WO2019046703A1

WO2019046703A1 - Methods for improving genome editing in fungi

Info

Publication number: WO2019046703A1
Application number: PCT/US2018/049066
Authority: WO
Inventors: Jeffrey Shasky; Suchindra Maiyuran; Jan Lehmbeck
Original assignee: Novozymes A/S
Priority date: 2017-09-01
Filing date: 2018-08-31
Publication date: 2019-03-07

Abstract

The present disclosure relates to methods for modifying the genome of a fungal cell, comprising: (A) introducing into the fungal cell: (i) a first nucleic acid construct comprising a polynucleotide encoding an RNA-guided DNA endonuclease; (ii) a second nucleic acid construct comprising (a) a U6 promoter sequence operably linked to (1) a transfer RNA encoding sequence, (2) a single guide RNA encoding sequence, and (b) a U6 terminator sequence; and optionally (iii) a third nucleic acid construct comprising a donor DNA for modifying the genome; and (B) selecting a transformant of the fungal cell, wherein the genome is modified.

Description

METHODS FOR IMPROVING GENOME EDITING IN FUNGI

Reference to a Sequence Listing

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

Background of the Disclosure

Field of the Disclosure

The present disclosure relates to methods for improving genome editing in fungi.

Description of the Related Art

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas systems have evolved in bacteria as adaptive immune defenses that can introduce double strand breaks in DNA in a sequence-specific manner. Such systems perform this function via the activity of a ribonucleoprotein complex that includes short RNA sequences known as tracrRNA and crRNA and an RNA dependent endonuclease (Cas endonuclease) that targets a specific DNA sequence via homology in a gene of interest to a portion of the crRNA, called the variable targeting domain, and generates double strand breaks in the target DNA sequence.

The CRISPR/Cas9 endonuclease system has been described extensively for editing genomes in a variety of eukaryotes (Doudna et al., 2014, Science 346: 1258096), E. coli (Jiang et ai, 2013, Nat. Biotechnol. 31 : 233-239), yeast (DiCarlo et ai, 2013, Nucleic Acids Res. 41 : 4336-4343), Lactobacillus (Oh et al., 2014, Nucleic Acids Res. 42: e131), and filamentous fungi such as Trichoderma reesei (Liu et al., 2015, Cell Discovery 1 : 15007), Aspergillus fumigatus (Fuller et al., 2015, Eukaryot. Cell. 14(1 1): 1073-1080; Zhang et ai., 2015, Fungal Genet. Biol. 86: 47-57), and M. oryzae (Arazoe et al. , 2015, Biotechnol. Bioeng. 112(12): 2543- 2549).

The power of the CRISPR/Cas9 endonuclease system lies in its simplicity to target and edit a single base pair or more 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, and silence or activate genes. The CRISPR-Cas9 endonuclease is a dual-RNA guided endonuclease protein (Jinek ef al., 2012, Science 337: 816-821). Further development of CRISPR-Cas9 as a genome editing tool has led to engineering of a single guide RNA molecule that guides the endonuclease to its DNA target. The single guide RNA retains the critical features necessary for interacting with the Cas9 endonuclease and targeting to the desired nucleotide sequence. When complexed with the RNA molecule, the Cas9 endonuclease will bind to a 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 strand cleavage activity.

Schwartz et al., 2016, ACS Synthetic Biology 5 (7): 754-764, disclose synthetic RNA polymerase III promoters for facilitating high-efficiency CRISPR-Cas9-mediated genome editing in the yeast Yarrowia lipolytica.

WO 2015/131101 discloses the use of U2, U3, U5, U6, and 7SL promoters for driving expression of a single guide RNA.

There is a need in the art for developing more efficient Cas-based genome editing systems for use in fungal cells.

The present disclosure provides improved methods for modifying the genome of a fungal cell.

Summary of the Disclosure

The present disclosure relates to methods for modifying the genome of a fungal cell, the method comprising the steps of:

(A) introducing into the fungal cell:

(i) a first nucleic acid construct comprising a polynucleotide encoding an RNA-guided DNA endonuclease for introducing a double-strand break at a target site in the genome of the fungal cell, wherein the fungal cell comprises a protospacer adjacent motif sequence for the RNA-guided DNA endonuclease immediately following the 3' end of the target site;

(ii) a second nucleic acid construct comprising (a) a U6 promoter sequence operably linked at the 5' end of (1) a sequence encoding a transfer RNA and (2) a sequence encoding a single guide RNA at the 3' end of the transfer RNA sequence, and (b) a U6 transcriptional terminator sequence operably linked at the 3' end of the sequence encoding the single guide RNA, wherein the single guide RNA directs the RNA-guided DNA endonuclease to the target site to introduce the double-strand break, and wherein the second nucleic acid construct increases the frequency of the RNA- guided DNA endonuclease in producing the double-strand break at the target site; and

(iii) a third nucleic acid construct comprising a donor DNA comprising a nucleotide sequence of interest for modifying the target site, wherein the third nucleic acid construct is incorporated into the target site by endogenous DNA repair, and

(B) selecting a transformant of the fungal cell, wherein the target site is modified with the nucleotide sequence of interest. In one embodiment of the method, the donor DNA further comprises a 5' homology sequence and a 3' homology sequence flanking the nucleotide sequence of interest, wherein the 5' homology sequence and the 3' homology sequence correspond to an upstream region and a downstream region, respectively, of the target site for incorporation of the donor DNA into the double-strand break by homologous recombination.

The present disclosure also relates to methods for modifying the genome of a fungal cell, the method comprising the steps of:

(A) introducing into the fungal cell:

(i) a first nucleic acid construct comprising a polynucleotide encoding an RNA-guided DNA endonuclease for introducing a double-strand break at a target site in the genome of the fungal cell, wherein the fungal cell comprises a protospacer adjacent motif sequence for the RNA-guided DNA endonuclease immediately before the 5' end or immediately following the 3' end of the target site; and

(B) selecting a transformant of the fungal cell, wherein the target site is modified. The present disclosure also relates to a fungal cell obtained by such methods.

The present disclosure also relates to a fungal cell, comprising:

(i) a first nucleic acid construct comprising a polynucleotide encoding an RNA- guided DNA endonuclease for introducing a double-strand break at a target site in the genome of the fungal cell, wherein the fungal cell comprises a protospacer adjacent motif sequence for the RNA-guided DNA endonuclease immediately before the 5' end or immediately following the 3' end of the target site;

(ii) a second nucleic acid construct comprising (a) a U6 promoter sequence operably linked at the 5' end of (1) a sequence encoding a transfer RNA and (2) a sequence encoding a single guide RNA at the 3' end of the transfer RNA sequence, and (b) a U6 transcriptional terminator sequence operably linked at the 3' end of the sequence encoding the single guide RNA, wherein the single guide RNA directs the RNA-guided DNA endonuclease to the target site to introduce the double-strand break, and wherein the second nucleic acid construct increases the frequency of the RNA-guided DNA endonuclease in producing the double-strand break at the target site; and

(iii) a third nucleic acid construct comprising a donor DNA comprising a nucleotide sequence of interest for modifying the target site, wherein the third nucleic acid construct is incorporated into the target site by endogenous DNA repair.

In one embodiment of the fungal cell, the donor DNA further comprises a 5' homology sequence and a 3' homology sequence flanking the nucleotide sequence of interest, wherein the 5' homology sequence and the 3' homology sequence correspond to an upstream region and a downstream region, respectively, of the target site for incorporation of the donor DNA into the double-strand break by homologous recombination.

The present disclosure also relates to a fungal cell, comprising:

(ii) a second nucleic acid construct comprising (a) a U6 promoter sequence operably linked at the 5' end of (1) a sequence encoding a transfer RNA and (2) a sequence encoding a single guide RNA at the 3' end of the transfer RNA sequence, and (b) a U6 transcriptional terminator sequence operably linked at the 3' end of the sequence encoding the single guide RNA, wherein the single guide RNA directs the RNA-guided DNA endonuclease to the target site to introduce the double-strand break, and wherein the second nucleic acid construct increases the frequency of the RNA-guided DNA endonuclease in producing the double-strand break at the target site.

The present disclosure also relates to a nucleic acid construct comprising (a) a U6 promoter sequence operably linked at the 5' end of (1) a sequence encoding a transfer RNA and (2) a sequence encoding a single guide RNA at the 3' end of the transfer RNA sequence, and (b) a U6 transcriptional terminator sequence operably linked at the 3' end of the sequence encoding the single guide RNA, wherein the single guide RNA directs an RNA-guided DNA endonuclease to a target site in a fungal cell to introduce a double-strand break, and wherein the nucleic acid construct increases the frequency of the RNA-guided DNA endonuclease in producing the double-strand break at the target site.

Brief Description of the Drawings

Figure 1 shows a map of plasmid pSMai271.

Figure 2 shows a map of plasmid pSMai268.

Figure 3 shows a map of plasmid pSMai274. Figure 4 shows a map of plasmid pSMai280.

Figure 5 shows a map of plasmid pJfyS238.

Figure 6 shows a map of plasmid pJfyS242.

Figure 7 shows a map of plasmid pJfyS254.

Figure 8 shows a map of plasmid pJfyS244.

Figure 9 shows a map of plasmid pJfyS245.

Figure 10 shows a map of plasmid pJfyS247.

Figure 1 1 shows a map of plasmid pSMai279

Figure 12 shows a map of plasmid pJfyS249.

Figure 13 shows a map of plasmid pJfyS250.

Figure 14 shows a map of plasmid pJfyS251.

Figure 15 shows a map of plasmid pJfyS253.

Figure 16 shows a map of plasmid pAT1 153.

Figure 17 shows a map of plasmid pAT1 154.

Figure 18 shows a map of plasmid pJfyS259.

Figure 19 shows a map of plasmid pCHSN2.

Figure 20 shows a map of pSMai322a.

Definitions

Cas endonuclease: The term "Cas endonuclease" means an RNA-guided DNA endonuclease associated with CRISPR that cleaves a target DNA sequence when coupled with a single guide RNA. The Cas endonuclease is guided by the single guide RNA(s) to recognize and cleave a specific target site in double stranded DNA in the genome of a cell. CRISPR-Cas systems are currently classified as Type I, Type II, and Type III CRISPR-Cas systems (Liu and Fan, 2014, Plant Mol. Biol. 85: 209-218). For purposes of the present disclosure, the CRISPR-Cas system is a Type II CRISPR-Cas system employing a Cas9 endonuclease or variant thereof (including, for example, a Cas9 nickase). The Cas9 endonuclease comprises two nuclease domains, an HNH (McrA-like) nuclease domain that cleaves the complementary DNA strand and a RuvC-like nuclease domain that cleaves the noncomplementary DNA strand. Target recognition and cleavage by the Cas9 endonuclease requires a chimeric single guide RNA consisting of a fusion of crRNA (a 20-nucleotide guide sequence and a partial direct repeat) and tracrRNA (frans-activating crRNA) and a short conserved sequence motif downstream of the crRNA binding region, called a protospacer adjacent motif (PAM). In the CRISPR-Cas9 system derived from the bacterium Streptococcus pyogenes, the target DNA immediately precedes a 5-NGG PAM. The RNA-guided Cas9 endonuclease activity creates site-specific double strand breaks, which are then repaired by either non-homologous end joining (NHEJ) or homology-directed repair (HDR). It is understood that the term "Cas endonuclease" encompasses variants thereof.

Cas9 nickase: The term "Cas9 nickase" means a Cas9 endonuclease that introduces a single-strand nick into a target double stranded DNA sequence when coupled with a chimeric single guide RNA. Cas9 nickases can be generated recombinantly by inactivating one of the two nuclease domains in a parent Cas9 endonuclease (e.g., by site-directed mutagenesis). A non-limiting example of a Cas9 nickase is the Cas9 nickase in which the RuvC domain is inactivated by a D10A mutation in the Cas9 endonuclease from Streptococcus pyogenes (Sander and Joung, 2013, Nature Biotechnology 1-9). Two guide RNAs designed on opposite DNA strands are required with a Cas9 nickase to create a double stranded break.

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.

Codon-optimized gene: The term "codon-optimized gene" means a gene having its frequency of codon usage optimized to the frequency of preferred codon usage of a host cell. The nucleic acid changes made to codon-optimize a gene do not change the amino acid sequence of the encoded polypeptide of the parent gene.

Control sequences: The term "control sequences" means nucleic acid sequences necessary for expression of a polynucleotide comprising a non-coding RNA or a polynucleotide encoding a polypeptide. Each control sequence may be native (i.e., from the same gene) or heterologous (i.e., from a different gene) to the polynucleotide encoding the polypeptide or native or heterologous 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 a transcriptional stop signal. 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.

Cpf endonuclease: The term "Cpf endonuclease" means an RNA-guided DNA endonuclease associated with CRISPR that cleaves a target DNA sequence when coupled with a single guide RNA. The Cpf endonuclease is guided by the single guide RNA(s) to recognize and cleave a specific target site in double stranded DNA in the genome of a cell. For purposes of the present disclosure, the CRISPR-Cpf system employs an Acidaminococcus sp. Cpf1 endonuclease, a Lachnospiraceae sp. Cpf1 endonuclease, or a Francisella novicide Cpf1 endonuclease or variant thereof. The Cpf1-crRNA complex cleaves target DNA by identification of a protospacer adjacent motif (PAM) 5'-TTTN for the Acidaminococcus sp. Cpf1 endonuclease and Lachnospiraceae sp. Cpf1) endonuclease, and a PAM sequence 5'-TTN for the Francisella novicide Cpf Ί . After identification of the PAM, Cpf1 introduces sticky-end DNA double-stranded break of 4-5 nucleotides overhang distal to the 3' end of the targeted PAM which is then repaired by either non-homologous end joining (NHEJ) or homology-directed repair (HDR). It is understood that the term "Cpf1 endonuclease" encompasses variants thereof.

Donor DNA: The term "donor DNA" means a polynucleotide that comprises a nucleotide sequence of interest for modifying a target site in the genome of a fungal cell. The donor DNA can be double-stranded DNA. The nucleotide sequence of the donor DNA can be any nucleotide sequence such as a gene or a region of a gene, one or more nucleotides for introducing a mutation into a gene, a gene disruption sequence, etc. In one aspect, the donor DNA further comprises a first region of homology and a second region of homology to corresponding regions of the target site for incorporation of the donor DNA into the double- strand break by homologous recombination, i.e., the donor DNA has a high degree of homology to the sequence immediately upstream and downstream of the intended editing site. The term "donor DNA" is also understood herein to mean "DNA repair template".

