WO2017191210A1

WO2017191210A1 - Genome editing by crispr-cas9 in filamentous fungal host cells

Info

Publication number: WO2017191210A1
Application number: PCT/EP2017/060570
Authority: WO
Inventors: Dorte M.K. KLITGAARD
Original assignee: Novozymes A/S
Priority date: 2016-05-04
Filing date: 2017-05-03
Publication date: 2017-11-09

Abstract

The present invention relates to methods for modifying at least one target sequence in the genome of a filamentous fungal host cell by employing a Class II Cas9 enzyme, e.g., the S. pyogenes Cas9, together with a suitable guide RNA for each target sequence to generate a site-specific cut or nick in at least one genome target sequence, followed by the repair of the cut(s) and/or nick(s) via integration of one or more modified modified donor part of the filamentous fungal host cell genome through double recombination on each side of the nick(s).

Description

Genome Editing by CRISPR-Cas9 in Filamentous Fungal Host Cells

Reference to a Sequence Listing

This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference.

Field of the Invention

The invention provides methods for modifying the genome of a filamentous fungal host cell by employing a Class II Cas9 enzyme, e.g., the S. pyogenes Cas9.

Background of the Invention

The so-called CRISPR (clustered regularly interspaced short galindromic repeats) Cas9 genome editing system originally isolated from Streptomyces pyogenes has been widely used as a tool to modify the genomes of a number of eukaryotes.

The Cas9 enzyme has two RNA-guided DNA endonuclease domains capable of targeting specific genomic sequences. The system has been described extensively for editing genomes in a variety of eukaryotes (Doudna, J.A. and E. Charpentier, Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science, 2014. 346(6213): p.1258096), E. coli (Jiang, W., et al., RNA-guided editing of bacterial genomes using CRISPR- Cas systems. Nat Biotechnol, 2013. 31 (3): p. 233-9), yeast (DiCarlo, J.E., et al., Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Res, 2013. 41 (7): p. 4336-43), Lactobacillus (Oh, J.H. and J. P. van Pijkeren, CRISPR-Cas9-assisted recombineering in Lactobacillus reuteri. Nucleic Acids Res, 2014. 42(17): p. e131 ) and filamentous fungi such as Trichoderma reesei (Liu, R., et al., Efficient genome editing in filamentous fungus Trichoderma reesei using the CRISPR/Cas9 system. Cell Discovery, 2015.

1 )-

The power of the Cas9 system lies in its simplicity to target and edit up to a single base pair in a specific gene of interest. In addition, it is possible to target multiple genes for modification (multiplexing) in a single reaction, generate insertions and deletions, as well as silence or activate genes. In 2012, The CRISPR-Cas9 protein was shown to be a dual-RNA guided endonuclease protein (Jinek, M., et al., A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science, 2012. 337(6096): p. 816-21.). Further development for utilization the CRISPR-Cas9 as a genome editing tool has led to the engineering of a single guided RNA molecule that guides the endonuclease to its DNA target. The single guide RNA retains the critical features necessary for both interaction with the Cas9 protein and further targeting to the desired nucleotide sequence. When complexed with the

RNA molecule, the Cas9 protein will bind DNA sequence and create a double stranded break using two catalytic domains. When engineered to contain a single amino acid mutation in either catalytic domain, the Cas9 protein functions as a nickase, a variant protein with single stranded cleavage activity. Genome editing in Clostridium cellulyticum via CRISPR-Cas9 nickase was recently demonstrated by Xu et al. (Xu, T., et al., Efficient Genome Editing in Clostridium cellulolyticum via CRISPR-Cas9 Nickase. Appl Environ Microbiol, 2015. 81 (13): p. 4423-31.).

Many scientific publications and published patent applications relating to the CRISPR- Cas9 genome editing technology have become available over the last 5-10 years. More recently, a general method for transforming a replicative plasmid carrying the S. pyogenes Cas9-encoding gene into Aspergillus niger was described by N0dvig et al. (A CRISPR-Cas9 System for Genetic Engineering of Filamentous Fungi. 2015. PLoS ONE 10(7): e0133085. doi:10.1371/journal. pone.0133085).

An efficient double split-marker or bi-partite selection marker filamentous fungal host cell transformation system was disclosed in Nielsen et al. (Efficient PCR-based gene targeting with a recyclable marker for Aspergillus nidulans, Fungal Genetics and Biology, 2006(43) 54 to 64).

Summary of the Invention

The invention provides methods for modifying at least one target sequence in the genome of a filamentous fungal host cell by employing a Class II Cas9 enzyme, e.g., the S. pyogenes Cas9, together with a suitable guide RNA for each target sequence to generate a site-specific cut or nick in at least one genome target sequence, followed by the repair of the cut(s) and/or nick(s) via integration of one or more modified modified donor part of the filamentous fungal host cell genome through double recombination on each side of the cut(s) or nick(s), preferably while also employing a double split-marker system to ensure the correct specific genomic integration of the modified donor part.

Accordingly, in a first aspect the invention relates to methods for modifying the genome of a filamentous fungal host cell, said methods comprising the steps of:

A) providing a filamentous fungal host cell comprising at least one genome target sequence to be modified, wherein the at least one genome target sequence is flanked by a functional protospacer adjacent motif sequence for a Class-ll Cas9 enzyme;

B) transforming the filamentous fungal host cell with:

i) a non-replicating polynucleotide construct comprising a polynucleotide encoding a Class-ll Cas9 enzyme and a polynucleotide encoding a single-guide RNA or a guide RNA complex for the at least one target sequence to be modified, said single-guide RNA or guide RNA complex comprising:

a) a first RNA comprising 20 or more nucleotides that are at least 80% complementary to and capable of hybridizing to the at least one genome target sequence to be modified and comprising a tracr mate sequence, and b) a second RNA comprising a tracr sequence complementary to and capable of hybridizing with the tracr mate sequence of (a); and

ii) at least one polynucleotide construct comprising one or more modified donor part of the filamentous fungal host cell genome, said donor part comprising the at least one genome target sequence having one or more desired nucleotide modification(s) as well as at least 15 unmodified nucleotides flanking the modification(s) on each side;

wherein the 20 or more nucleotides of the first RNA hybridize with the at least one genome target sequence and wherein the variant Class-ll Cas9 enzyme interacts with the single-guide RNA or the guide RNA complex and cuts or nicks the at least one genome target sequence,

whereafter the one or more modified donor part of the filamentous fungal host cell genome is inserted into the genome by a recombination event on each side of the cut or nick, thereby introducing the one or more desired mofication(s) into the genome; and

C) selecting a filamentous fungal host cell, wherein the at least one genome target sequence has been modified.

Brief Description of the Figures

Figure 1 shows a schematic drawing of the plasmid pDMKP1 136 the construction of which is described in Example 1 below.

Figure 2 shows a schematic drawing of the plasmid pCRISPR1 -4h which is identical to plasmid pFC332 disclosed in N0dvig et al. 2015 {vide supra).

Figure 3 shows a schematic drawing of the plasmid DMKP1 145 the construction of which is described in Example 4 below.

