endonuclease in the genome of the microorganism host cell;
• wherein the polynucleotide-guided endonuclease interacts with the guide polynucleotide and with a protospacer-complementary sequence in the genome and cuts or nicks the genome,
• and wherein the at least one single-stranded oligonucleotide directs DNA repair across the cut or nick, thereby introducing the one or more desired modification(s) into the target sequence of the genome with an efficiency of at least:
• 70%, preferably at least 80% when the cut or nick is located 10-20 nucleotides from the desired nucleotide modification(s),
• 60% when the cut or nick is located 21-30 nucleotides from the desired nucleotide modification(s),
• 50% when the cut or nick is located 31-43 nucleotides from the desired nucleotide modification(s),
• 40% when the cut or nick is located 44-52 nucleotides from the desired nucleotide modification(s), or
• 30% when the cut or nick is located at least 53 nucleotides from the desired nucleotide modification(s).

20:

• The method of claim 19, wherein the microorganism host cell is selected from the species of Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, and Trichoderma viride.