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. The term "expression" also means production of a non-coding RNA (e.g., a single guide RNA).

Expression vector: The term "expression vector" means a linear or circular DNA molecule that comprises a polynucleotide encoding a polypeptide or a non-coding polynucleotide (e.g., a single guide RNA) and is operably linked to control sequences that provide for its expression.

Genome: The term "genome" means the complete set of genetic information in a fungal cell which is present as long molecules of DNA called chromosomes and extrachromosomal elements of DNA (e.g., plasmids) and RNA.

Guide RNA or single guide RNA: The term "guide RNA" (gRNA) or "single guide RNA" (sgRNA) means an engineered single-stranded RNA, involving (1) the targeting function of the CRISPR RNA (crRNA) sequence (for Mad7 and Cpf1), or (2) the targeting function of the CRISPR RNA (crRNA) and the nuclease-binding function of the transactivating CRISPR RNA (tracrRNA) sequence (for Cas9). For the Cas9 endonuclease, the crRNA sequence is an approximately 20 nucleotide sequence that defines the genomic target of interest for modification via homology and directs Cas9 endonuclease activity. The 20 nucleotide sequence acts as a "guide", which recruits the Cas9/gRNA complex to a specific DNA target site based on the crRNA sequence, directly upstream of a protospacer adjacent motif (PAM), through RNA-DNA base pairing. The PAM is required for cleavage, but is not part of the gRNA or sgRNA sequence. The Cas9 endonuclease will cleave approximately 3 bases upstream of the PAM. For the Mad7 and Cpf1 endonucleases, they are guided by a single CRISPR RNA (crRNA) and does not require a transactivating CRISPR RNA (tracrRNA). The Mad7 and Cpf 1 endonucleases cleave DNA distal to its PAM after the +18/+23 position of the protospacer creating a staggered DNA overhang.

Homologous recombination: The term "homologous recombination" means the exchange of DNA fragments between two DNA molecules at sites of homology via a classical Campbell-type homologous recombination event.

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. 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.

Hybridization: The term "hybridization" means the pairing of substantially complementary strands of nucleic acids through base pairing. A nucleic acid sequence is considered to be hybridizable to a reference nucleic acid sequence if the two sequences specifically hybridize to one another under medium to very high stringency conditions, using, for example, standard Southern blotting procedures. Hybridization may be performed under medium, medium-high, high or very high stringency conditions. Medium stringency conditions means prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 35% formamide for 12 to 24 hours, followed by washing three times each for 15 minutes using 0.2X SSC, 0.2% SDS at 55°C. Medium- high stringency conditions means prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 35% formamide for 12 to 24 hours, followed by washing three times each for 15 minutes using 0.2X SSC, 0.2% SDS at 60°C. High stringency conditions means prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide for 12 to 24 hours, followed by washing three times each for 15 minutes using 0.2X SSC, 0.2% SDS at 65°C. Very high stringency conditions means prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide for 12 to 24 hours, followed by washing three times each for 15 minutes using 0.2X SSC, 0.2% SDS at 70°C. High and very high stringency conditions can be used to identify sequences having strict identity or near- strict identity with a hybridization probe, while medium to medium-high stringency conditions can be used to identify homologs.

Introduce or introducing: The term "introduce" or "introducing" means inserting a polynucleotide into a cell (e.g., a recombinant nucleic acid construct) by any method known in the art such as transfection, transformation, transduction, electroporation, particle bombardment, cell fusion techniques, or the like.

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).

Mad endonuclease: The term "Mad endonuclease" means an RNA-guided DNA endonuclease associated with CRISPR that cleaves a target DNA sequence when coupled with a single guide RNA. The Mad endonuclease is guided by the single guide RNA(s) to recognize and cleave a specific target site in double stranded DNA in the genome of a cell. CRISPR-Mad systems are closely related to the Type V (Cpf1-like) of Class-2 family of CAS enzymes. For purposes of the present disclosure, the CRISPR-Mad system employs an Eubacterium rectale Mad7 endonuclease or variant thereof. The Mad7-crRNA complex cleaves target DNA by identification of a protospacer adjacent motif (PAM) 5'-YTTN. After identification of the PAM, Mad7 introduces sticky-end DNA double-stranded break of 4-5 nucleotides overhang to the 3' end of the targeted PAM which is then repaired by either nonhomologous end joining (NHEJ) or homology-directed repair (HDR). It is understood that the term "Mad endonuclease" encompasses variants thereof.

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 (Bendtsen et a/., 2004, J. Mol. Biol. 340: 783-795). 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 biological activity. In one aspect, the mature polypeptide coding sequence is based on the signal peptide prediction program SignalP (Bendtsen et al., 2004, supra).

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

Nucleic acid construct: The term "nucleic acid construct" means 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 may comprise one or more control sequences.

Non-replicating nucleic acid construct: The term "non-replicating nucleic acid construct" means a nucleic acid construct that does not comprise an 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 a polynucleotide such that the control sequence directs expression of the polynucleotide.

Promoter: The term "promoter" means a DNA sequence that defines where transcription of a gene by an RNA polymerase begins. A promoter is located directly upstream or at the 5' end of the transcription start site of a gene. RNA polymerase and the necessary transcription factors bind to the promoter sequence and initiate transcription.

Protospacer adjacent motif: The term "protospacer adjacent motif" or "PAM" means a 2-6 base pair DNA sequence immediately downstream or upstream of the target site in the genome, which is recognized directly by an RNA-guided DNA endonuclease, e.g., a Cas9, Mad7, or Cpf1 endonuclease, to promote cleavage of the target site by the RNA-guided DNA endonuclease. The Cas9 endonuclease from Streptococcus pyogenes recognizes 5'-NGG on the 3' end of the gRNA sequence. The Mad7 endonuclease from Eubacterium rectale recognizes 5'-YTTN on the 5' end of the gRNA sequence. The Cpf1 endonuclease from Acidaminococcus sp. and Lachnospiraceae sp. recognize 5'-TTTN and the Cpf1 endonuclease Francisella novicide recognizes 5'-TTN-3' on the 5' end of the gRNA.

RNA-guided DNA endonuclease: The term "RNA-guided DNA endonuclease" refers to any nuclease that can form a complex with a guide RNA (gRNA) molecule in which the gRNA contains a sequence homologous to a target region in the genome of a host cell. The complex of the "RNA-guided DNA endonuclease" with its corresponding gRNA is capable of cleaving DNA within, proximal, or distal to the homologous target sequence contained within the gRNA depending on the mechanism of the specific nuclease. The homologous region in the genome is proximal or distal to a Protospacer Adjacent Motif or PAM sequence, which is specific for each type of RNA-guided endonuclease. The RNA-guided endonuclease activity creates site-specific double strand breaks, which are then repaired by either non-homologous end joining (NHEJ) or homology-directed repair (HDR). Examples of RNA-guided endonucleases described to date include Cas9, Cpf1 , and Mad7. It is understood that the term "RNA-guided DNA endonuclease" encompasses variants thereof.

RNA polymerase III: The term "RNA polymerase III" means a nucleotidyl transferase that polymerizes ribonucleotides using DNA genes as templates (Paule and White, 2000, Nucleic Acids Res. 28(6): 1283) to produce small ribonucleic (RNA) molecules including, but not limited to, aminoacyl transfer RNAs, 5S ribosomal RNAs, splicecomal RNAs (snRNAs), and U6 small nuclear RNAs.

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 disclosure, 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 disclosure, 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)

Target site: The term "target site" means a polynucleotide sequence in the genome of a fungal cell comprising a protospacer sequence immediately adjacent to a PAM sequence, wherein an RNA-guided DNA endonuclease, e.g., a Cas9, Mad7, and Cpf1 endonuclease, recognizes the PAM sequence and catalyzes cleavage within the protospacer sequence. The target site can be any segment of the genome of a fungal cell. In one embodiment, the target site is a gene or region thereof, e.g., an open reading frame, a protein coding sequence, an intron site, an intron enhancing motif, a mRNA splice site, a promoter, a transcriptional regulatory element, a transcriptional terminator, and a translational regulatory element.

Transcriptional terminator: The term "transcriptional terminator" means a DNA sequence downstream of the polynucleotide sequence of a gene which is recognized by RNA polymerase as a signal to stop synthesizing and release nascent RNA from the transcriptional complex.

Transfer RNA: The term "transfer RNA" means a molecule composed of RNA, typically 73 to 94 nucleotides in length, that serves as the physical link between the nucleotide sequence of nucleic acids and the amino acid sequence of proteins. Transfer RNA carries an amino acid to the protein synthetic machinery of a cell (ribosome) as directed by a three- nucleotide sequence (codon) in a messenger RNA (mRNA) and attaches the correct amino acid to a protein chain that is being synthesized at the ribosome cell when the anticodon of the tRNA pairs with a codon on the mRNA being translated into the protein. There are at least 20 species of transfer RNA, each species capable of combining with a specific amino acid. Each type of transfer RNA molecule can be attached to only one type of amino acid, so each organism has many types of transfer RNA. Since the genetic code contains multiple codons that specify the same amino acid, there are many transfer RNA molecules bearing different anticodons which also carry the same amino acid. There are often multiple species of tRNA for each codon and as a result there can be more than one hundred tRNA genes within the genome of a particular fungal cell. For example, see Hani and Feldman, 1998, Nucleic Acids Res. 26: 689-696. The terms "transfer RNA" and "tRNA" are used interchangeably herein.

U6 promoter: The term "U6 promoter" means a promoter obtained from a U6 small nuclear RNA (snRNA) gene and transcribed by RNA polymerase III. Variant: The term "variant" means a polypeptide comprising an alteration, i.e., a substitution, an insertion, and/or a 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.

In accordance with this detailed disclosure, the following definitions apply. Note that the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

Detailed Description of the Disclosure

The present disclosure relates to methods for modifying the genome of a fungal cell employing a guide RNA/RNA-guided DNA endonuclease, e.g., a Cas9, Mad7, or Cpfl endonuclease system, wherein a nucleic acid construct comprising a U6 promoter and U6 transcriptional terminator operably linked to (1) a sequence encoding a transfer RNA and (2) a sequence encoding a single guide RNA, increases the frequency of the RNA-guided DNA endonuclease in producing a double-strand break at a target site in the genome of the fungal cell.

In one aspect, the present disclosure relates to a method for modifying the genome of a fungal cell, the method comprising the steps of:

(A) introducing into the fungal cell:

(i) a first nucleic acid construct comprising a polynucleotide encoding an RNA-guided DNA endonuclease for introducing a double-strand break at a target site in the genome of the fungal cell, wherein the fungal cell comprises a protospacer adjacent motif sequence for the RNA-guided DNA endonuclease immediately before the 5' end or immediately following the 3' end of the target site;

(ii) a second nucleic acid construct comprising (a) a U6 promoter sequence operably linked at the 5' end of (1) a sequence encoding a transfer RNA and (2) a sequence encoding a single guide RNA at the 3' end of the transfer RNA sequence, and (b) a U6 transcriptional terminator sequence operably linked at the 3' end of the sequence encoding the single guide RNA, wherein the single guide RNA directs the

RNA-guided DNA endonuclease to the target site to introduce the double-strand break, and wherein the second nucleic acid construct increases the frequency of the RNA- guided DNA endonuclease in producing the double-strand break at the target site; and

(iii) a third nucleic acid construct comprising a donor DNA comprising a nucleotide sequence of interest for modifying the target site, wherein the third nucleic acid construct is incorporated into the target site by endogenous DNA repair; and (B) selecting a transformant of the fungal cell, wherein the target site is modified with the nucleotide sequence of interest.

In one embodiment of the method, the donor DNA further comprises a 5' homology sequence and a 3' homology sequence flanking the nucleotide sequence of interest, wherein the 5' homology sequence and the 3' homology sequence correspond to an upstream region and a downstream region, respectively, of the target site for incorporation of the donor DNA into the double-strand break by homologous recombination.

In another aspect, the present disclosure relates to a method for modifying the genome of a fungal cell, the method comprising the steps of:

(A) introducing into the fungal cell:

(i) a first nucleic acid construct comprising a polynucleotide encoding an RNA-guided DNA endonuclease for introducing a double-strand break at a target site of the fungal cell, wherein the fungal cell comprises a protospacer adjacent motif sequence for the RNA-guided DNA endonuclease immediately before the 5' end or immediately following the 3' end of the target site; and

(ii) a second nucleic acid construct comprising (a) a U6 promoter sequence operably linked at the 5' end of (1) a sequence encoding a transfer RNA, (2) a sequence encoding a single guide RNA at the 3' end of the transfer RNA sequence, and (b) a U6 transcriptional terminator sequence operably linked at the 3' end of the sequence encoding the single guide RNA, wherein the single guide RNA directs the

RNA-guided DNA endonuclease to the target site to introduce the double-strand break, and wherein the second nucleic acid construct increases the frequency of the RNA- guided DNA endonuclease in producing the double-strand break at the target site; and (B) selecting a transformant of the fungal cell, wherein the target site is modified. In another aspect, the present disclosure relates to a fungal cell, comprising:

(i) a first nucleic acid construct comprising a polynucleotide encoding an RNA- guided DNA endonuclease for introducing a double-strand break at a target site of the fungal cell to be modified, wherein the fungal cell comprises a protospacer adjacent motif sequence for the RNA-guided DNA endonuclease immediately before the 5' end or immediately following the 3' end of the target site;

In another aspect, the present disclosure also relates to a fungal cell, comprising:

It has been shown that several genomic loci can be modified simultaneously by employing a guide RNA together with an RNA-guided DNA endonuclease. Logically, it should be possible to modify several different genome loci simultaneously by employing different corresponding guide RNAs.

Accordingly, in a preferred embodiment of the present disclosure, at least two genomic loci in a fungal cell are modified by at least one insertion, deletion and/or substitution of one or more nucleotides, codons, coding sequences or regulatory sequences.