Figure 4 shows a schematic drawing of the plasmid DMKP1 149 the construction of which is described in Example 6 below.

Definitions

cDNA: The term "cDNA" means a DNA molecule that can be prepared by reverse transcription from a mature, spliced, mRNA molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA. The initial, primary RNA transcript is a precursor to mRNA that is processed through a series of steps, including splicing, before appearing as mature spliced mRNA.

Coding sequence: The term "coding sequence" means a polynucleotide, which directly specifies the amino acid sequence of a polypeptide. The boundaries of the coding sequence are generally determined by an open reading frame, which begins with a start codon such as ATG, GTG, or TTG and ends with a stop codon such as TAA, TAG, or TGA. The coding sequence may be a genomic DNA, cDNA, synthetic DNA, or a combination thereof. Control sequences: The term "control sequences" means nucleic acid sequences necessary for expression of a polynucleotide encoding a mature polypeptide of the present invention. Each control sequence may be native (i.e., from the same gene) or foreign (i.e., from a different gene) to the polynucleotide encoding the polypeptide or native or foreign to each other. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the polynucleotide encoding a polypeptide.

Expression: The term "expression" includes any step involved in the production of a polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

Expression vector: The term "expression vector" means a linear or circular DNA molecule that comprises a polynucleotide encoding a polypeptide and is operably linked to control sequences that provide for its expression.

Host cell: The term "host cell" means any cell type that is susceptible to transformation, transfection, transduction, or the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.

Isolated: The term "isolated" means a substance in a form or environment that does not occur in nature. Non-limiting examples of isolated substances include (1 ) any non-naturally occurring substance, (2) any substance including, but not limited to, any enzyme, variant, nucleic acid, protein, peptide or cofactor, that is at least partially removed from one or more or all of the naturally occurring constituents with which it is associated in nature; (3) any substance modified by the hand of man relative to that substance found in nature; or (4) any substance modified by increasing the amount of the substance relative to other components with which it is naturally associated (e.g., recombinant production in a host cell; multiple copies of a gene encoding the substance; and use of a stronger promoter than the promoter naturally associated with the gene encoding the substance).

Mature polypeptide: The term "mature polypeptide" means a polypeptide in its final form following translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc. In one aspect, the mature polypeptide is based on the signal peptide prediction program SignalP (Nielsen et al., 1997, Protein Engineering 10: 1 -6). It is known in the art that a host cell may produce a mixture of two of more different mature polypeptides (i.e., with a different C-terminal and/or N-terminal amino acid) expressed by the same polynucleotide. It is also known in the art that different host cells process polypeptides differently, and thus, one host cell expressing a polynucleotide may produce a different mature polypeptide (e.g., having a different C-terminal and/or N-terminal amino acid) as compared to another host cell expressing the same polynucleotide.

Mature polypeptide coding sequence: The term "mature polypeptide coding sequence" means a polynucleotide that encodes a mature polypeptide having activity. In one aspect, the mature polypeptide coding sequence is based on the signal peptide prediction program SignalP (Nielsen et al., 1997, supra).

Nucleic acid construct: The term "nucleic acid construct" or "polynucleotide construct" mean a nucleic acid or polynucleotide molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic, which comprises one or more control sequences.

Non-replicating polynucleotide construct: The term "non-replicating polynucleotide construct" in the present context is a polynucleotide construct that does not comprise a filamentous fungal autonomous replication initiation sequence, such as, the well-known AMA1 sequence.

Operably linked: The term "operably linked" means a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide such that the control sequence directs expression of the coding sequence.

Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity".

For purposes of the present invention, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:

(Identical Residues x 100)/(Length of Alignment - Total Number of Gaps in Alignment)

For purposes of the present invention, the sequence identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows: (Identical Deoxyribonucleotides x 100)/(Length of Alignment - Total Number of Gaps in Alignment)

Variant: The term "variant" means a polypeptide comprising an alteration, i.e., a substitution, insertion, and/or deletion, at one or more (e.g., several) positions. A substitution means replacement of the amino acid occupying a position with a different amino acid; a deletion means removal of the amino acid occupying a position; and an insertion means adding an amino acid adjacent to and immediately following the amino acid occupying a position.

DETAILED DESCRIPTION

In a first aspect the invention relates to methods for modifying the genome of a filamentous fungal host cell, said methods comprising the steps of:

B) transforming the filamentous fungal host cell with:

whereafter the one or more modified donor part of the filamentous fungal host cell genome is inserted into the genome by a recombination event on each side of the cut or nick, thereby introducing the one or more desired mofication(s) into the genome; and C) selecting a filamentous fungal host cell, wherein the at least one genome target sequence has been modified.

Filamentous Fungal Host Cells

The filamentous fungal cell of the instant invention includes all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra). The filamentous fungi are generally characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic.

The filamentous fungal host cell may be an Acremonium, Aspergillus, Aureobasidium,

Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cell.

For example, the filamentous fungal host cell may be an 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, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se. Suitable procedures for transformation of Aspergillus and Trichoderma host cells are described in EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81 : 1470-1474, and Christensen et al., 1988, Bio/Technology 6: 1419-1422. Suitable methods for transforming Fusarium species are described by Malardier et al., 1989, Gene 78: 147-156, and WO 96/00787. It is advantageous in the methods of the present invention to employ a filamentous fungal host cell that is unable to quickly repair the nicked target sequence(s) without integration of the modified donor part of the genome.

Accordingly, it is preferred that the filamentous fungal host cell provided in step (A) of the first aspect of the invention comprises an inactivated non-homologous end joining (NHEJ) system; preferably the cell comprises an inactivated DNA Ligase D (LigD) and/or DNA-end- binding protein Ku; even more preferably the cell comprises inactivated ligD, ku70 and or ku80 gene or homologoue(s) thereof. Genomic modifications

Any genomic coding sequence or gene that encodes a polypeptide may be modified at the nucleotide sequence level. Such modifications may not alter the amino acid sequence of the encoded polypeptide or they may lead to changes in the amino acid sequence, such as, deletions, insertions or substitutions.

If an amino acid is substituted for another amino acid with similar characteristics it may be termed a conservative substitution. Examples of conservative substitutions are within the groups of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methionine). Amino acid substitutions that do not generally alter specific activity are known in the art and are described, for example, by H. Neurath and R.L. Hill, 1979, In, The Proteins, Academic Press, New York. Common substitutions are Ala/Ser, Val/lle, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/lle, LeuA al, Ala/Glu, and Asp/Gly.

Alternatively, the amino acid changes are of such a nature that the physico-chemical properties of the polypeptides are altered. For example, amino acid changes may improve the thermal stability of the polypeptide, alter the substrate specificity, change the pH optimum, and the like.

Essential amino acids in a polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081 -1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for activity to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271 : 4699-4708. The active site of the enzyme or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224: 899- 904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. The identity of essential amino acids can also be inferred from an alignment with a related polypeptide.

Single or multiple amino acid substitutions, deletions, and/or insertions can be made and tested using known methods of mutagenesis, recombination, and/or shuffling, followed by a relevant screening procedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241 : 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991 , Biochemistry 30: 10832-10837; U.S. Patent No. 5,223,409; WO 92/06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner ei a/., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17: 893-896). Mutagenized DNA molecules that encode active polypeptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide.