The present disclosure demonstrates in the Examples of the present disclosure that a nucleic acid construct comprising a combination of (1) a U6 promoter sequence, (2) a transfer RNA sequence, (3) a sequence encoding a single guide RNA, and (4) a U6 transcriptional terminator sequence operably linked to each other increases the frequency of an RNA-guided DNA endonuclease in producing a double-strand break at a target site. Each of the nucleic acid constructs described above for the RNA-guided DNA endonuclease, the single guide RNA, and the donor DNA can be introduced into a fungal cell in any manner known in the art, including, for example, transfection, transformation, transduction, electroporation, particle bombardment, cell fusion techniques, or the like. The constructs can be introduced sequentially as desired or simultaneously. In one embodiment, the first, second, and third constructs are on separate fragments. In another embodiment, the first, second, and third constructs are on the same fragment. In another embodiment, the first and second constructs are on the same fragment. In another embodiment, the first and third constructs are on the same fragment. In another embodiment, the second and third constructs are on the same fragment.

When editing a genome with a donor DNA, each nucleic acid construct is preferably a non-replicating nucleic acid construct in that the construct does not comprise an autonomous replication initiation sequence. When editing a genome without a donor DNA, each nucleic acid construct is preferably a replicating nucleic acid construct comprising an autonomous replication initiation sequence to prevent integration of the construct.

The advantages of using donor DNA is that you can introduce specifically by homologous recombination the desired insertion, deletion or point mutation, whereas without donor DNA the repair of the double stranded break occurs by non-homologous-end-joining and results in a non-specific mutation (deletion or in some cases a small insertion) around the double stranded break.

RNA-Guided Endonucleases

In the methods of the present disclosure, any RNA-guided DNA endonuclease can be used.

The RNA-guided DNA endonuclease can be a Cas endonuclease, a Mad endonuclease, or a Cpf endonuclease.

In one aspect, the Cas endonuclease can be any Cas endonuclease or a functional fragment thereof useful in the methods of the present disclosure. In one embodiment, the Cas endonuclease is a Cas9 endonuclease. Examples of Cas9 endonucleases are the Cas9 endonucleases from the following bacterial species: Streptococcus sp. (e.g., S. pyogenes, S. mutans, and S. thermophilus), Campylobacter sp. (e.g. , C. jejuni), Neisseria sp. (e.g., N. meningitidis), Francisella sp. (e.g., F. novicida), and Pasteurella sp. (e.g., P. multocida). For a discussion of Cas9 endonucleases, see Makarova et al., 2015, Nature 13: 722-736.

In another embodiment, the Cas9 endonuclease is a Streptococcus pyogenes Cas9 endonuclease (e.g., SEQ ID NO: 1 1). In another embodiment, the Cas9 endonuclease is a

Streptococcus mutans Cas9 endonuclease (e.g., SEQ ID NO: 52). In another embodiment, the Cas9 endonuclease is a Streptococcus thermophilus Cas9 endonuclease {e.g., SEQ ID NO: 54). In another embodiment, the Cas9 endonuclease is a Campylobacter jejuni Cas9 endonuclease (e.g., SEQ ID NO: 56). In another embodiment, the Cas9 endonuclease is a Neisseria meningitidis Cas9 endonuclease (e.g., SEQ ID NO: 58). In another embodiment, the Cas9 endonuclease is a Francisella novicida Cas9 endonuclease (e.g., SEQ ID NO: 60). In another embodiment, the Cas9 endonuclease is a Pasteurella multocida Cas9 endonuclease (e.g., SEQ ID NO: 62).

In another embodiment, the Cas9 endonuclease is a variant of a parent Cas9 endonuclease. In one embodiment, the Cas9 endonuclease variant is a Cas9 nickase in which the RuvC domain is inactivated by a D10A mutation in the Cas9 endonuclease from Streptococcus pyogenes (Sander and Joung, 2013, Nature Biotechnology 1-9). It is expected that other Class-ll Cas9 enzymes may be modified similarly.

In another embodiment, the Cas9 endonuclease is a Streptomyces pyogenes Cas9 endonuclease (SEQ ID NO: 1 1). In another embodiment, the Cas9 endonuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1 1. In another embodiment, the Cas9 endonuclease is encoded by a polynucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 10.

In another embodiment, the Cas9 endonuclease variant has only one active nuclease domain. In a more preferred embodiment, the Cas9 endonuclease variant comprises a substitution with alanine in the amino acid position corresponding to position 10 of the Streptomyces pyogenes Cas9 amino acid sequence. In a most preferred embodiment, the Cas9 endonuclease variant comprises a substitution of aspartic acid for alanine at position 10, D10A, of the Streptomyces pyogenes Cas9 amino acid sequence.

In another aspect, the Mad endonuclease can be any Mad endonuclease or a functional fragment thereof useful in the methods of the present disclosure. In one embodiment, the Mad endonuclease is a Mad7 endonuclease. An example of a Mad7 endonuclease is the Mad7 endonuclease from Eubacterium rectale. For a discussion of the Mad7 endonuclease, see WO 2018/071672.

In another embodiment, the Mad7 endonuclease is a Eubacterium Mad7 endonuclease. In another embodiment, the Eubacterium Mad7 endonuclease is an Eubacterium rectale Mad7 endonuclease. In another embodiment, the Eubacterium rectale Mad7 endonuclease comprises the amino acid sequence of SEQ ID NO: 94.

In another embodiment, the Mad7 endonuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 94. In another preferred embodiment, the Mad7 endonuclease is encoded by a polynucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 93.

In one aspect, the Cpf endonuclease can be any Cpf endonuclease or a functional fragment thereof useful in the methods of the present disclosure. In one embodiment, the Mad endonuclease is a Cpf1 endonuclease. Examples of Cpf1 endonucleases are the Cpf1 endonucleases from Acidaminococcus sp., Lachnospiraceae sp., and Francisella novicide. For a discussion of the Cpf1 endonuclease, see Zetsche et al. , 2015, Cell 163(3): 759-771.

In another embodiment, the Cpf1 endonuclease is an Acidaminococcus Cpfl endonuclease. In another embodiment, the Acidaminococcus Cpf1 comprises the amino acid sequence of SEQ ID NO: 105. In another embodiment, the Cpf1 endonuclease is a Lachnospiraceae Cpf1 endonuclease. In another embodiment, the Lachnospiraceae Cpf1 comprises the amino acid sequence of SEQ ID NO: 107. In another embodiment, the Cpf1 endonuclease is a Francisella Cpf1 endonuclease. In another embodiment, the Cpf1 endonuclease is a Francisella novicide Cpf1 endonuclease. In another embodiment, the Francisella novicide Cpf1 comprises the amino acid sequence of SEQ ID NO: 109.

In another embodiment, the Cpf1 endonuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 105. In another preferred embodiment, the Cpf1 endonuclease is encoded by a polynucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 104.

In another embodiment, the Cpf1 endonuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 107. In another preferred embodiment, the Cpf1 endonuclease is encoded by a polynucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 106.

In another embodiment, the Cpf1 endonuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 109. In another preferred embodiment, the Cpf1 endonuclease is encoded by a polynucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 108.

In another embodiment, a gene encoding the RNA-guided DNA endonuclease is a codon-optimized synthetic sequence for expression in a fungal cell.

In another embodiment, the RNA-guided DNA endonuclease gene is operably linked to one or more polynucleotides encoding nuclear localization signals so the expressed endonuclease is efficiently transported from the cytoplasm to the nucleus. Examples of nuclear localization signals are the SV40 nuclear localization signal, Aspergillus nidulans GATA transcription factor (AreA), Trichoderma reesei transcriptional regulator for cellulase and hemicellulase gene expression (XYR1), Trichoderma reesei blue light regulator 2 (blr2), Xenopus laevis oocyte Nucleoplasmin nuclear localization signal, Caenorhabditis elegans transcription factor EGL-13 nuclear localization signal, homo sapiens transcription factor c- Myc nuclear localization signal, and Escherichia coli replication fork arresting protein (TUS- protein) nuclear localization signal.

Guide RNA

The guide RNA (gRNA) in CRISPR-Cas9 genome editing constitutes the re- programmable part that makes the system so versatile. In the natural Streptomyces pyogenes system, the guide RNA is a complex of two RNA polynucleotides, a crRNA containing about 20 nucleotides that determine the specificity of the Cas9 enzyme and a tracrRNA which hybridizes to the crRNA to form an RNA complex that interacts with the Cas9 endonuclease. See Jinek et ai, 2012, Science 337: 816-821.

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

In the methods of the present disclosure, any guide RNA system can be used.

In one embodiment, the guide RNA is the natural Streptomyces pyogenes system (Jinek et ai, 2012, Science 337(6096): 816-821).

In another embodiment, the guide RNA, known as a single guide RNA (sgRNA), is an engineered single-stranded chimeric RNA, which combines the scaffolding function of the bacterial transactivating CRISPR RNA (tracrRNA) with the specificity of the bacterial CRISPR RNA (crRNA). The last 20 bp at the 5' end of the crRNA acts as a "guide", which recruits the Cas9/gRNA complex to a specific DNA target site, directly upstream of a protospacer adjacent motif (PAM), through RNA-DNA base pairing.

In another embodiment, the single guide RNA comprises a first RNA comprising 20 or more nucleotides that are at least 85%, e.g., 90%, 95%, 96%, 97%, 98%, 99% or 100%, complementary to and capable of hybridizing to the target sequence.

In another embodiment, the first RNA comprising the 20 or more nucleotides are at least 90%, 95%, 97%, 98%, 99% or even 100% complementary to and capable of hybridizing to the target sequence.

In another embodiment, the single guide RNA is a Streptomyces pyogenes Cas9 guide RNA (SEQ I D NO: 15). In another embodiment, the single guide RNA comprises a polynucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 15. In another embodiment, the guide RNA is an Eubacterium rectale Mad7 guide RNA (SEQ ID NO: 110). In another embodiment, the guide RNA comprises a polynucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1 10.

In another embodiment, the guide RNA is a Cpf1 guide RNA (SEQ ID NO: 11 1 , 112, 113, 1 14, or 1 15). In another embodiment, the guide RNA comprises a polynucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 11 1 , 1 12, 1 13, 1 14, or 115.

Promoters

The second nucleic acid construct comprises a U6 promoter sequence operably linked at the 5' end of a sequence encoding a transfer RNA and a sequence encoding a single guide RNA at the 3' end of the transfer RNA sequence.

The U6 promoter can be any such promoter that is functional in driving expression of the transfer RNA sequence and the single guide RNA sequence in a fungal cell.

In one aspect, the U6 promoter is an Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chalara, Chrysosporium, Coprinus, Cordyceps, Coriolus, Cryphonectria, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Nectria, Neocallimastix, Neosartorya, Neurospora, Oidiodendron, Ophiostoma, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Podospora, Schizophyllum, Sodiomyces, Talaromyces, Thermoascus, Thermomyces, Thielavia, Tolypocladium, Trametes, Trichoderma, or Verticillium U6 promoter.

In another aspect, the U6 promoter is an Acremonium alcalophilum, Aspergillus awamori, Aspergillus clavatus, Aspergillus flavus, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Aspergillus terreus, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chaetomium globosum, Chalara longipe, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Cordyceps militaris, Coriolus hirsutus, Cryphonectria parasitica, 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, Fusarium verticillioides, Humicola insolens, Humicola lanuginosa, Magnaporthe grisea, Magnaporthe oryzae, Magnaporthiopsis poae, Mucor miehei, Myceliophthora thermophila, Nectria haematococca, Neosartorya fischeri, Neurospora crassa, Neurospora discrete, Neurospora tetrasperma, Oidiodendron maius, Ophiostoma piceae, Penicillium chrysogenum, Penicillium brevicompactum, Penicillium expansum, Penicillium fellutanum, Penicillium lanosocoeruleum, Penicillium oxalicum, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Podospora anserine, Sodiomyces alkalinus, Talaromyces aculeatus, Talaromyces emersonii, Thermoascus aurantiacus, Thermomyces lanuginosus, Thielavia arenaria, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma atroviride, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, Trichoderma virens, Trichoderma viride, Verticillium alfalfa, or Verticillium dahlia U6 promoter.

In one embodiment, the U6 promoter is an Aspergillus fumigatus U6-1 promoter (e.g., SEQ ID NO: 20). In another embodiment, the U6 promoter is an Aspergillus fumigatus U6-2 promoter (e.g., SEQ ID NO: 27). In another embodiment, the U6 promoter is an Aspergillus fumigatus U6-3 promoter (e.g., SEQ ID NO: 30). In another embodiment, the U6 promoter is an Aspergillus oryzae U6-2 promoter (e.g., SEQ ID NO: 33). In another embodiment, the U6 promoter is a Magnaporthe oryzae U6 promoter (e.g., SEQ ID NO: 13). In another embodiment, the U6 promoter is a Trichoderma reesei U6 promoter (e.g., SEQ ID NO: 37).

In another embodiment, the U6 promoter comprises a polynucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 13.

In another embodiment, the U6 promoter comprises a polynucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 20.

In another embodiment, the U6 promoter comprises a polynucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 27.

In another embodiment, the U6 promoter comprises a polynucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 30.

In another embodiment, the U6 promoter comprises a polynucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 33.

In another embodiment, the U6 promoter comprises a polynucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 37.

Terminators

The second nucleic acid construct comprises (a) a U6 promoter sequence operably linked at the 5' end of (1) a sequence encoding a transfer RNA and (2) a sequence encoding a single guide RNA at the 3' end of the transfer RNA sequence, and (b) a U6 transcriptional terminator sequence operably linked at the 3' end of the sequence encoding the single guide RNA.

The U6 terminator can be any such terminator that is functional in terminating expression of the transfer RNA sequence and the single guide RNA sequence in a fungal cell.

In one aspect, the U6 terminator is an Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chalara, Chrysosporium, Coprinus, Cordyceps, Coriolus, Cryphonectria, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Nectria, Neocallimastix, Neosartorya, Neurospora, Oidiodendron, Ophiostoma, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Podospora, Schizophyllum, Sodiomyces, Talaromyces, Thermoascus, Thermomyces, Thielavia, Tolypocladium, Trametes, Trichoderma, or Verticillium U6 terminator.

In another aspect, the U6 terminator is an Acremonium alcalophilum, Aspergillus awamori, Aspergillus clavatus, Aspergillus flavus, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Aspergillus terreus, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chaetomium globosum, Chalara longipe, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Cordyceps militaris, Coriolus hirsutus, Cryphonectria parasitica, 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, Fusarium verticillioides, Humicola insolens, Humicola lanuginosa, Magnaporthe grisea, Magnaporthe oryzae, Magnaporthiopsis poae, Mucor miehei, Myceliophthora thermophila, Nectria haematococca, Neosartorya fischeri, Neurospora crassa, Neurospora discrete, Neurospora tetrasperma, Oidiodendron maius, Ophiostoma piceae, Penicillium chrysogenum, Penicillium brevicompactum, Penicillium expansum, Penicillium fellutanum, Penicillium lanosocoeruleum, Penicillium oxalicum, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Podospora anserine, Sodiomyces alkalinus, Talaromyces aculeatus, Talaromyces emersonii, Thermoascus aurantiacus, Thermomyces lanuginosus, Thielavia arenaria, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma atroviride, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, Trichoderma virens, Trichoderma viride, VerticiHium alfalfa, or VerticiHium dahlia U6 terminator.

In another embodiment, the U6 terminator is an Aspergillus fumigatus U6-1 terminator (e.g., SEQ ID NO: 16). In another embodiment, the U6 terminator is an Aspergillus fumigatus U6-2 terminator (e.g., SEQ ID NO: 28). In another embodiment, the U6 terminator is an Aspergillus fumigatus U6-3 terminator (e.g., SEQ ID NO: 31). In another embodiment, the U6 terminator is an Aspergillus oryzae U6-2 terminator (e.g., SEQ ID NO: 34). In another embodiment, the U6 terminator is a Magnaporthe oryzae U6 terminator (e.g., SEQ ID NO: 18). In another embodiment, the U6 terminator is a Trichoderma reesei U6 terminator (e.g., SEQ ID NO: 44).

In another embodiment, the U6 terminator comprises a polynucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 16.

In another embodiment, the U6 terminator comprises a polynucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 18.

In another embodiment, the U6 terminator comprises a polynucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 28.

In another embodiment, the U6 terminator comprises a polynucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 31.

In another embodiment, the U6 terminator comprises a polynucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 34.

In another embodiment, the U6 terminator comprises a polynucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 44. Transfer RNA

The second nucleic acid construct comprises a U6 promoter sequence operably linked at the 5' end of a sequence encoding a transfer RNA (tRNA).

In practicing the methods of the present disclosure, the tRNA can be any useful tRNA. There are at least 20 species of tRNA, each species capable of combining with a specific amino acid. Each type of tRNA molecule can be attached to only one type of amino acid, so each organism has many types of tRNA. Since the genetic code contains multiple codons that specify the same amino acid, there are many tRNA molecules bearing different anticodons which also carry the same amino acid.

In one aspect, the tRNA sequence is from a fungal tRNA gene. In a preferred aspect, the tRNA sequence is from a filamentous fungal tRNA gene. In another preferred aspect, the tRNA sequence is from a yeast tRNA gene.

In one embodiment, the tRNA sequence is from an Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chalara, Chrysosporium, Coprinus, Cordyceps, Coriolus, Cryphonectria, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Nectria, Neocallimastix, Neosartorya, Neurospora, Oidiodendron, Ophiostoma, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Podospora, Schizophyllum, Sodiomyces, Talaromyces, Thermoascus, Thermomyces, Thielavia, Tolypocladium, Trametes, Trichoderma, or Verticillium tRNA gene

In a preferred embodiment, the tRNA sequence is from an Acremonium alcalophilum, Aspergillus awamori, Aspergillus clavatus, Aspergillus flavus, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Aspergillus terreus, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chaetomium globosum, Chalara longipe, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Cordyceps militaris, Coriolus hirsutus, Cryphonectria parasitica, 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, Fusarium verticillioides, Humicola insolens, Humicola lanuginosa, Magnaporthe grisea, Magnaporthe oryzae, Magnaporthiopsis poae, Mucor miehei, Myceliophthora thermophila, Nectria haematococca, Neosartorya fischeri, Neurospora crassa, Neurospora discrete, Neurospora tetrasperma, Oidiodendron maius, Ophiostoma piceae, Penicillium chrysogenum, Penicillium brevicom pactum, Penicillium expansum, Penicillium fellutanum, Penicillium lanosocoeruleum, Penicillium oxalicum, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Podospora anserine, Sodiomyces alkalinus, Talaromyces aculeatus, Talaromyces emersonii, Thermoascus aurantiacus, Thermomyces lanuginosus, Thielavia arenaria, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma atroviride, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, Trichoderma virens, Trichoderma viride, Verticillium alfalfa, or Verticillium dahlia tRNA gene.

In one embodiment, the tRNA sequence is from a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia tRNA gene.

In a preferred embodiment, the tRNA sequence is from a Kluyveromyces lactis,

Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica tRNA gene.

In another preferred embodiment, the tRNA sequence is from an Aspergillus fumigatus tRNA gene. In a more preferred embodiment, the tRNA sequence is SEQ ID NO: 22 from Aspergillus fumigatus. In another more preferred embodiment, the tRNA sequence is SEQ ID NO: 25 from Aspergillus fumigatus.

Target Site

The target site can be anywhere in the genome of a fungal cell as long as the site includes an adjacent protospacer adjacent motif (PAM). The protospacer adjacent motif is a 2-6 base pair DNA sequence immediately before or following the target site, which is recognized directly by an RNA-guided DNA endonuclease to promote cleavage of the target site. For the S. pyogenes Cas9 endonuclease, the sequence of the protospacer adjacent motif is NGG (5'→3') where N is A, G, C, or T. Other Cas9 endonucleases have different protospacer adjacent motifs. For the Eubacterium rectale Mad7 endonuclease, the sequence of the protospacer adjacent motif is YTTN. For the Acidaminococcus sp. and Lachnospiraceae sp. Cpf1 endonucleases, the sequence of the protospacer adjacent motif is TTTN and for the Francisella novicide Cpf1 endonuclease, the sequence of the protospacer adjacent motif is TTN.

Eubacterium rectale recognizes 5'-YTTN on the 5' end of the gRNA sequence. The Cpf1 endonuclease from Acidaminococcus sp. and Lachnospiraceae sp. recognize 5'-TTTN and the Cpf1 endonuclease Francisella novicide recognizes 5'-TTN-3' on the 5' end of the gRNA

For an overview of other PAM sequences, see, for example, Shah et ai, 2013, RNA

Biol. 10(5): 891-899. The target site is at least 20 nucleotides in length in order to allow its hybridization to the corresponding 20 nucleotide sequences of the guide RNA.

The target site can be native to the fungal cell or heterologous to the fungal cell. The target site can be located within or near a gene of interest or a region thereof; within a non- coding region; between two genes; or any region within the host cell genome. The region can be, for example, an open reading frame, a protein coding sequence, an intron site, an intron enhancing motif, a mRNA splice site, a promoter, a transcriptional regulatory element, a transcriptional terminator, and a translational regulatory element. The gene of interest can be a hydrolase, an isomerase, a ligase, a lyase, an oxidoreductase, or a transferase. Non-limiting examples of genes include genes encoding an acetylesterase, alpha-galactosidase, alpha- glucosidase, aminopeptidase, amylase, beta-galactosidase, beta-glucosidase, beta- xylosidase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endonuclease, esterase, glucoamylase, hexose oxidase, invertase, laccase, lipase, mannosidase, mutanase, oxidase, pectin depolymerase, pectin methylesterase, pectinolytic enzyme, peroxidase, phenoloxidase, phytase, polygalacturonase, polyphenoloxidase, protease, rhamnogalacturonase, ribonuclease, transglutaminase, and xylanase.

Donor DNA

A donor DNA, also known as a DNA repair template, comprises a nucleotide sequence of interest, for modifying or editing a target site of a fungal cell, and additional homologous sequence corresponding to immediately upstream and downstream of the target site (termed "5' homology sequence" and "3' homology sequence") for incorporation of the donor DNA into the double-strand break at the target site. The length of each homology sequence is dependent on the size of the modification being made. The donor DNA can be a double- stranded oligonucleotide, circular double-stranded DNA plasmid, or linear double-stranded DNA plasmid.

Non-limiting examples for modifying a target site are deleting a gene or a portion thereof, disrupting a gene, altering a nucleotide or nucleotides within a gene, replacing a gene with a heterologous gene encoding a protein with improved biological activity, e.g. , a homolog or variant, introducing a mutation into a gene, replacing a gene with a heterologous gene encoding a protein with different biological activity, inserting a gene, or repairing a gene.

In an embodiment, the nucleotide sequence of interest for modifying the target site comprises at least 1 , 5, 10, 20, 40, 60, 100, 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 10,000 nucleotides In one aspect, the nucleotide sequence of interest is a gene.

The gene of interest can be. an oxidoreductase, a transferase, a hydrolase, a lyase, an isomerase, or a ligase. In another aspect, the polypeptide is an acetylmannan esterase, acetylxylan esterase, aminopeptidase, alpha-amylase, arabinanase, arabinofuranosidase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, coumaric acid esterase, cyclodextrin glycosyltransferase, cutinase, cyclodextrin glycosyltransferase, deamidase, deoxyribonuclease, dispersin, endoglucanase, esterase, feruloyl esterase, GH61 polypeptide having cellulolytic enhancing activity, alpha-galactosidase, beta-galactosidase, glucocerebrosidase, glucose oxidase, alpha-glucosidase, beta-glucosidase, glucuronidase, glucuronoyl esterase, haloperoxidase, hemicellulase, invertase, isomerase, laccase, ligase, lipase, lysozyme, mannanase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase, phosphodiesterase, phospholipase, phytase, phenoloxidase, polyphenoloxidase, proteolytic enzyme, ribonuclease, alpha-1 ,6-transglucosidase, transglutaminase, urokinase, xanthanase, xylanase, or beta-xylosidase

In another aspect, the nucleotide sequence of interest is a region of a gene.

The region can be, for example, an open reading frame, a protein coding sequence, an intron site, an intron enhancing motif, a mRNA splice site, a promoter, a transcriptional regulatory element, a transcriptional terminator, and a translational regulatory element.

Techniques used to isolate or clone a gene as the nucleotide sequence of interest are known in the art and include isolation from genomic DNA or cDNA, or a combination thereof. The cloning of the gene from genomic DNA can be affected, 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 ai, 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.

Any gene that encodes, for example, a polypeptide may be modified at the nucleotide sequence level to serve as the nucleotide sequence of interest. 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 with 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, Leu/Val, Ala/Glu, and Asp/Gly.

Alternatively, the amino acid changes can be 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 ef al., 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.

In another aspect, the nucleotide sequence of interest is one or more nucleotides for introducing a mutation into a gene, e.g., a codon.

In another aspect, the nucleotide sequence of interest is a mutated sequence encoding a variant.

A gene as the nucleotide sequence of interest may be manipulated in a variety of ways to provide for expression of the polypeptide in a fungal cell by operably linking the coding sequence to one or more heterologous control sequences that direct the expression of the coding sequence in the fungal cell. 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. 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 a nucleotide sequence of interest in a filamentous fungal host cell are promoters obtained from the genes for Aspergillus nidulans acetamidase, Aspergillus nidulans translation elongation factor, 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 endonuclease I, Trichoderma reesei endonuclease II, Trichoderma reesei endonuclease III, Trichoderma reesei endonuclease 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. 6,011 , 147.

In a yeast host, useful promoters are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1 , ADH2/GAP), Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomyces cerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful promoters for yeast host cells are described by Romanos et ai, 1992, Yeast 8: 423-488. 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 methods of the present disclosure.

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 endonuclease I, Trichoderma reesei endonuclease II, Trichoderma reesei endonuclease III, Trichoderma reesei endonuclease V, Trichoderma reesei xylanase I, Trichoderma reesei xylanase II, Trichoderma reesei xylanase III, Trichoderma reesei beta- xylosidase, and Trichoderma reesei translation elongation factor.

Preferred terminators for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are described by Romanos et ai, 1992, supra.

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.

Suitable leaders for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

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. Useful polyadenylation sequences for yeast host cells are described by Guo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

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 heterologous to the coding sequence. A heterologous signal peptide coding sequence may be required where the coding sequence does not naturally contain a signal peptide coding sequence. Alternatively, a heterologous 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 endonuclease V, Humicola lanuginosa lipase, and Rhizomucor miehei aspartic proteinase.

Useful signal peptides for yeast host cells are obtained from the genes for

Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase. Other useful signal peptide coding sequences are described by Romanos et al., 1992, supra.

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 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 yeast, the ADH2 system or GAL1 system may be used. In filamentous fungi, the Aspergillus n/^'gerglucoamylase 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.

The methods of the present disclosure can be used to reduce or eliminate expression of a gene in a fungal cell by insertion, disruption, substitution or deletion of one or more nucleotides into the gene or a regulatory element required for transcription or translation thereof.

In one embodiment, a gene of a fungal cell is modified so expression of the gene is reduced or eliminated. 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. A gene may be modified by gene replacement, gene deletion, or gene disruption to eliminate or reduce expression of the gene. For example, in the gene disruption method, a polynucleotide sequence corresponding to the gene is mutagenized in vitro to produce a defective polynucleotide sequence that is then introduced into a fungal cell to produce a defective gene. The gene may be disrupted with a selectable marker that then may be used for selection of transformants in which the gene has been modified.

The modification of a gene may involve 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.

When it is desirable to delete a portion of a gene at the target site, the donor DNA is composed of the gene with the portion deleted.

When it is desirable to delete a gene in its entirety at the target site, the donor DNA is composed of a first region of homology and a second region of homology flanking the gene to be deleted so homologous recombination can occur.

The methods of the present disclosure rely on the introduction of the donor DNA into the target site by endogenous DNA repair. In one embodiment, the endogenous DNA repair is by homologous recombination of the 3' region and the 5' region of the donor DNA with corresponding regions of homology of the target site. In another embodiment, the endogenous DNA repair is by non-homologous end joining.

Homologous recombination requires sufficient donor DNA flanking the nucleotide sequence of interest to enable effective recombination, which requires at least 5 nucleotides of identical sequences to allow homologous recombination between the target site and the donor DNA. Consequently, the donor DNA should contain the nucleotide sequence of interest and at least 5 nucleotides on each side for successful double recombination.