The polypeptide may be a hybrid polypeptide in which a region of one polypeptide is fused at the N-terminus or the C-terminus of a region of another polypeptide.

The polypeptide may be a fusion polypeptide or cleavable fusion polypeptide in which another polypeptide is fused at the N-terminus or the C-terminus of the polypeptide of the present invention. A fusion polypeptide is produced by fusing a polynucleotide encoding another polypeptide to a polynucleotide of the present invention. Techniques for producing fusion polypeptides are known in the art, and include ligating the coding sequences encoding the polypeptides so that they are in frame and that expression of the fusion polypeptide is under control of the same promoter(s) and terminator. Fusion polypeptides may also be constructed using intein technology in which fusion polypeptides are created post- translationally (Cooper et al., 1993, EMBO J. 12: 2575-2583; Dawson et al., 1994, Science 266: 776-779).

A fusion polypeptide can further comprise a cleavage site between the two polypeptides. Upon secretion of the fusion protein, the site is cleaved releasing the two polypeptides. Examples of cleavage sites include, but are not limited to, the sites disclosed in Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000, J. Biotechnol. 76: 245-251 ; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13: 498-503; and Contreras et al., 1991 , Biotechnology 9: 378-381 ; Eaton et al., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995, Biotechnology 13: 982- 987; Carter et al., 1989, Proteins: Structure, Function, and Genetics 6: 240-248; and Stevens, 2003, Drug Discovery World 4: 35-48. Genome target sequence

The at least one genome target sequence to be modified by the methods of the invention is at least 20 nucleotides in length in order to allow its hybridization to the corresponding 20 nucleotide sequence of the guide RNA. The at least one genome target sequence to be modified can be located anywhere in the genome but will often be within a coding sequence or open reading frame.

The at least one genome target sequence to be modified need to have a suitable protospacer adjacent motif (PAM) located next to it to allow the corresponding Class-ll Cas9 nickase enzyme to bind a nick the target. The PAM for the S. pyogenes Cas9 enzyme has been reported to be a ccc triplet on the guide RNA complementary strand (the hybridizing strand of the target sequence). See Jinek M. et al., A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012;337:816-21 .

For an overview of other PAM sequences, see, for example, Shah, S.A. et al, Protospacer recognition motifs, RNA Biol. 2013 May 1 ; 10(5): 891-899.

Accordingly, in a preferred embodiment of the invention, the at least one genome target sequence to be modified comprises at least 20 nucleotides; preferably the at least one genome target sequence to be modified is comprised in an open reading frame encoding a polypeptide.

Class-ll Cas9 enzyme

Several Class-ll Cas9 analogues or homologues are known and more are being discovered almost monthly as the scientific interest has surged over the last few years; a review is provided in Makarova K.S. et al, An updated evolutionary classification of CRISPR- Cas systems, 2015, Nature vol. 13: 722-736.

The Cas9 enzyme of Streptomyces pyogenes is a model Class-ll Cas9 enzyme and it is to-date the best characterized.

A variant of this enzyme was developed which has only one active nuclease domain (as opposed to the two active domains in the wildtype enzyme) by substituting a single amino acid, aspartic acid for alanine, in position 10: D10A. It is expected that other Class-ll Cas9 enzymes may be modified similarly.

Accordingly, in a preferred embodiment, the variant of the Class-ll Cas9 enzyme having only one active nuclease domain comprises a substitution of aspartic acid for alanine in the amino acid position corresponding to position 10, D10A, in the Streptomyces pyogenes Cas9 amino acid sequence. Guide RNA

The guide RNA in CRISPR-Cas9 genome editing constitutes the re-programmable part that makes the system so versatile. In the natural S. pyogenes system the guide RNA is actually a complex of two RNA polynucleotides, a first crRNA containing about 20 nucleotides that determine the specificity of the Cas9 enzyme as well as the tracr RNA which hybridizes to the cr RNA to form an RNA complex that interacts with Cas9. See Jinek M. et al., A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012;337:816-21 . The terms crRNA and tracrRNA are used interchangeably with the terms tracr-mate RNA and tracr RNA herein.

Since the discovery of the CRISPR-Cas9 system single polynucleotide guide RNAs have been developed and successfully applied just as effectively as the natural two part guide RNA complex.

In a preferred embodiment, the single-guide RNA or RNA complex comprises a first RNA comprising 20 or more nucleotides that are at least 85% complementary to and capable of hybridizing to the at least one genome target sequence; preferably the 20 or more nucleotides are at least 90%, 95%, 97%, 98%, 99% or even 100% complementary to and capable of hybridizing to the at least one genome target sequence.

In another preferred embodiment, the host cell comprises a single-guide RNA comprising the first and second RNAs in the form of a single polynucleotide and wherein the tracr mate sequence and the tracr sequence form a stem-loop structure when hybridized with each other.

Modified donor part of the genome

The methods of the intant invention rely on the integration of a modified piece of genomic DNA back into the genome to replace a DNA section in the genome that contains the nicked or cut target sequence. This integration probably happens via a classical Campbell-type recombination event on each side of the nicked or cut target sequence, or actually just on each side of the nick or cut. This double recombination requires sufficient wildtype donor genomic DNA flanking the modified sequence to enable effective recombination. In our experience, this requires approximately at least 15 nucleotides of identical sequences to allow recombination between the genome and an incoming construct. So the modified donor DNA for integration should contain the actual modification plus around 15 nucleotides on each side for successful double recombination.

Accordingly, in a preferred embodiment the one or more modified donor part of the filamentous fungal host cell genome comprises at least 15 nucleotides; preferably at least 16 nucleotides; preferably at least 17, 18, 19, 20, 25, 30, 35, 40, 45, 50 or 100 nucleotides; more preferably at least 150; 200; 250; 300; 350; 400; 450; 500; 550; 600; 650; 700; 750; 800; 850; 900; 950; 1 ,000; 2,000; 4,000; 6,000; 8,000 or at least 10,000 nucleotides. Multiplexing

In a preferred embodiment at least one genome target sequence in the host cell selected in step (B) has been modified by at least one insertion, deletion and/or substitution of one or more nucleotide, codon, coding sequence or regulatory sequence.

It has been shown that several genome target sequences can be mofided simultaneously by employing a guide RNA together with Cas9. Logically, it should be possible to modify several different genome target sequences simultaneously by employing different corresponding guide RNAs or RNA complexes.

Accordingly, in a preferred embodiment of the invention, at least two genome target sequences in the host cell selected in step (B) have been modified by at least one insertion, deletion and/or substitution of one or more nucleotide, codon, coding sequence or regulatory sequence.

Polynucleotides

Techniques used to isolate or clone a polynucleotide are known in the art and include isolation from genomic DNA or cDNA, or a combination thereof. The cloning of the polynucleotides from genomic DNA can be effected, e.g., by using the well known polymerase chain reaction (PCR) or antibody screening of expression libraries to detect cloned DNA fragments with shared structural features. See, e.g., Innis et al., 1990, PCR: A Guide to Methods and Application, Academic Press, New York. Other nucleic acid amplification procedures such as ligase chain reaction (LCR), ligation activated transcription (LAT) and polynucleotide-based amplification (NASBA) may be used.