Accordingly, the third nucleic acid construct is incorporated into the target site of the fungal cell by homologous recombination of the 3' region and the 5' region of the donor DNA via corresponding regions of homology of the target site. The first and second regions of homology can flank the nucleotide sequence of interest or can be present in the nucleotide sequence of interest. The first and second regions share sufficient homology to the corresponding regions of the target site.

"Sufficient homology" indicates that the nucleotide sequences of the regions have sufficient structural similarity to undergo homologous recombination. The structural similarity includes overall length of each region, as well as sequence similarity of the regions. Sequence similarity is preferably described by percent sequence identity over the whole length of the regions.

The region of homology can be any length sufficient for promoting homologous recombination at the target site. For example, the region of homology can comprise at least 5 or more bases in length to undergo homologous recombination with the corresponding genomic region.

The amount of homology shared by the first and second regions of a donor DNA with the corresponding regions of homology of the target site can range from about 5-20 bp, 20-50 bp, 50-100 bp, 75-150 bp, 100-250 bp, 150-300 bp, 200-400 bp, 250-500 bp, 300-600 bp, 350-750 bp, 400-800 bp, 450-900 bp, 500-1000 bp, 600-1250 bp, 700-1500 bp, 800-1750 bp, 900-2000 bp, 1-2.5 kb, 1.5-3 kb, 2-4 kb, 2.5-5 kb, 3-6 kb, 3.5-7 kb, 4-8 kb, 5-10 kb, or up to and including the total length of the locus. The amount of homology is preferably described by percent sequence identity over the full aligned length of each region of homology to the corresponding region of homology of the target site, such as a sequence identity of at least 50%, 55%, 60%, 65%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

Sufficient homology can also be described by the predicted ability of two polynucleotides to specifically hybridize under high or very high stringency conditions. Fungal Cells

The fungal cell may be a yeast cell or a filamentous fungal cell. In one embodiment, the fungal cell is a yeast cell. In another embodiment, the fungal cell is a filamentous fungal cell.

"Yeast" as used herein includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes). Since the classification of yeast may change in the future, for the purposes of this disclosure, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, Passmore, and Davenport, editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

In one embodiment, the yeast cell can be a Candida, Hansenula, Kluyveromyces,

Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell.

In a preferred embodiment, the fungal cell can be a Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica cell.

"Filamentous fungi" include all filamentous forms from the phyla Ascomycota, Basidiomucota, Chytridiomycota, Eumycota, Oomycota, and Zygomycota (as defined by Hawksworth et ai, 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.

In one embodiment, the filamentous fungal cell can 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, Thermomyces, Thielavia, Tolypocladium, Trametes, or Trichoderma cell.

In a preferred embodiment, the filamentous fungal cell can 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, Talaromyces emersonii, Thermomyces lanuginosus, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cell.

Filamentous 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 disclosure to employ a fungal cell that is unable to quickly repair the cleaved target site before the donor DNA comprising a nucleotide sequence of interest is introduced into the target site. Accordingly, it is preferred that the fungal cell 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 HgD, kulO and or ku80 gene or homolog(s) thereof. Nucleic Acid Constructs

The methods of the present disclosure relate to several nucleic acid constructs that are used for modifying a target site in the genome of a fungal cell.

In one aspect, the nucleic acid construct comprises a polynucleotide encoding an RNA- guided DNA endonuclease, e.g., a Cas9, or Mad7 endonuclease, for introducing a double- strand break at a target site in the genome of a fungal cell, wherein the fungal cell comprises a protospacer adjacent motif sequence for the RNA-guided DNA endonuclease immediately before the 5' end or immediately following the 3' end of the target site.

In another aspect, the nucleic acid construct comprises (a) a U6 promoter sequence operably linked at the 5' end of (1) a sequence encoding a transfer RNA and (2) a sequence encoding a single guide RNA at the 3' end of the transfer RNA sequence, and (b) a U6 transcriptional terminator sequence operably linked at the 3' end of the sequence encoding the single guide RNA, wherein the single guide RNA directs the RNA-guided DNA endonuclease, e.g., a Cas9, Mad7, or Cpf1 endonuclease, to a target site in the genome of a fungal cell to introduce a double-strand break, and wherein the nucleic acid construct increases the frequency of the RNA-guided DNA endonuclease in producing the double- strand break at the target site.

In another aspect, the nucleic acid construct comprises a donor DNA comprising a nucleotide sequence of interest for modifying a target site in the genome of a fungal cell, wherein the third nucleic acid construct is incorporated into the target site by endogenous DNA repair.

In another aspect, the nucleic acid construct comprises a donor DNA comprising a nucleotide sequence of interest for modifying a target site in the genome of a fungal cell and a 5' homology sequence and a 3' homology sequence flanking the nucleotide sequence of interest, wherein the 5' homology sequence and the 3' homology sequence correspond to an upstream region and a downstream region, respectively, of the target site for incorporation of the donor DNA into the double-strand break, wherein the third nucleic acid construct is incorporated into the target site by endogenous DNA repair.

In a preferred embodiment, the nucleic acid construct comprising the polynucleotide encoding the RNA-guided DNA endonuclease, e.g., the Cas9, Mad7, or Cpf1 endonuclease, and the nucleic acid construct comprising (a) a U6 promoter sequence operably linked at the 5' end of (1) a sequence encoding a transfer RNA, (2) a sequence encoding a single guide RNA at the 3' end of the transfer RNA sequence, and (b) a U6 transcriptional terminator sequence operably linked at the 3' end of the sequence encoding the single guide RNA are on a single DNA fragment or a single vector. Expression Vectors

The nucleic acid constructs of the present disclosure may be inserted into an appropriate vector 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 fungal 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), pyr (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, pyr4 (orotidine-5^!-phosphate decarboxylase), pyr2 (orotate phosphoribosyltransferase), and ptrA (pyrithiamine resistance) 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.

More than one copy of a nucleic acid construct of the present disclosure 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 disclosure are well known to one skilled in the art (see, e.g., Sambrook et ai, 1989, Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, New York).

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

Strains

Trichoderma reesei strain BTR213 is a classical and spontaneous mutant of T. reesei strain RutC30 (Montenecourt and Eveleigh, 1977, Appl. Environ. Microbiol. 34(6): 777-82)

Trichoderma reesei strain JfyS139/144-1 OB is a ku70- strain derived from T. reesei RutC30. (U.S. Published Application 2014/0234914).

Trichoderma reesei strain JfyS154-78-3C is a HgD- strain derived from RutC30 Aspergillus oryzae Jal_355 is described in WO 2005/070962 Example 10.

Trichoderma reesei strain GMer62-1A9 is a ku70 disrupted and paracelsin synthetase

(parS) deleted strain of T. reesei BTR213.

Media and Solutions

COVE plates were composed of 218 g of sorbitol, 20 g of agar, 20 ml COVE salts solution, 10 mM acetamide, 15 mM CsCI₂, and deionized water to 1 liter. The solution was adjusted to pH 7.0 before autoclaving.

COVE salts solution was composed of 26 g of KCI, 26 g of MgS0₄-7H₂0, 76 g of KH₂P0₄, 50 ml COVE trace metals solution, and deionized water to 1 liter.

COVE trace metals solution was composed of 0.04 g of Na₂B₄Oy 10H₂O, 0.4 g of CuS0₄-5H₂0, 1.2 g of FeS0₄-7H₂0, 0.7 g of MnS0₄ H₂0, 0.8 g of Na₂Mo0₂-2H₂0, 10 g of ZnS0₄-7H₂0, and deionized water to 1 liter.

LB + Amp medium was composed of 10 g of tryptone, 5 g of yeast extract, 5 g of sodium chloride, 50 mg of ampicillin (filter sterilized, added after autoclaving), and deionized water to 1 liter.

PEG buffer was composed of 60% polyethylene glycol (PEG) 4000, 10 mM Tris-HCI pH 7.5, and 10 mM CaCI₂ in deionized water.

SOC medium was composed of 20 g of tryptone, 5 g of yeast extract, 0.5 g of NaCI, 10 ml of 250 mM KCI, and deionized water to 1 liter.

STC was composed of 1 M sorbitol, 10 mM Tris pH 7.5, and 10 mM CaCI₂ in deionized water.

TAE buffer was composed of 4.84 g of Tris base, 1.14 ml of glacial acetic acid, 2 ml of 0.5 M EDTA pH 8.0, and deionized water to 1 liter.

2XYT + amp plates were composed of 16 g of tryptone, 10 g of yeast extract, 5 g of NaCI, 15 g of Bacto agar, 1 ml of ampicillin at 100 mg/ml, and deionized water to 1 liter.

YP medium was composed of 1 % yeast extract and 2% peptone in deionized water.

STC was composed of 1.2 M sorbitol, 10 mM CaCI₂, 10 mM Tris-HCI pH 7.5. Example 1 : Trichoderma reesei strain BTR213 genomic DNA extraction

T. reesei strain BTR213 was grown in 50 ml of YP medium supplemented with 2% glucose in a baffled shake flask at 28°C for 2 days with agitation at 200 rpm. Mycelia were harvested by filtration using MIRACLOTH® (Calbiochem), washed twice in deionized water, and frozen under liquid nitrogen. Frozen mycelia were ground, by mortar and pestle, to a fine powder, and total DNA was isolated using a DNEASY® Plant Maxi Kit (QIAGEN Inc.).

Example 2: Trichoderma reesei protoplast generation and transformation

Protoplast preparation and transformation were performed using a protocol similar Penttila et al., 1987, Gene 61 : 155-164 modified as follows. Briefly, T. reesei strain JfyS139/144-10B was cultivated in 25 ml of YP medium supplemented with 2% (w/v) glucose and 10 mM uridine at 27°C for 17 hours with gentle agitation at 90 rpm. Mycelia were collected by filtration using a Vacuum Driven Disposable Filtration System (Millipore) and washed twice with deionized water and twice with 1.2 M sorbitol. Protoplasts were generated by suspending the washed mycelia in 20 ml of 1.2 M sorbitol containing 15 mg of GLUCANEX® 200 G (Novozymes A/S) per ml and 0.36 units of chitinase (Sigma Chemical Co.) per ml for 15-25 minutes at 34°C with gentle shaking at 90 rpm. Protoplasts were collected by centrifuging at 400 x g for 7 minutes and washed twice with cold 1.2 M sorbitol. The protoplasts were counted using a haemocytometer and re-suspended to a final concentration of 1x10⁸ protoplasts per ml of STC. Excess protoplasts were stored in a Cryo 1°C Freezing Container (Nalgene) at - 80°C.

For each reaction, an RNA-guided DNA endonuclease plasmid, e.g. , Cas9/gRNA, Mad7/gRNA, or Cpf1/gRNA, plasmid and a wA gene deletion plasmid were added to 100 μΙ of the protoplast solution and mixed gently. PEG buffer (250 μΙ) was added and mixed, and the transformation was incubated at 34°C for 30 minutes. STC (3 ml) was then added and mixed, and spread onto COVE plates supplemented with 1 M sucrose. The plates were incubated at 30°C for 7-10 days. Example 3: Construction of donor plasmid pSMai271 to delete the native Trichoderma reesei polyketide synthase (wA) gene

A donor plasmid, pSMai271 (Figure 1), was constructed by combining three DNA segments using an IN-FUSION® Advantage PCR Cloning Kit (Takara Bio USA, Inc.) for deleting the native T. reesei polyketide synthase (wA) gene [(SEQ ID NO: 1 [DNA sequence] and SEQ ID NO: 2 [deduced amino acid sequence]).

The Aspergillus nidulans amdS selectable marker gene was amplified from pMJ09 (U.S. Patent No. 8,318,458) using the forward and reverse primers shown below.

Forward primer:

5'-GAGCACCCGTTTTCATGCATTCTACGCCAGGACCGAGCAA-3' (SEQ ID NO: 3) Reverse primer:

5'-GCATTGGCGGCCTATGCATCTGGAAACGCAACCCTGAAGG-3' (SEQ I D NO: 4)

For amplification of the Aspergillus nidulans amdS gene, the reaction was composed of 10 ng of pMJ09, 200 μΜ dNTPs, 1 μΜ of each primer, 1X PHUSION® High-Fidelity Hot Start DNA Polymerase Buffer (New England Biolabs, Inc.), and 1.0 unit of PHUSION® High- Fidelity Hot Start DNA Polymerase (New England Biolabs, Inc.) in a final volume of 50 μΙ. The amplification reaction was incubated in a thermocycler programmed for 1 cycle at 98°C for 3 minutes; 30 cycles each at 98°C for 10 seconds, 58°C for 30 seconds, and 72°C for 1.5 minutes; and 1 cycle at 72°C for 15 minutes. The PCR product was isolated by 1% agarose gel electrophoresis using TAE buffer where a 2758 bp fragment was excised from the gel and extracted using a NUCLEOSPIN® Gel and PCR Clean-up Kit (Takara Bio USA, Inc.).

A 400 bp fragment of the upstream non-coding region of the T. reesei wA gene was amplified from T. reesei BTR213 genomic DNA using the forward and reverse primers shown below.

Forward primer:

5'-GTCGACCTGCAGGCATGCGTTTAAACCAAAGACCTTACAATTAGCT-3' (SEQ ID NO: 5) Reverse primer:

5'-GGTCCTGGCGTAGAATGCATGAAAACGGGTGCTCCAAATA-3' (SEQ ID NO: 6)

A 413 bp fragment of the downstream non-coding region of the T. reesei wA gene was amplified from T. reesei BTR213 genomic DNA using the forward and reverse primers shown below. T. reesei BTR213 genomic DNA was prepared according to the procedure described in Example 1.