Modification of a polynucleotide encoding a polypeptide of the present invention may be necessary for synthesizing polypeptides substantially similar to the polypeptide. The term "substantially similar" to the polypeptide refers to non-naturally occurring forms of the polypeptide. These polypeptides may differ in some engineered way from the polypeptide isolated from its native source, e.g., variants that differ in specific activity, thermostability, pH optimum, or the like. The variants may be constructed on the basis of the polynucleotide presented as the mature polypeptide coding sequence, e.g., a subsequence thereof, and/or by introduction of nucleotide substitutions that do not result in a change in the amino acid sequence of the polypeptide, but which correspond to the codon usage of the host organism intended for production of the enzyme, or by introduction of nucleotide substitutions that may give rise to a different amino acid sequence. For a general description of nucleotide substitution, see, e.g., Ford et al., 1991 , Protein Expression and Purification 2: 95-107.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs that are transformed into the filamentous fungal host cell in the first aspect of the invention: The polynucleotide construct of (i) in the first aspect is a non-replicating polynucleotide comprising a polynucleotide encoding a Class-ll Cas9 enzyme and a polynucleotide encoding a single-guide RNA or a guide RNA complex for the at least one target sequence to be modified, said single-guide RNA or guide RNA complex comprising:

a) a first RNA comprising 20 or more nucleotides that are at least 80% complementary to and capable of hybridizing to the at least one genome target sequence to be modified and comprising a tracr mate sequence, and

b) a second RNA comprising a tracr sequence complementary to and capable of hybridizing with the tracr mate sequence of (a).

In a preferred embodiment, the non-replicating polynucleotide construct of (i) is a plasmid or a linear construct. It is also preferred that the construct of (i) does not comprise a selectable antibiotic resistance marker.

In another preferred embodiment, the non-replicating polynucleotide construct does not comprise a filamentous fungal autonomous replication initiation sequence, such as, the well- known AMA1 sequence.

The polynucleotide construct of (ii) in the first aspect comprises one or more modified donor part of the filamentous fungal host cell genome, said donor part comprising the at least one genome target sequence having one or more desired nucleotide modification(s) as well as at least 15 unmodified nucleotides flanking the modification(s) on each side;

The 20 or more nucleotides of the first RNA in the construct of (i) hybridize with the at least one genome target sequence in the method of the first aspect and the variant Class-ll Cas9 enzyme interacts with the single-guide RNA or the guide RNA complex and cuts or nicks the at least one genome target sequence in the method of the first aspect, whereafter the one or more modified donor part of the filamentous fungal host cell genome in the construct of (ii) is inserted into the genome by a recombination event on each side of the cut or nick, thereby introducing the one or more desired mofication(s) into the genome.

A polynucleotide to be expressed is operably linked to one or more control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences. The polynucleotide may be manipulated in a variety of ways to provide for expression of the polypeptide. Manipulation of the polynucleotide prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying polynucleotides utilizing recombinant DNA methods are well known in the art.

The control sequence may be a promoter, a polynucleotide that is recognized by a host cell for expression of a polynucleotide encoding a polypeptide of the present invention. The promoter contains transcriptional control sequences that mediate the expression of the polypeptide. The promoter may be any polynucleotide that shows transcriptional activity in the host cell including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.

Examples of suitable promoters for directing transcription of the nucleic acid constructs of the present invention in a filamentous fungal host cell are promoters obtained from the genes for Aspergillus nidulans acetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase {glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Fusarium oxysporum trypsin-like protease (WO 96/00787), Fusarium venenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO 00/56900), Fusarium venenatum Quinn (WO 00/56900), Rhizomucor miehei lipase, Rhizomucor miehei aspartic proteinase, Trichoderma reesei beta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I, Trichoderma reesei xylanase II, Trichoderma reesei xylanase III, Trichoderma reesei beta-xylosidase, and Trichoderma reesei translation elongation factor, as well as the NA2-tpi promoter (a modified promoter from an Aspergillus neutral alpha-amylase gene in which the untranslated leader has been replaced by an untranslated leader from an Aspergillus triose phosphate isomerase gene; non-limiting examples include modified promoters from an Aspergillus niger neutral alpha- amylase gene in which the untranslated leader has been replaced by an untranslated leader from an Aspergillus nidulans or Aspergillus oryzae triose phosphate isomerase gene); and mutant, truncated, and hybrid promoters thereof. Other promoters are described in U.S. Patent No. 6,01 1 ,147.

The control sequence may also be a transcription terminator, which is recognized by a host cell to terminate transcription. The terminator is operably linked to the 3'-terminus of the polynucleotide encoding the polypeptide. Any terminator that is functional in the host cell may be used in the present invention.

Preferred terminators for filamentous fungal host cells are obtained from the genes for Aspergillus nidulans acetamidase, Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase, Fusarium oxysporum trypsin-like protease, Trichoderma reesei beta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I, Trichoderma reesei xylanase II, Trichoderma reesei xylanase III, Trichoderma reesei beta-xylosidase, and Trichoderma reesei translation elongation factor. The control sequence may also be an mRNA stabilizer region downstream of a promoter and upstream of the coding sequence of a gene which increases expression of the gene.

The control sequence may also be a leader, a nontranslated region of an mRNA that is important for translation by the host cell. The leader is operably linked to the 5'-terminus of the polynucleotide encoding the polypeptide. Any leader that is functional in the host cell may be used.

Preferred leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.

The control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3'-terminus of the polynucleotide and, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence that is functional in the host cell may be used.

Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes for Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.

The control sequence may also be a signal peptide coding region that encodes a signal peptide linked to the N-terminus of a polypeptide and directs the polypeptide into the cell's secretory pathway. The 5'-end of the coding sequence of the polynucleotide may inherently contain a signal peptide coding sequence naturally linked in translation reading frame with the segment of the coding sequence that encodes the polypeptide. Alternatively, the 5'-end of the coding sequence may contain a signal peptide coding sequence that is foreign to the coding sequence. A foreign signal peptide coding sequence may be required where the coding sequence does not naturally contain a signal peptide coding sequence. Alternatively, a foreign signal peptide coding sequence may simply replace the natural signal peptide coding sequence in order to enhance secretion of the polypeptide. However, any signal peptide coding sequence that directs the expressed polypeptide into the secretory pathway of a host cell may be used.

Effective signal peptide coding sequences for filamentous fungal host cells are the signal peptide coding sequences obtained from the genes for Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicola insolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucor miehei aspartic proteinase.

The control sequence may also be a propeptide coding sequence that encodes a propeptide positioned at the N-terminus of a polypeptide. The resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases). A propolypeptide is generally inactive and can be converted to an active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding sequence may be obtained from the genes for Bacillus subtilis alkaline protease {aprE), Bacillus subtilis neutral protease (nprT), Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor miehei aspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide sequences are present, the propeptide sequence is positioned next to the N-terminus of a polypeptide and the signal peptide sequence is positioned next to the N-terminus of the propeptide sequence.

It may also be desirable to add regulatory sequences that regulate expression of the polypeptide relative to the growth of the host cell. Examples of regulatory sequences are those that cause expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. In filamentous fungi, the Aspergillus niger glucoamylase promoter, Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzae glucoamylase promoter, Trichoderma reesei cellobiohydrolase I promoter, and Trichoderma reesei cellobiohydrolase II promoter may be used. Other examples of regulatory sequences are those that allow for gene amplification. In eukaryotic systems, these regulatory sequences include the dihydrofolate reductase gene that is amplified in the presence of methotrexate, and the metallothionein genes that are amplified with heavy metals. In these cases, the polynucleotide encoding the polypeptide would be operably linked to the regulatory sequence. Expression Vectors

The present invention also relates to recombinant expression vectors comprising a polynucleotide of the present invention, a promoter, and transcriptional and translational stop signals. The various nucleotide and control sequences may be joined together to produce a recombinant expression vector that may include one or more convenient restriction sites to allow for insertion or substitution of the polynucleotide encoding the polypeptide at such sites. Alternatively, the polynucleotide may be expressed by inserting the polynucleotide or a nucleic acid construct comprising the polynucleotide into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can bring about expression of the polynucleotide. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may be a linear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one that, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, or a transposon, may be used.

The vector preferably contains one or more selectable markers that permit easy selection of transformed, transfected, transduced, or the like cells. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like.

Selectable markers for use in a filamentous fungal host cell include, but are not limited to, adeA (phosphoribosylaminoimidazole-succinocarboxamide synthase), adeB (phosphoribosyl-aminoimidazole synthase), amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents thereof. Preferred for use in an Aspergillus cell are Aspergillus nidulans or Aspergillus oryzae amdS and pyrG genes and a Streptomyces hygroscopicus bar gene. Preferred for use in a Trichoderma cell are adeA, adeB, amdS, hph, and pyrG genes.

The selectable marker may be a dual selectable marker system as described in WO 2010/039889. In one aspect, the dual selectable marker is an hph-tk dual selectable marker system.

The vector preferably contains an element(s) that permits integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on the polynucleotide's sequence encoding the polypeptide or any other element of the vector for integration into the genome by homologous or non-homologous recombination. Alternatively, the vector may contain additional polynucleotides for directing integration by homologous recombination into the genome of the host cell at a precise location(s) in the chromosome(s). To increase the likelihood of integration at a precise location, the integrational elements should contain a sufficient number of nucleic acids, such as from around 30 to around 10,000 base pairs, or from around 400 to around 10,000 base pairs, or from around 800 to around 10,000 base pairs, which have a high degree of sequence identity to the corresponding target sequence to enhance the probability of homologous recombination. The integrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell. Furthermore, the integrational elements may be non-encoding or encoding polynucleotides. On the other hand, the vector may be integrated into the genome of the host cell by nonhomologous recombination. More than one copy of a polynucleotide of the present invention may be inserted into a host cell to increase production of a polypeptide. An increase in the copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the polynucleotide where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the polynucleotide, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.

The procedures used to ligate the elements described above to construct the recombinant expression vectors of the present invention are well known to one skilled in the art (see, e.g., Sambrook et at., 1989, supra).

Reducing or Eliminating Gene Expression

Reducing or eliminating expression of the polynucleotide using, for example, one or more nucleotide insertion, disruption, substitution or deletion, is well known in the art.

In a method of the first aspect of invention, in a preferred embodiment, the genome of the host cell is modified to ensure that expression of a polynucleotide is reduced or eliminated, for example by modification, inactivation or full/partial deletion. The polynucleotide to be modified, inactivated or deleted may be, for example, the coding region or a part thereof essential for activity, or a regulatory element required for expression of the coding region. An example of such a regulatory or control sequence may be a promoter sequence or a functional part thereof, i.e., a part that is sufficient for affecting expression of the polynucleotide. Other control sequences for possible modification include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, signal peptide sequence, transcription terminator, and transcriptional activator.

Modification or inactivation of the polynucleotide may be performed by subjecting the parent cell to mutagenesis and selecting for mutant cells in which expression of the polynucleotide has been reduced or eliminated. The mutagenesis, which may be specific or random, may be performed, for example, by use of a suitable physical or chemical mutagenizing agent, by use of a suitable oligonucleotide, or by subjecting the DNA sequence to PCR generated mutagenesis. Furthermore, the mutagenesis may be performed by use of any combination of these mutagenizing agents.

Examples of a physical or chemical mutagenizing agent suitable for the present purpose include ultraviolet (UV) irradiation, hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), O-methyl hydroxylamine, nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formic acid, and nucleotide analogues.

When such agents are used, the mutagenesis is typically performed by incubating the parent cell to be mutagenized in the presence of the mutagenizing agent of choice under suitable conditions, and screening and/or selecting for mutant cells exhibiting reduced or no expression of the gene.

Modification or inactivation of the polynucleotide may be accomplished by insertion, substitution, or deletion of one or more nucleotides in the gene or a regulatory element required for transcription or translation thereof. For example, nucleotides may be inserted or removed so as to result in the introduction of a stop codon, the removal of the start codon, or a change in the open reading frame. Such modification or inactivation may be accomplished by site-directed mutagenesis or PCR generated mutagenesis in accordance with methods known in the art. Although, in principle, the modification may be performed in vivo, i.e., directly on the cell expressing the polynucleotide to be modified, it is preferred that the modification be performed in vitro as exemplified below.

An example of a convenient way to eliminate or reduce expression of a polynucleotide is based on techniques of gene replacement, gene deletion, or gene disruption. For example, in the gene disruption method, a nucleic acid sequence corresponding to the endogenous polynucleotide is mutagenized in vitro to produce a defective nucleic acid sequence that is then transformed into the parent cell to produce a defective gene. By recombination, the defective nucleic acid sequence replaces the endogenous polynucleotide. It may be desirable that the defective polynucleotide also encodes a marker that may be used for selection of transformants in which the polynucleotide has been modified or destroyed. In an aspect, the polynucleotide is disrupted with a selectable marker such as those described herein.

The polypeptide-deficient mutant cells are particularly useful as host cells for expression of native and heterologous polypeptides. Therefore, the present invention further relates to methods of producing a native or heterologous polypeptide, comprising (a) cultivating the mutant cell under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide. The term "heterologous polypeptides" means polypeptides that are not native to the host cell, e.g., a variant of a native protein. The host cell may comprise more than one copy of a polynucleotide encoding the native or heterologous polypeptide.

The present invention is further described by the following examples that should not be construed as limiting the scope of the invention.

Double Split-Marker System

The double split-marker system or bi-partite split marker system, as it is also called, was disclosed in Nielsen et al. (vide supra). The system was developed to secure efficient tranformation and correct site-specific genomic integration of polynucleotide(s) of interest in filamentous fungal host cells.