Forward primer:

5'-GGTTGCGTTTCCAGATGCATAGGCCGCCAATGCTCTTGACAAA-3' (SEQ ID NO: 7) Reverse primer:

5'-GCTATGACCATGATTACGCCGTTTAAACCGCAGCCATGAGTATCACGC-3' (SEQ ID NO: 8)

For amplification of the non-coding region of the T. reesei wA gene upstream sequence or downstream sequence, the reactions were composed of 150 ng of T. reesei BTR213 genomic DNA, 200 μΜ dNTPs, 1 μΜ of each primer (SEQ ID NOs: 5 and 6 or 7 and 8, respectively), 1X PHUSION® High-Fidelity Hot Start DNA Polymerase Buffer, and 1.0 unit of PHUSION® High-Fidelity Hot Start DNA Polymerase in a final volume of 50 μΙ. The amplification reactions were incubated in a thermocycler programmed for 1 cycle at 98°C for 3 minutes; 30 cycles each at 98°C for 10 seconds, 58°C for 30 seconds, and 72°C for 1.5 minutes; and 1 cycle at 72°C for 15 minutes. The PCR products were isolated by 1% agarose gel electrophoresis using TAE buffer where 400 bp and 413 bp fragments were separately excised from the gels and extracted using a NUCLEOSPIN® Gel and PCR Clean-up Kit.

A fourth DNA segment was generated by restriction enzyme digestion of pUC19 (New

England BioLabs Inc.) with Hind III. The reaction was composed of 5 μg of pUC19, 20 units of Hind III, and 5 μΙ of CUTSMART® Buffer (New England Biolabs, Inc.) in a total volume of 50 μΙ. The reaction was incubated for 4 hours at 37°C and then separated by 1 % agarose gel electrophoresis using TAE buffer where a 2.7 kb fragment was excised from the gel and extracted using a NUCLEOSPIN® Gel and PCR Clean-up Kit.

The three PCR products of 2758 bp, 400 bp, and 413 bp were inserted into Hind III digested pUC19 using an IN-FUSION® HD Cloning Kit (Takara Bio USA, Inc.) according to the manufacturer's protocol. The IN-FUSION® reaction was composed of 2 μΙ of 5X INFUSION® HD Enzyme Premix, 100 ng of the Hind III digested pUC19, 50 ng of the 400 bp wA gene upstream PCR product, 50 ng of the 413 bp wA gene downstream PCR product, and 50 ng of the 2758 bp Aspergillus nidulans amdS gene PCR product in a 10 μΙ reaction volume. The reaction was incubated for 15 minutes at 50°C. After the incubation period, a 2 μΙ aliquot was transformed into 50 μΙ of E. coli STELLAR™ competent cells (Takara Bio USA, Inc.) according to the manufacturer's protocol. The cells were heat shocked at 42°C for 45 seconds and then 450 μΙ of SOC medium, pre-heated to 42°C, were added. The cells were incubated at 37°C with shaking at 200 rpm for 60 minutes and then spread onto a 150 mm diameter 2XYT plus ampicillin plate and incubated at 37°C overnight. The resulting E. coli transformants were individually inoculated into 3 ml of LB + Amp medium in 14 ml round-bottom polypropylene tubes and incubated at 37°C overnight with shaking at 200 rpm. Plasmid DNA was isolated using a BIOROBOT® 9600 (QIAGEN Inc.). The insert was confirmed by DNA sequencing with a Model 377 XL Automated DNA Sequencer (Applied Biosystems Inc.) using dye-terminator chemistry (Giesecke et al., 1992, J. Virol. Methods 38(1): 47-60). One plasmid containing the insert with no PCR errors was identified and designated pSMai271. Example 4: Elements of CRISPR/Cas9 backbone vector pSMai268

Plasmid pSMai268 (SEQ ID NO: 9, Figure 2) is a CRISPR/Cas9 expression plasmid lacking elements for single guide RNA expression, but containing a S. pyogenes Cas9 protein coding sequence [(SEQ ID NO: 10 [DNA sequence] and SEQ ID NO: 1 1 [deduced amino acid sequence]) codon-optimized (SEQ ID NO: 12) for use in Aspergillus, under control of the Aspergillus nidulans tefl promoter from pFC330-333 (N0dvig et ai, 2015, PLoS One 10(7): 1-18). For selection in T. reesei plasmid pSMai268 contains the hygromycin phosphotranferase gene from pHT1 (Cummings et ai, 1999, Curr. Genet. 36: 371), conferring resistance to hygromycin B. Plasmid pSMai268 has the pUC19 backbone (Yanisch-Perron et al., 1985, Gene. 33 (1): 103-1 19) as well as a multiple cloning site in between Hind III restriction sites. In order to clone in different promoters and other elements for single guide RNA (gRNA) expression, plasmid pSMai268 was digested with Hind III liberating the multiple cloning site region located between the restriction sites. The digestion was purified by 1 % agarose gel electrophoresis using TAE buffer where a 9.7 kb band was excised and agarose was extracted using a NUCLEOSPIN® II Gel and PCR Clean-up Kit (Takara Bio USA, Inc.). Briefly agarose was dissolved in 2 volumes of NTI buffer (Takara Bio USA, Inc.) and applied to the Kit supplied column using a vacuum manifold. The DNA was washed with 750 μΙ of wash buffer NT3 wash buffer (Takara Bio USA, Inc.) and eluted with 15 μΙ of elution buffer NE (Takara Bio USA, Inc.).

Example 5: Construction of plasmid pSMai274 containing the Magnaporthe oryzae U6- 2 promoter, a wA protospacer, a Streptomyces pyogenes single guide RNA sequence, and a poly-T terminator

The Magnaporthe oryzae U6-2 promoter (SEQ ID NO: 13) was identified by searching the gene annotations of the Magnaporthe grisea (Magnaporthe oryzae) strain 70-15 (MG8) genome sequence database from the Joint Genome Institute (JGI). A synthetic DNA sequence containing 500 bp of the M. oryzae U6-2 promoter, a 20 bp protospacer region targeting the T. reesei wA gene (SEQ ID NO: 14), a sequence encoding a S. pyogenes single guide RNA (SEQ ID NO: 15), 5 bp of a synthetic poly-T terminator (TTTTT), and flanking homologous sequences for insertion into plasmid pSMai268 was synthesized as a STRING™ DNA fragment (SEQ ID NO: 17) by GENEART® (Thermo Fisher Scientific). The DNA fragment was resuspended in 10 mM Tris pH 8 buffer at a concentration of 25 ng/μΙ and 1 μΙ was used to insert the fragment into Hind Ill-digested pSMai268 using an IN-FUSION® HD Cloning Kit. The reaction was composed of 100 ng of Hind Ill-digested pSMai268, 25 ng of the DNA fragment, and 1X IN-FUSION® HD Premix in a 10 μΙ reaction volume. After incubating the mixture for 15 minutes at 50°C, 2 μΙ of the reaction were transformed into 50 μΙ of E. coli STELLAR™ competent cells (Takara Bio USA, Inc.). The cells were heat shocked at 42°C for 45 seconds after which 450 μΙ of SOC medium were added. The cells were incubated at 37°C with shaking at 200 rpm for 30 minutes and the total volume was spread onto a 150 mm 2XYT + Amp plate and incubated at 37°C overnight. The resulting E. coli transformants were individually inoculated into 3 ml of LB + Amp medium in 14 ml round-bottom polypropylene tubes and incubated at 37°C overnight with shaking at 200 rpm. Plasmid DNA was isolated using a BIOROBOT® 9600. The insert was confirmed by DNA sequencing with a Model 377 XL Automated DNA Sequencer using dye-terminator chemistry (Giesecke et ai, 1992, supra). One plasmid designated pSMai274 (Figure 3) was selected for analyzing the Magnaporthe oryzae U6-2 promoter for single guide RNA expression in T. reesei. Example 6: Construction of plasmid pSMai280 containing the Magnaporthe oryzae U6- 2 promoter, wA protospacer, Streptomyces pyogenes single guide RNA sequence, and Magnaporthe oryzae U6-2 terminator

The Magnaporthe oryzae U6-2 terminator (SEQ ID NO: 18) was identified by searching the gene annotations of the Magnaporthe grisea (Magnaporthe oryzae) strain 70- 15 (MG8) genome sequence database from the Joint Genome Institute (JGI). A synthetic DNA sequence containing 500 bp of the M. oryzae U6-2 promoter, the 20 bp protospacer region targeting the T. reesei wA gene, the S. pyogenes single guide RNA sequence, 215 bp of the M. oryzae U6-2 terminator, and flanking homologous sequences for insertion into plasmid pSMai268 was synthesized as a STRING™ DNA fragment (SEQ ID NO: 19) by GENEART®. The DNA fragment was resuspended in 10 mM Tris pH 8 at a concentration of 25 ng/μΙ and 1 μΙ was used to insert the fragment into Hind Ill-digested pSMai268 using an IN-FUSION® HD Cloning Kit. The reaction was composed of 100 ng of Hind Ill-digested pSMai268, 25 ng of the DNA fragment, and 1X IN-FUSION® HD Premix in a 10 μΙ reaction volume. After incubating the mixture for 15 minutes at 50°C, 2 μΙ of the reaction were transformed into 50 μΙ of E. coli STELLAR™ competent cells (Takara Bio USA, Inc.). The cells were heat shocked at 42°C for 45 seconds after which 450 μΙ of SOC medium were added. The cells were incubated at 37°C with shaking at 200 rpm for 30 minutes and the total volume was spread onto a 150 mm 2XYT + Amp plate and incubated at 37°C overnight. The resulting E. coli transformants were individually inoculated into 3 ml of LB + Amp medium in 14 ml round-bottom polypropylene tubes and incubated at 37°C overnight with shaking at 200 rpm. Plasmid DNA was isolated using a BIOROBOT® 9600. The insert was confirmed by DNA sequencing with a Model 377 XL Automated DNA Sequencer using dye-terminator chemistry (Giesecke et al., 1992, supra). One plasmid designated pSMai280 (Figure 4) was selected for analyzing the Magnaporthe oryzae U6-2 promoter with its terminator for single guide RNA expression in T. reesei.

Example 7: Construction of plasmid pJfyS238 containing the Aspergillus fumigatus U6- 1 promoter, wA protospacer, Streptomyces pyogenes single guide RNA sequence, and poly-T terminator

The Aspergillus fumigatus U6-1 promoter (SEQ ID NO: 20) was identified by searching the gene annotations of the Aspergillus fumigatus strain 293 genome sequence database from the Joint Genome Institute (JGI). A synthetic DNA sequence containing 500 bp of the A. fumigatus U6-1 promoter, the 20 bp protospacer region targeting the T. reesei wA gene, the S. pyogenes single guide RNA sequence, the poly-T terminator, and flanking homologous sequences for insertion into plasmid pSMai268 was synthesized as a STRING™ DNA fragment (SEQ ID NO: 21) by GENEART®. The DNA fragment was resuspended in 10 mM Tris pH 8 at a concentration of 30 ng/μΙ and 2 μΙ was used to insert the fragment into Hind Ill-digested pSMai268 using an IN-FUSION® HD Cloning Kit. The reaction was composed of 125 ng of Hind III digested pSMai268, 60 ng of the DNA fragment, and 1X IN-FUSION® HD Premix in a 10 μΙ reaction volume. After incubating the mixture for 15 minutes at 50°C, 2 μΙ of the reaction were transformed into 50 μΙ of E. coli STELLAR™ competent cells. The cells were heat shocked at 42°C for 45 seconds after which 100 μΙ of SOC medium were added and the total volume was spread onto a 150 mm 2XYT + Amp plate and incubated at 37°C overnight. The resulting E. coli transformants were individually inoculated into 3 ml of LB + Amp medium in 14 ml round-bottom polypropylene tubes and incubated at 37°C overnight with shaking at 200 rpm. Plasmid DNA was isolated using a BIOROBOT® 9600. The insert was confirmed by DNA sequencing with a Model 377 XL Automated DNA Sequencer using dye-terminator chemistry (Giesecke et al., 1992, supra). One plasmid designated pJfyS238 (Figure 5) was selected for analyzing the A. fumigatus U6-1 promoter for single guide RNA expression in T. reesei.

Example 8: Construction of plasmid pJfyS242 containing the Aspergillus fumigatus U6- 1 promoter, Aspergillus fumigatus tRNAgly(GCC)1 -6 gene sequence, wA protospacer, Streptomyces pyogenes single guide RNA sequence, and poly-T terminator

The Aspergillus fumigatus tRNAgly(GCC)1-6 gene sequence (SEQ ID NO: 22) was identified ((chr4:3650 53-3650223 (+)) by searching the gene annotations of the Aspergillus fumigatus strain 293 genome sequence database from the Joint Genome Institute (JGI). This specific tRNAgly(GCC)1-6 sequence was selected as the upstream sequence beginning with the "G" base, which is required for transcript initiation of U6 promoters (Goomer and Kunkel, 1992, Nucleic Acids Research 20(18): 4903-4912). A synthetic DNA sequence containing the 500 bp of the A. fumigatus U6-1 promoter (Example 7), the tRNAgly(GCC)1-6 sequence with the 3' trailer sequence removed (nucleotides 92 to 1 11 of SEQ ID NO: 22), the 20 bp protospacer region targeting the T. reesei wA gene, the S. pyogenes single guide RNA sequence, the poly-T terminator, and flanking homologous sequences for insertion into plasmid pSMai268 was synthesized as a STRING™ DNA fragment (SEQ ID NO: 23) by GENEART®. The DNA fragment was resuspended in 10 mM Tris pH 8 at a concentration of 30 ng/μΙ and 2 μΙ was used to insert the fragment into Hind Ill-digested pSMai268 using an IN- FUSION® HD Cloning Kit. The reaction was composed of 125 ng of Hind III digested pSMai268, 60 ng of the DNA fragment, and 1X IN-FUSION® HD Premix in a 10 μΙ reaction volume. After incubating the mixture for 15 minutes at 50°C, 2 μΙ of the reaction were transformed into 50 μΙ of E. coli STELLAR™ competent cells. The cells were heat shocked at 42°C for 45 seconds after which 100 μΙ of SOC medium were added and the total volume was spread onto a 150 mm 2XYT + Amp plate and incubated at 37°C overnight. The resulting E. coli transformants were individually inoculated into 3 ml of LB + Amp medium in 14 ml round- bottom polypropylene tubes and incubated at 37°C overnight with shaking at 200 rpm. Plasmid DNA was isolated using a BIOROBOT® 9600. The insert was confirmed by DNA sequencing with a Model 377 XL Automated DNA Sequencer using dye-terminator chemistry (Giesecke et al., 1992, supra). One plasmid designated pJfyS242 (Figure 6) was selected for analyzing the A. fumigatus U6-1 tRNAgly(GCC)1-6 sequence for single guide RNA expression in T. reesei. Example 9: Construction of plasmid pJfyS254 containing the Aspergillus fumigatus tRNAhis(GTG)1-2 promoter, Aspergillus fumigatus tRNAhis(GTG)1-2, wA protospacer, Streptomyces pyogenes single guide RNA sequence, and poly-T terminator

The Aspergillus fumigatus tRNAhis(GTG)1-2 and corresponding promoter were identified by searching the gene annotations of the Aspergillus fumigatus strain 293 genome sequence database from the Joint Genome Institute (JGI). The tRNA sequence was annotated and the 481 bp region upstream of the tRNAhis(GTG)1-2 annotation was selected as the promoter.