The present inventors found that the co-transformation of a non-replicative construct encoding Cas9 and modified donor DNA construct significantly improved the transformation efficiency compared to the system reported by N0dvig et al (vide supra). However, to eliminate any possibility of ectopic integration, the Cas9 and modified donor DNA co-transformation protocol was adapted to include a set split-markers in the filamentous fungal host cell flanking the target sequence to be modified as well as a set of corresponding split-markers flanking the modified donor DNA on the incoming construct. This way, only the correct genomic integration would lead to two functional selectable markers in the host cell genome. Surprisingly, this was much more efficient - both when a plasmid was employed as well as when a linear construct was employed. The latter construct was by far the most efficient.

Accordingly, in a preferred embodiment of the first aspect, the at least one genome target sequence to be modified in the filamentous fungal host cell is flanked on each side by a non-functional split selection marker gene, and wherein the at least one polynucleotide construct comprising one or more modified donor part of the filamentous fungal host cell genome also comprises a corresponding non-functional split selection marker gene on each side of the one or more modified donor part, whereby only the correct intended insertion of the modified donor part into the filamentous fungal host cell genome via double recombination events will restore the functionality of said selection marker genes. Preferably, the selection marker genes comprise niaD and nii^'A and only the correct insertion of the donor part via double recombination in the split niaD and niaA genes, respectively, will restore the functionality of both genes and both genes are required for the cell to grow on a sodium nitrate-containing medium.

EXAMPLES

All DNA for transformation was amplified using a proof-reading polymerase, Phusion (Thermo Fisher Scientific), according to the manufacturer's instructions. DNA was purified using the QIAquick kit (QIAGEN), according to the manufacturer's instructions.

DNA for sequencing was amplified using a proof-reading polymerase, KAPA HotStart ReadyMix (KAPA Biosystems), according to the manufacturer's instructions.

USER™ cloning relies on a commercial kit from New England Biolabs (Ipswich, MA, USA), according to the manufacturers instructions.

Two plasmids are employed in the examples below that were kindly provided by N0dvig et al. (see N0dvig et al. (A CRISPR-Cas9 System for Genetic Engineering of Filamentous Fungi. 2015. PLoS ONE 10(7): e0133085. doi:10.1371/journal. pone.0133085); the plasmids are identical except they carry different antibiotic selection markers:

• pFC330 (pCRISPR1 -4p) which carries a pyrG selection marker.

• pFC332 (pCRISPR1 -4h; figure 2) which carries a hph selection marker. Example 1. Preparation of donor DNA for deletion of the wA gene in A. oryzae

The purpose of this experiment was to construct a piece of donor DNA (gene targeting substrate) for deletion of wA in A. oryzae. The donor DNA comprises an expression construct for Thermomyces lanoginosus lipase (TLL), a pyrG marker from Aspergillus fumigatus, and 2000 bp flanks that are homologous to the upstream and downstream regions of wA. The nucleotide sequence of the wA gene is available from the Aspergillus genome database as accession number: AO090102000545.

Construction of plasmid pDMKP1 136

The plasmid pDMKP1 136 (figure 1 ) was constructed by PCR amplification of five PCR fragments that were subsequently assembled via USER™ cloning. The reactions were performed using an EPPENDORF^® MASTERCYCLER^® 5333 system programmed for 1 cycle at 95°C for 5 minutes; 35 cycles each at 98°C for 30 seconds, 58°C for 30 seconds, and 72°C for 4 minutes; and a final elongation at 72°C for 5 minutes. Subsequently, the two fragments were assembled via USER™ cloning, and verified by restriction analysis.

Table 1 .

Preparation of donor DNA for deletion of wA

Donor DNA with 2000 bp homologous flanks was PCR amplified from pDMKP1 136 using the primers: 5'-tgatcaacctcacctttggtcgg (forward) (SEQ ID NO:1 1 ) and

5'-aaaggtctcatatatactcgacctcgtgag (reverse) (SEQ ID NO: 12).

Example 2. Co-transformation of plasmid pCRISPR1 -4p and donor DNA

The purpose of this experiment was to show that co-transformation of the replicating

CRISPR/Cas9 plasmid pCRISPR1 -4p (wherein the "p" signifies the pyrG selection marker), which effectuates a double-stranded cut in the wA gene of A. oryzae, and the donor DNA of Example 1 for deletion of wA in A. oryzae, increases the transformation efficiency compared to transformation with donor DNA of Example 1 by itself.

This experiment is outlined in principle in figure 1 of N0dvig et al. 2015 (vide supra), where it was also carried out in several non-homologous end-joining (NHEJ) proficient Aspergillus species.

Transformation of COLS1392 (//qPA) and TOC1512 (liqP+, Ku70+, Ku80+)

The substrates were transformed into the two pyrG^~ strains COLS1392 and TOC1512 as shown below, and the number of transformants was counted after 3 days incubation at 30°C.

The ligD gene has been deleted in the host strain COLS1392, which rendered it severely impaired in its NHEJ capability.

Correctly integrated transformants were visually identifiable, as deletion of wA results in a color shift of the conidia from green to white.

Table 2.

Results

Everything was as expected. In both strains of Aspergillus oryzae co-transformation with donor DNA and a CRISPR plasmid with pyrG selection (pCRISPR1 -4p) was more efficient than transformation with donor DNA only. The double-stranded break induced by the CRISPR-Cas9 system was conducive to the introduction of the donor DNA via double recombination, as expected.

The NHEJ-impaired COLS1392 strain had a very low transformation efficiency when it was transformed with pCRISPR1 -4p alone. Without donor DNA to repair the double stranded break induced by the CRISPR-Cas9 system through recombination, and without a fully functioning NHEJ repair system, the transformed cells mostly died.

The NHEJ proficient TOC1512 host strain was clearly able to repair repair the double stranded break induced by the CRISPR-Cas9 system when it was transformed with pCRISPR1 -4p alone.

Example 3. Co-transformation of plasmid pCRISPR1 -4h and donor DNA

The purpose of this experiment was to show that co-transformation of the replicating CRISPR/Cas9 plasmid pCRISPR1 -4h (the "h" signifies a hph (hygromycin phosphatase) selection marker which was not selected for in this experiment; see figure 2) and donor DNA for deletion of wA in Aspergillus oryzae increases the transformation efficiency compared to transformation with donor DNA only, even in the absence of antibiotic selection for the pCRISPR1 -4h plasmid in the experiment.

Transformation of COLS1392 (//qPA) and TOC1512 (liqP+, Ku70+, Ku80+)

The substrates were transformed into the two pyrG^~ strains COLS1392 and TOC1512 as shown below, and the number of transformants was counted after 3 days incubation at 30°C. Correctly integrated transformants were visually identifiable, as deletion of wA results in a color shift of the conidia from green to white. Table 3.

Results

In both strains of Aspergillus oryzae, co-transformation with donor DNA and a CRISPR plasmid without any antibiotic selection was more efficient than transformation with donor DNA only. Example 4. Construction of the non-replicating plasmid pDMKP1145

The published CRISPR/Cas9 plasmids pCRISPR1 -4p and pCRISPR1 -4h (figure 2) contain a large, inverted repeat (AMA1 ) enabling autonomous replication of the plasmid in Aspergillus. In this experiment a CRISPR/Cas9 plasmid was constructed without the AMA1 sequence, thus abolishing the ability to replicate within Aspergillus.

Construction of the plasmid pDMKP1 145 (figure 3)

pCRISPR1 -4h was used as the template for PCR amplification of the entire vector except AMA1. The large vector was PCR amplified as two fragments as shown below. The reactions were performed using an EPPENDORF^® MASTERCYCLER^® 5333 system programmed for 1 cycle at 95°C for 5 minutes; 35 cycles each at 98°C for 30 seconds, 58°C for 30 seconds, and 72°C for 4 minutes; and a final elongation at 72°C for 5 minutes. Subsequently, the two fragments were assembled via USER cloning to create plasmid pDMKP1 145 (figure 3) which was verified by restriction analysis.

Table 4.

Example 5. Co-transformation of plasmid pDMKP1145 and donor DNA

The purpose of this experiment was to show that co-transformation of the non- replicating CRISPR/Cas9 plasmid pDMKP1 145 (described in the example above) and donor DNA for deletion of wA in Aspergillus oryzae increases the transformation efficiency significantly, even in the absence of antibiotic selection for the pDMKP1 145 plasmid which was not able to replicate in Aspergillus.

Transformation of COLS1392 (//qPA)

The substrates were transformed into the pyrG^~ strain COLS 1392 as shown below, and the number of transformants was counted after 3 days incubation at 30°C. Correctly integrated transformants were visually identifiable, as deletion of wA results in a color shift of the conidia from green to white. Table 5.

Results

Co-transformation with donor DNA and a non-replicating CRISPR plasmid without any antibiotic selection was 6-fold more efficient than transformation with donor DNA only.

The co-transformation of donor DNA + pCRISPR1 -4h worked better in this experiment than in Example 3 above, but the two results are not directly comparable as different ratios as well as total amounts of DNA were used. The two examples also used different batches of competent cells (protoplasts).

Surprisingly, co-transformation with a non-replicating plasmid was found to be about twice as efficient than co-transformation with a replicating (AMA1 -containing) plasmid.

Example 6. Construction of the non-replicating marker-free plasmid pDMKP1149

The CRISPR/Cas9 plasmids employed herein are quite large as they contain many different elements, e.g. an antibiotic selection marker which is not always applied. In this experiment we constructed a smaller non-replicating and markerless plasmid denoted pDMKP1 149 (figure 4).

Construction of the plasmid pDMKP1 149

pDMKP1 145 (figure 3) was used as the template for PCR amplification of the entire vector except the marker. The large vector was PCR amplified as two fragments as shown below. The reactions were performed using an EPPENDORF^® MASTERCYCLER^® 5333 system programmed for 1 cycle at 95°C for 5 minutes; 35 cycles each at 98°C for 30 seconds, 58°C for 30 seconds, and 72°C for 4 minutes; and a final elongation at 72°C for 5 minutes. Subsequently, the two fragments were assembled via USER™ cloning to create plasmid pDMKP1 149 (figure 4) which was verified by restriction analysis.

Table 6.

Example 7. Co-transformation of plasmid pDMKP1149 and donor DNA

The purpose of this experiment was to investigate if co-transformation of the non- replicating and marker-free CRISPR/Cas9 plasmid pDMKP1 149 (described in Example 6) and donor DNA for deletion of wA in Aspergillus oryzae might have an increased transformation efficiency.

The expectation was that the transformation efficiency of pDMKP1 149 would be on par its parent pDMKP1 145 and donor DNA (as described in Example 5 above), however, it turned out to be even higher.

Transformation of COLS1392 (//qPA)

The substrates were transformed into the pyrG^~ strain COLS1392 as shown below, and the number of transformants was counted after 3 days incubation at 30°C. Correctly integrated transformants were visually identifiable as deletion of wA results in a color shift of the conidia from green to white.

Table 7.

Results

In Aspergillus oryzae COLS1392, co-transformation with donor DNA and a CRISPR plasmid without any selection marker and without the ability to replicate was more efficient than transformation with donor DNA only. Surprisingly, co-transformation with the smaller marker- free plasmid pDMKP1 149 was found to be more efficient than co-transformation with the pDMKP1 145 plasmid, containing a marker which was not selected for.

Example 8. Co-transformation of a linear fragment and donor DNA

The purpose of this experiment was to investigate the co-transformation of a linear piece of DNA containing the same functional elements as the non-replicating marker-free CRISPR/Cas9 plasmid pDMKP1 149 (described in Example 6) and donor DNA for deletion of wA in Aspergillus oryzae. Preparation of a linear CRISPR fragment

A linear piece of DNA, containing all the functional CRISPR/Cas9 elements from pDMKP1 149 was made by PCR amplification, using the primers:

5'-taagctccctaattggccc (forward) (SEQ ID NO:21 )

5'-cttgtattgggatgaattttgtatgcac (reverse) (SEQ ID NO:22)

Transformation of COLS1392 (HQDA)

The substrates were transformed into the pyrG^~ strain COLS1392 as shown below, and the number of transformants was counted after 3 days incubation at 30°C. Correctly integrated transformants were visually identifiable as deletion of wA resulting in a color shift of the conidia from green to white.

Table 8.

Results

In Aspergillus oryzae COLS1392, co-transformation with donor DNA and a non- replicating linear CRISPR/Cas9 PCR fragment without a selection marker was more efficient than transformation with donor DNA alone, but plasmids had a higher transformation efficiency in this example.

Example 9. Construction of a plasmid guiding Cas9 towards amdS

The CRISPR/Cas9 constructs used in Example 1 -8 above contain a protospacer that guides the Cas9 nuclease towards wA, thus, inducing a double-stranded break at a specific site in wA. In this experiment, a CRISPR/Cas9 plasmid, pDMKP1 148, was constructed that contains another protospacer which targets amdS instead of wA.

Besides targeting the amdS gene, in this experiment we also rely on a so-called double split-marker system (DSMS) or bi-partite selection marker system (Nielsen et al. 2006, vide supra). The double split marker system herein consists of the two split markers in the Aspergillus oryzae COLS1300 strain: niaDA and niiAA, which flank the amdS selection marker. Only the correct insertion of the donor DNA via double recombination in the partial niaD and niaA genes, respectively, in host cell COLS1300 will restore the functionality of both genes and both genes are required for the cell to grow on a sodium nitrate-containing medium.

Construction of the plasmid pDMKP1 148

A protospacer was designed to target the amdS gene: the protospacer consisted of 20 nucleotides: 5' ggccgaactgaagatcacag (SEQ ID NO:23), followed by a CRISPR/Cas9 proteospacer adjacent motif (PAM) sequence: agg.

pDMKP1 145 was used as the template for PCR amplification of the entire vector. The large vector was PCR amplified as two fragments as shown below. The new protospacer was inserted as an overlap between fragment 1 and fragment 2. The reactions were performed using an EPPENDORF^® MASTERCYCLER^® 5333 system programmed for 1 cycle at 95°C for 5 minutes; 35 cycles each at 98°C for 30 seconds, 58°C for 30 seconds, and 72°C for 4 minutes; and a final elongation at 72°C for 5 minutes. Subsequently, the two fragments were assembled via USER cloning to create plasmid pDMKP1 148 which was verified by restriction analysis and sequencing.