A synthetic DNA fragment containing 481 bp of the Aspergillus fumigatus tRNAhis(GTG)1-2 promoter (SEQ ID NO: 24), tRNAhis(GTG)1-2 sequence (SEQ ID NO: 25) with the 3' leader sequence removed (nucleotides 92 to 11 1 of SEQ ID NO: 25), the 20 bp protospacer region targeting the T. reesei wA gene, the S. pyogenes single guide RNA sequence, the poly-T terminator, and flanking homologous sequences for insertion into plasmid pSMai268 was synthesized as a STRING™ DNA fragment (SEQ ID NO: 26) by GENEART®. The DNA fragment was resuspended in 10 mM Tris pH 8 at a concentration of 30 ng/μΙ and 2 μΙ was used to insert the fragment into Hind Ill-digested pSMai268 using an INFUSION® HD Cloning Kit. The reaction was composed of 125 ng of Hind III digested pSMai268, 60 ng of the DNA fragment, and 1X IN-FUSION® HD Premix in a 10 μΙ reaction volume. After incubating the mixture for 15 minutes at 50°C, 2 μΙ of the reaction were transformed into 50 μΙ of E. coli STELLAR™ competent cells. The cells were heat shocked at 42°C for 45 seconds after which 100 μΙ of SOC medium were added and the total volume was spread onto a 150 mm 2XYT + Amp plate and incubated at 37°C overnight. The resulting E. coli transformants were individually inoculated into 3 ml of LB + Amp medium in 14 ml round- bottom polypropylene tubes and incubated at 37°C overnight with shaking at 200 rpm. Plasmid DNA was isolated using a BIOROBOT® 9600. The insert was confirmed by DNA sequencing with a Model 377 XL Automated DNA Sequencer using dye-terminator chemistry (Giesecke et al., 1992, supra). One plasmid designated pJfyS254 (Figure 7) was selected for analyzing the tRNAhis(GTG)1-2 promoter and the tRNAhis(GTG)1-2 sequence for single guide RNA and tRNA expression in T. reesei.

Example 10: Construction of plasmid pJfyS244 containing the Aspergillus fumigatus U6-2 promoter, wA protospacer, Streptomyces pyogenes single guide RNA sequence, and A. fumigatus U6-2 terminator

The Aspergillus fumigatus U6-2 promoter (SEQ ID NO: 27) and terminator (SEQ ID NO: 28) were identified by searching the gene annotations of the Aspergillus fumigatus strain 293 genome sequence database from the Joint Genome Institute (JGI). A synthetic DNA sequence containing 500 bp of the A. fumigatus U6-2 promoter, the 20 bp protospacer region targeting the T. reesei wA gene, the S. pyogenes single guide RNA sequence, 209 bp of the A. fumigatus U6-2 terminator, and flanking homologous sequences for insertion into plasmid pSMai268 was synthesized as a STRING™ DNA fragment (SEQ ID NO: 29) by GENEART®. The DNA fragment was resuspended in 10 mM Tris pH 8 at a concentration of 30 ng/μΙ and 2 μΙ was used to insert the fragment into Hind Ill-digested pSMai268 using an IN-FUSION® HD Cloning Kit. The reaction was composed of 125 ng Hind Ill-digested pSMai268, 60 ng of the DNA fragment, and 1X IN-FUSION® HD Premix in a 10 μΙ reaction volume. After incubating the mixture for 15 minutes at 50°C, 2 μΙ of the reaction were transformed into 50 μΙ of E. coli STELLAR™ competent cells. The cells were heat shocked at 42°C for 45 seconds after which 100 μΙ of SOC medium were added and the total volume was spread onto a 150 mm 2XYT + Amp plate and incubated at 37°C overnight. The resulting E. coli transformants were individually inoculated into 3 ml of LB + Amp medium in 14 ml round-bottom polypropylene tubes and incubated at 37°C overnight with shaking at 200 rpm. Plasmid DNA was isolated using a BIOROBOT® 9600. The insert was confirmed by DNA sequencing with a Model 377 XL Automated DNA Sequencer using dye-terminator chemistry (Giesecke et al., 1992, supra). One plasmid designated pJfyS244 (Figure 8) was selected for analyzing the A. fumigatus U6- 2 promoter and terminator for single guide RNA expression in T. reesei.

Example 11 : Construction of plasmid pJfyS245 containing the Aspergillus fumigatus U6-3 promoter, wA protospacer, Streptomyces pyogenes single guide RNA sequence, and A. fumigatus U6-3 terminator The Aspergillus fumigatus U6-3 promoter (SEQ ID NO: 30) and terminator (SEQ ID NO: 31)] were identified by searching the gene annotations of the Aspergillus fumigatus strain 293 genome sequence database from the Joint Genome Institute (JGI). A synthetic DNA sequence containing 500 bp of the A. fumigatus U6-3 promoter, a 20 bp protospacer region targeting the T. reesei wA gene, the S. pyogenes single guide RNA sequence, 209 bp of the A. fumigatus U6-3 terminator, and flanking homologous sequences for insertion into plasmid pSMai268 was synthesized as a STRING™ DNA fragment (SEQ ID NO: 32) by GENEART®. The DNA fragment was resuspended in 10 mM Tris pH 8 at a concentration of 30 ng/μΙ and 2 μΙ was used to insert the fragment into Hind Ill-digested pSMai268 using an IN-FUSION® HD Cloning Kit. The reaction was composed of 125 ng of Hind Ill-digested pSMai268, 60 ng of the DNA fragment, and 1X IN-FUSION® HD Premix in a 10 μΙ reaction volume. After incubating the mixture for 15 minutes at 50°C, 2 μΙ of the reaction were transformed into 50 μΙ of E. coli STELLAR™ competent cells. The cells were heat shocked at 42°C for 45 seconds after which 100 μΙ of SOC medium were added and the total volume was spread onto a 150 mm 2XYT + Amp plate and incubated at 37°C overnight. The resulting E. coli transformants were individually inoculated into 3 ml of LB + Amp medium in 14 ml round-bottom polypropylene tubes and incubated at 37°C overnight with shaking at 200 rpm. Plasmid DNA was isolated using a BIOROBOT® 9600. The insert was confirmed by DNA sequencing with a Model 377 XL Automated DNA Sequencer using dye-terminator chemistry (Giesecke et ai, 1992, supra). One plasmid designated pJfyS245 (Figure 9) was selected for analyzing the A. fumigatus U6-3 promoter and terminator for single guide RNA expression in T. reesei.

Example 12: Construction of plasmid pJfyS247 containing the Aspergillus oryzae U6-2 promoter, wA protospacer, Streptomyces pyogenes single guide RNA sequence, and Aspergillus oryzae U6-2 terminator

A synthetic DNA sequence containing 508 bp of the Aspergillus oryzae U6-2 promoter (Katayama et ai, 2016, Biotechnol. Lett. 38: 637-642; SEQ ID NO: 33;, the 20 bp protospacer region targeting the T. reesei wA gene, the S. pyogenes single guide RNA sequence, 138 bp of the A. oryzae U6-2 terminator (Katayama et ai, 2016, supra; SEQ ID NO: 34), and flanking homologous sequences for insertion into plasmid pSMai268 was synthesized as a STRING™ DNA fragment (SEQ ID NO: 35) by GENEART®. The DNA fragment was resuspended in 10 mM Tris pH 8 at a concentration of 30 ng/μΙ and 2 μΙ was used to insert the fragment into Hind Ill-digested pSMai268 using an IN-FUSION® HD Cloning Kit. The reaction was composed of 125 ng of Hind III digested pSMai268, 60 ng of the DNA fragment, and 1X IN-FUSION® HD Premix in a 10 μΙ reaction volume. After incubating the mixture for 15 minutes at 50°C, 2 μΙ of the reaction were transformed into 50 μΙ of E. coli STELLAR™ competent cells. The cells were heat shocked at 42°C for 45 seconds after which 100 μΙ of SOC medium were added and the total volume was spread onto a 150 mm 2XYT + Amp plate and incubated at 37°C overnight. The resulting E. coli transformants were individually inoculated into 3 ml of LB + Amp medium in 14 ml round-bottom polypropylene tubes and incubated at 37°C overnight with shaking at 200 rpm. Plasmid DNA was isolated using a BIOROBOT® 9600. The insert was confirmed by DNA sequencing with a Model 377 XL Automated DNA Sequencer using dye-terminator chemistry (Giesecke et al., 1992, supra). One plasmid designated pJfyS247 (Figure 10) was selected for analyzing the A. oryzae U6-2 promoter and terminator for single guide RNA expression in T. reesei.

Example 13: Construction of plasmid pSMai279 containing the Trichoderma reesei U6 small nuclear RNA promoter, Streptomyces pyogenes single guide RNA sequence, Trichoderma reesei U6 terminator, and wA protospacer

A predicted T. reesei U6 small nuclear RNA gene was identified by performing BLAST searches of the T. reesei RutC30 genome sequence (JGI) using the Homo sapiens U6-1 small nuclear RNA gene sequence as query (SEQ ID NO: 36). Approximately 175 bp of the DNA sequence corresponding to the T. reesei U6 small nuclear RNA promoter (SEQ ID NO: 37) was amplified from T. reesei BTR213 genomic DNA using the forward and reverse primers shown below (SEQ ID NOs: 38 and 39). Similarly, a 130 bp fragment of the 20 bp protospacer region targeting the T. reesei wA gene and the S. pyogenes single guide RNA were amplified from pSMai274 using the forward and reverse primers shown below (SEQ ID NOs: 40 and 41). Approximately 307 bp of the DNA sequence predicted to be RNA polymerase Ill-based U6 terminator (SEQ ID NO: 44) was amplified from T. reesei BTR213 genomic DNA using the forward and reverse primers shown below (SEQ ID NOs: 42 and 43). T. reesei BTR213 genomic DNA was prepared according to the procedure described in Example 1.

Forward primer:

5'-CCTGCAGG C ATG C A AG CTTT ATAGTA AT AAA AG CTT AG C- 3' (SEQ ID NO: 38)

Reverse primer:

5'-GGTATTCTCATCCAGCGATAGACTACCATCAAACAG-3' (SEQ ID NO: 39)

Forward primer:

5'-TTGATGGTAGTCTATCGCTGGATGAGAATACCCTCG-3' (SEQ ID NO: 40)

Reverse primer:

5'-TGGAAGAGAAAAAAAAAAGCACCGACTCGGTGCCAC-3' (SEQ ID NO: 41)

Forward primer:

5'-CACCGAGTCGGTGCTTTTTTTTTTCTCTTCCAAGTCGTG-3' (SEQ ID NO: 42)

Reverse primer: 5'-TGATTCTGCTGTCTCGAAGCTTCGAAGACGGGCTGCCGAGGA-3' (SEQ ID NO: 43)

For amplification of the T. reesei U6 small nuclear RNA gene promoter, the reaction was composed of 150 ng of T. reesei BTR213 genomic DNA, 200 μΜ dNTPs, 1 μΜ of each primer (SEQ ID NO: 38 and 39), 1X PHUSION® High-Fidelity Hot Start DNA Polymerase Buffer, and 1.0 unit of PHUSION® High-Fidelity Hot Start DNA Polymerase in a final volume of 50 μΙ. The amplification reaction was incubated in a thermocycler programmed for 1 cycle at 98°C for 3 minutes; 30 cycles each at 98°C for 10 seconds, 56°C for 30 seconds, and 72°C for 30 seconds; and 1 cycle at 72°C for 15 minutes. The PCR product was isolated by 1 % agarose gel electrophoresis using TAE buffer where a 212 bp fragment was excised from the gel and extracted using a NUCLEOSPIN® Gel and PCR Clean-up Kit.

For amplification of the 20 bp protospacer region targeting the T. reesei wA gene and the S. pyogenes single guide RNA, the reactions were composed of 10 ng of pSMai274, 200 μΜ dNTPs, 1 μΜ of each primer (SEQ ID NOs: 40 and 41), 1X PHUSION® High-Fidelity Hot Start DNA Polymerase Buffer, and 1.0 unit of PHUSION® High-Fidelity Hot Start DNA Polymerase in a final volume of 50 μΙ. The amplification reactions were incubated in a thermocycler programmed for 1 cycle at 98°C for 3 minutes; 30 cycles each at 98°C for 10 seconds, 56°C for 30 seconds, and 72°C for 30 seconds; and 1 cycle at 72°C for 15 minutes. The PCR products were isolated by 1 % agarose gel electrophoresis using TAE buffer where a 130 bp fragment was excised from the gel and extracted using a NUCLEOSPIN® Gel and PCR Clean-up Kit.

For amplification of the T. reesei U6 small nuclear RNA gene terminator, the reaction was composed of 150 ng of T. reesei BTR213 genomic DNA, 200 μΜ dNTPs, 1 μΜ of each primer (SEQ ID NO: 42 and 43), 1X PHUSION® High-Fidelity Hot Start DNA Polymerase Buffer, and 1.0 unit of PHUSION® High-Fidelity Hot Start DNA Polymerase in a final volume of 50 μΙ. The amplification reaction was incubated in a thermocycler programmed for 1 cycle at 98°C for 3 minutes; 30 cycles each at 98°C for 10 seconds, 56°C for 30 seconds, and 72°C for 30 seconds; and 1 cycle at 72°C for 15 minutes. The PCR product was isolated by 1 % agarose gel electrophoresis using TAE buffer where a 345 bp fragment was excised from the gel and extracted using a NUCLEOSPIN® Gel and PCR Clean-up Kit.