Table 9.

Example 10. Co-transformation of plasmid pDMKP1148 and donor DNA using a DSMS

As explained above, the double split marker system (DSMS) of this experiment relies on an amdS selection marker inserted between the two split markers in the Aspergillus oryzae niaDA, niiAA strain COLS1300.

The purpose of this experiment was to investigate if co-transformation of donor DNA with a CRISPR/Cas9 construct targeting amdS in combination with a double split-marker system to eliminate ectopic integration increases the transformation efficiency. Transformation of COLS1300 (//qPA)

The substrates were transformed into the niaDA, niiAA strain COLS1300 as shown below, and the number of transformants was counted after 3 days incubation at 30°C. Correctly integrated transformants were selected for on minimal medium with sodium nitrate as sole nitrogen source. Due to the design of the DSMS, ectopic integration of the donor DNA would not result in growth on sodium nitrate.

Table 10.

Results

Evidently, co-transformation with donor DNA employing a double-split marker system for targeting as well as a non-replicating CRISPR/Cas9 plasmid was more efficient, even in the absence of any antibiotic selection for the plasmid, than any of the constructs in the above experiments.

Example 11. Co-transformation of a linear fragment and donor DNA using a DSMS

Like in the previous example, the double split marker system (DSMS) of this experiment relies on an amdS selection marker inserted between the two split markers in the Aspergillus oryzae niaDA, niiAA strain COLS1300.

The purpose of this experiment was to investigate if co-transformation of a donor DNA employing a double split-marker system and a linear CRISPR/Cas9 construct targeting amdS would increase the transformation efficiency.

Preparation of a linear CRISPR fragment

5'-taagctccctaattggccc (forward) (SEQ ID NO: 28) and

5'-cttgtattgggatgaattttgtatgcac (reverse) (SEQ ID NO: 29).

Transformation of COLS1300 (//qPA)

Table 1 1 .

Results

Surprisingly, the co-transformation with donor DNA employing a DSMS and a linear CRISPR/Cas9 fragment targeting amdS, was much more efficient than any of the other constructs tested herein.

Claims

1 . A method for modifying the genome of a filamentous fungal host cell, said method comprising the steps of:

B) transforming the filamentous fungal host cell with:

i) a non-replicating polynucleotide construct comprising a polynucleotide encoding a Class-ll Cas9 enzyme and a polynucleotide encoding a single-guide RNA or a guide RNA complex for the at least one target sequence to be modified, said single-guide

RNA or guide RNA complex comprising:

2. The method of claim 1 , wherein the filamentous fungal host cell is an Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cell.

3. The method of claim 1 or 2, wherein the filamentous fungal host cell is an 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, or Trichoderma viride cell.

4. The method of any of the preceding claims, wherein the filamentous fungal host comprises an inactivated non-homologous end joining (NHEJ) system; preferably the cell comprises an inactivated DNA Ligase D (LigD) and/or DNA-end-binding protein Ku; even more preferably the cell comprises an inactivated HgD, kulQ and/or ku80 gene or homologue(s) thereof.

5. The method of any of the preceding claims, wherein the non-replicating polynucleotide construct is a plasmid or a linear construct.

6. The method of any of the preceding claims, wherein the non-replicating polynucleotide construct does not comprise a selectable antibiotic resistance marker.

7. The method of any of the preceding claims, wherein the at least one genome target sequence to be modified in the filamentous fungal host cell is flanked on each side by a nonfunctional split selection marker gene, and wherein the at least one polynucleotide construct comprising one or more modified donor part of the filamentous fungal host cell genome also comprises a corresponding non-functional split selection marker gene on each side of the one or more modified donor part, whereby only the correct intended insertion of the modified donor part into the filamentous fungal host cell genome via double recombination events will restore the functionality of said selection marker genes.

8. The method of claim 7, wherein the selection marker genes comprise niaD and niiA and only the correct insertion of the donor part via double recombination in the split niaD and niaA genes, respectively, will restore the functionality of both genes and both genes are required for the cell to grow on a sodium nitrate-containing medium.

9. The method of any of the preceding claims, wherein the Class-ll Cas9 enzyme is a Streptomyces pyogenes Cas9 or a homologue thereof.

10. The method of any of the preceding claims, wherein the Class-ll Cas9 enzyme is a variant of a Class-ll Cas9 enzyme having only one active nuclease domain; preferably said variant comprises a substitution of aspartic acid for alanine in the amino acid position corresponding to position 10, D10A, in the Streptomyces pyogenes Cas9 amino acid sequence.

1 1 . The method of any of the preceding claims, wherein the at least one genome target sequence to be modified comprises at least 20 nucleotides; preferably the at least one genome target sequence to be modified is comprised in an open reading frame encoding a polypeptide.

12. The method of any of the preceding claims, wherein the single-guide RNA or RNA complex comprises a first RNA comprising 20 or more nucleotides that are at least 85% complementary to and capable of hybridizing to the at least one genome target sequence; preferably the 20 or more nucleotides are at least 90%, 95%, 97%, 98%, 99% or even 100% complementary to and capable of hybridizing to the at least one genome target sequence.

13. The method of any of the preceding claims, wherein the non-replicating polynucleotide construct comprises a polynucleotide encoding a single-guide RNA comprising the first and second RNAs in the form of a single polynucleotide, and wherein the tracr mate sequence and the tracr sequence form a stem-loop structure when hybridized with each other.

14. The method of any of the preceding claims, wherein the one or more modified donor part of the filamentous fungal host cell genome comprises at least 150 nucleotides; preferably at least 200 nucleotides; more preferably at least 250; 300; 350; 400; 450; 500; 550; 600; 650; 700; 750; 800; 850; 900; 950; 1 ,000; 2,000; 4,000; 6,000; 8,000 or at least 10,000 nucleotides.

15. The method of any of the preceding claims, wherein the at least one polynucleotide construct comprising one or more modified donor part of the filamentous fungal host cell genome comprises at least 15 unmodified nucleotides flanking the modified donor part comprising the at least one genome target sequence having one or more desired nucleotide modification(s) on each side; preferably at least 16 nucleotides; preferably at least 17, 18, 19, 20, 25, 30, 35, 40, 45, 50 or 100 nucleotides; more preferably at least 150; 200; 250; 300; 350; 400; 450; 500; 550; 600; 650; 700; 750; 800; 850; 900; 950; 1 ,000; 2,000; 4,000; 6,000; 8,000 or at least 10,000 nucleotides.

16. The method of any of the preceding claims, wherein at least one genome target sequence in the host cell selected in step (B) has been modified by at least one insertion, deletion and/or substitution of one or more nucleotide, codon, coding sequence, expression construct or regulatory sequence.

17. The method of any of the preceding claims, wherein at least two genome target sequences in the host cell selected in step (B) have been modified by at least one insertion, deletion and/or substitution of one or more nucleotide, codon, coding sequence, expression construct or regulatory sequence.