The fourth DNA segment was generated by restriction enzyme digestion of pSMai274 with Hind III. The reaction was composed of 5 μg of pSMai274, 40 units of Hind III, and 10 μΙ of CutSmart® Buffer in a total volume of 100 μΙ. The reaction was incubated at 37°C for 4 hours and then separated by 1 % agarose gel electrophoresis using TAE buffer where a 9.758 kb fragment was excised from the gel and extracted using a NUCLEOSPIN® Gel and PCR Clean-up Kit.

The three PCR products of 212 bp, 130 bp, and 345 bp were inserted into Hind III digested pSMai274 using an IN-FUSION® HD Cloning Kit according to the manufacturer's protocol. The reaction was composed of 2 μΙ 5X IN-FUSION® HD enzyme Premix, 100 ng of the Hind Ill-digested pSMai274, 50 ng of the 212 bp T. reesei U6 small nuclear RNA promoter PCR product, 50 ng of the 130 bp protospacer region targeting the T. reesei wA gene, the S. pyogenes single guide RNA PCR product, and 50 ng of the 345 bp T. reesei U6 small nuclear RNA gene terminator PCR product in a 10 μΙ reaction volume. The reaction was incubated for 15 minutes at 50°C. After the incubation period, 2 μΙ were transformed into 50 μΙ of E. coli STELLAR™ competent cells. The cells were heat shocked at 42°C for 45 seconds and then 450 μΙ of SOC medium, pre-heated to 42°C, were added. The cells were incubated at 37°C with shaking at 200 rpm for 60 minutes and then spread onto a 150 mm diameter 2XYT plus ampicillin plates and incubated at 37°C overnight. The resulting E. coli transformants were individually inoculated into 3 ml of LB + Amp medium in 14 ml round-bottom polypropylene tubes and incubated at 37°C overnight with shaking at 200 rpm. Plasmid DNA was isolated using a BIOROBOT® 9600. The insert was confirmed by DNA sequencing with a Model 377 XL Automated DNA Sequencer using dye-terminator chemistry (Giesecke et al., 1992, supra). One plasmid containing the insert with no PCR errors was identified and designated pSMai279 (Figure 1 1) and was used to analyze the T. reesei U6 small nuclear RNA promoter and terminator for single guide RNA expression in T. reesei. Example 14: Construction of plasmid pJfyS249 containing the Trichoderma reesei U6 small nuclear RNA promoter, Aspergillus fumigatus tRNAgly(GCC)1 -6 sequence, wA protospacer, Streptomyces pyogenes single guide RNA sequence, and T. reesei U6 small nuclear RNA terminator

A synthetic DNA sequence containing the 175 bp of the T. reesei U6 small nuclear RNA promoter (Example 13), the Aspergillus fumigatus tRNAgly(GCC)1-6 sequence with the 3' leader sequence removed (Example 13), the 20 bp protospacer region targeting the T. reesei wA gene, the S. pyogenes single guide RNA sequence, 307 bp of T. reesei U6 small nuclear RNA terminator, and flanking homologous sequences for insertion into plasmid pSMai268 was synthesized as a STRING™ DNA fragment (SEQ ID NO: 45) by GENEART®. The DNA fragment was resuspended in 10 mM Tris pH 8 at a concentration of 30 ng/μΙ and 2 μΙ was used to insert the fragment into Hind Ill-digested pSMai268 using an IN-FUSION® HD Cloning Kit. The reaction was composed of 125 ng of Hind \\\ digested pSMai268, 60 ng of the DNA fragment, and 1X IN-FUSION® HD Premix in a 10 μΙ reaction volume. After incubating the mixture for 15 minutes at 50°C, 2 μΙ of the reaction were transformed into 50 μΙ of E. coli STELLAR™ competent cells. The cells were heat shocked at 42°C for 45 seconds after which 100 μΙ of SOC medium were added and the total volume was spread onto a 150 mm 2XYT + Amp plate and incubated at 37°C overnight. The resulting E. coli transformants were individually inoculated into 3 ml of LB + Amp medium in 14 ml round-bottom polypropylene tubes and incubated at 37°C overnight with shaking at 200 rpm. Plasmid DNA was isolated using a BIOROBOT® 9600. The insert was confirmed by DNA sequencing with a Model 377 XL Automated DNA Sequencer using dye-terminator chemistry (Giesecke et al., 1992, supra). One plasmid designated pJfyS249 (Figure 12) was selected for analyzing the T. reesei U6 small nuclear RNA promoter and terminator for A. fumigatus tRNAgly(GCC)1-6 and single guide RNA expression in T. reesei. Example 15: Construction of plasmid pJfyS250 containing the Aspergillus oryzae U6-2 promoter, Aspergillus fumigatus tRNAgly(GCC)1-6, Streptomyces pyogenes single guide RNA sequence, Aspergillus oryzae U6-2 terminator, and wA protospacer

A synthetic DNA sequence containing 508 bp of the A. oryzae U6-2 promoter (Katayama et al., 2016, Biotechnol. Lett. 38: 637-642), the A. fumigatus tRNAgly(GCC)1-6 sequence with the 3' leader sequence removed (Example 8), the 20 bp protospacer region targeting the T. reesei wA gene, the S. pyogenes single guide RNA sequence, 138 bp of the A. oryzae U6-2 terminator (Katayama et al., 2016, supra), and flanking homologous sequences for insertion into plasmid pSMai268 was synthesized as a STRING™ DNA fragment (SEQ ID NO: 46) by GENEART®. The DNA fragment was resuspended in 10 mM Tris pH 8 at a concentration of 30 ng/μΙ and 2 μΙ was used to insert the fragment into Hind III digested pSMai268 using an IN-FUSION® HD Cloning Kit. The reaction was composed of 125 ng of Hind \\\ digested pSMai268, 60 ng of the DNA fragment, and 1X IN-FUSION® HD Premix in a 10 μΙ reaction volume. After incubating the mixture for 15 minutes at 50°C, 2 μΙ of the reaction were transformed into 50 μΙ of E. coli STELLAR™ competent cells. The cells were heat shocked at 42°C for 45 seconds after which 100 μΙ of SOC medium were added and the total volume was spread onto a 150 mm 2XYT + Amp plate and incubated at 37°C overnight. The resulting E. coli transformants were individually inoculated into 3 ml of LB + Amp medium in 14 ml round-bottom polypropylene tubes and incubated at 37°C overnight with shaking at 200 rpm. Plasmid DNA was isolated using a BIOROBOT® 9600. The insert was confirmed by DNA sequencing with a Model 377 XL Automated DNA Sequencer using dye-terminator chemistry (Giesecke et al., 1992, supra). One plasmid designated pJfyS250 (Figure 13) was selected for analyzing the A. oryzae U6-2 promoter for A. fumigatus tRNAgly(GCC)1-6 and single guide RNA expression in T. reesei Example 16: Construction of plasmid pJfyS251 containing the Magnaporthe oryzae U6- 2 promoter, A. fumigatus tRNAgly(GCC)1 -6 sequence, wA protospacer, Streptomyces pyogenes single guide RNA sequence, and polyT terminator

A synthetic DNA sequence containing 500 bp of the Magnaporthe oryzae U6-2 promoter (Example 5), the A. fumigatus tRNAgly(GCC)1-6 sequence with the 3' leader sequence removed, the 20 bp protospacer region targeting the T. reesei wA gene, the S. pyogenes single guide RNA sequence, 5 bp of the poly (T) terminator, and flanking homologous sequences for insertion into plasmid pSMai268 was synthesized as a STRING™ DNA fragment (SEQ ID NO: 47) by GENEART® cloned into a commercially supplied GENEART® standard cloning vector. The lyophilized plasmid DNA supplied by GENEART® containing the fragment was resuspended in water at a concentration of 30 ng/μΙ. The sequence containing the elements above was PCR amplified using PHUSION® Hot Start High Fidelity DNA Polymerase and the forward and reverse primers shown below.

Forward Primer:

5' - AG ATG A ATATTG CCTG C AG G- 3' (SEQ ID NO: 48)

Reverse Primer:

5'-TGATTCTGCTGTCTCGAAGC-3' (SEQ ID NO: 49)

The PCR was composed of 20 ng of the GENEART® plasmid as template, 200 μΜ dNTPs, 0.4 μΜ primers, 1X PHUSION® Reaction Buffer, and 2 units of PHUSION® Hot Start II High Fidelity DNA polymerase in a final volume of 50 μΙ. The reaction was incubated in a thermocycler programmed for 1 cycle at 98°C for 3 minutes; 30 cycles each at 98°C for 20 seconds, 52°C for 20 seconds, and 72°C for 50 seconds; and 1 cycle at 72°C for 2 minutes. One μΙ of Dpn I (10 Units) was added to the reaction and the reaction was incubated for 1 hour at 37°C to digest the plasmid template. The reaction was cleaned up using a NUCLEOSPIN® II Gel and PCR Clean-up Kit. Briefly 2 volumes of Kit-supplied NT buffer were added and the reaction was applied to the Clean-up column using a vacuum manifold. The column was rinsed with 750 μΙ of NTI wash buffer and eluted with 15 μΙ of NE buffer.

The fragment was inserted into Hind Ill-digested pSMai268 using an IN-FUSION® HD Cloning Kit. The reaction was composed of 125 ng of Hind III digested pSMai268, 60 ng of the PCR fragment, and 1X IN-FUSION® HD Premix in a 10 μΙ reaction volume. After incubating the mixture for 15 minutes at 50°C, 2 μΙ of the reaction were transformed into 50 μΙ of E. coli STELLAR™ competent cells. The cells were heat shocked at 42°C for 45 seconds after which 100 μΙ of SOC medium were added and the total volume was spread onto a 150 mm 2XYT + Amp plate and incubated at 37°C overnight. The resulting E. coli transformants were individually inoculated into 3 ml of LB + Amp medium in 14 ml round-bottom polypropylene tubes and incubated at 37°C overnight with shaking at 200 rpm. Plasmid DNA was isolated using a BIOROBOT® 9600. The insert was confirmed by DNA sequencing with a Model 377 XL Automated DNA Sequencer using dye-terminator chemistry (Giesecke et al., 1992, supra). One plasmid designated pJfyS251 (Figure 14) was selected for analyzing the Magnaporthe oryzae U6-2 promoter and terminator for single guide RNA with tRNAgly(GCC)1-6 expression in T. reesei. Example 17: Construction of plasmid pJfyS253 containing the Aspergillus fumigatus U6-3 promoter, Aspergillus fumigatus tRNAgly(GCC)1 -6 sequence, wA protospacer, Streptomyces pyogenes single guide RNA sequence, Aspergillus fumigatus U6-3 terminator

A synthetic DNA sequence containing 500 bp of the A. fumigatus U6-3 promoter, the Aspergillus fumigatus tRNAgly(GCC)1 -6 sequence, the 20 bp protospacer region targeting the T. reesei wA gene, the S. pyogenes single guide RNA sequence, 209 bp of the A. fumigatus U6-3 terminator, and flanking homologous sequences for insertion into plasmid pSMai268 was synthesized as a STRING™ DNA fragment (SEQ ID NO: 50) by GENEART® cloned into a commercially supplied GENEART® standard cloning vector. The lyophilized plasmid DNA supplied by GENEART® containing the fragment was resuspended in water at a concentration of 30 ng/μΙ. The sequence containing the elements above was PCR amplified using PHUSION® Hot Start High Fidelity DNA Polymerase and the forward and reverse primers shown in Example 17.

The PCR was composed of 20 ng of the GENEART® plasmid as template, 200 μΜ dNTPs, 0.4 μΜ primers, 1X PHUSION® Reaction Buffer, and 2 units of PHUSION® Hot Start II High Fidelity DNA polymerase in a final volume of 50 μΙ. The reaction was incubated in a thermocycler programmed for 1 cycle at 98°C for 3 minutes; 30 cycles each at 98°C for 20 seconds, 52°C for 20 seconds, and 72°C for 50 seconds; and 1 cycle at 72°C for 2 minutes. One μΙ of Dpn I (10 U) was added to digest the plasmid template and the reaction was incubated for 1 hour at 37°C. The reaction was cleaned up using a NUCLEOSPIN® II Gel and PCR Clean-up Kit. Briefly 2 volumes of Kit-supplied NT buffer were added and the reaction was applied to the Clean-up column using a vacuum manifold. The column was rinsed with 750 μΙ of NTI wash buffer and eluted with 15 μΙ NE buffer.

The fragment was inserted into Hind Ill-digested pSMai268 using an IN-FUSION® HD Cloning Kit. The reaction was composed of 125 ng of Hind III digested pSMai268, 60 ng of the PCR fragment, and 1X IN-FUSION® HD Premix in a 10 μΙ reaction volume. After incubating the mixture for 15 minutes at 50°C, 2 μΙ of the reaction were transformed into 50 μΙ of E. coli STELLAR™ competent cells. The cells were heat shocked at 42°C for 45 seconds after which 100 μΙ of SOC medium were added and the total volume was spread onto a 150 mm 2XYT + Amp plate and incubated at 37°C overnight. The resulting E. coli transformants were individually inoculated into 3 ml of LB + Amp medium in 14 ml round-bottom polypropylene tubes and incubated at 37°C overnight with shaking at 200 rpm. Plasmid DNA was isolated using a BIOROBOT® 9600. The insert was confirmed by DNA sequencing with a Model 377 XL Automated DNA Sequencer using dye-terminator chemistry (Giesecke et ai, 1992, supra). One plasmid designated pJfyS253 (Figure 15) was selected for analyzing the A. fumigatus U6-3 promoter for A. fumigatus tRNAgly(GCC)1-6 and single guide RNA expression in T. reesei.

Example 18: Analysis of promoter elements and/or tRNAs for single guide RNA expression to enhance homology-directed repair utilizing CRIPSR/Cas9

In each of the following experiments, the T. reesei strains were transformed with the wA deletion plasmid pSMai271 (Example 3) containing 400 bp of 5' flanking sequence and 400 bp of 3' flanking sequence of the wA gene in addition to the amdS selectable marker for selection of transformants. Strains in which the wA gene has been inactivated or removed display a non-pigmented white phenotype, in contrast to the wild-type green phenotype of T. reesei, and as such can be identified visually.

In each of the experiments, the deletion plasmid pSMai271 (Example 3) was transformed on its own using the method described in Example 2 and in low frequency. Due to the short flanking regions, the plasmid can integrate at the wA locus via the host cell's DNA repair machinery, effectively removing the gene coding sequence. These transformations represent the transformation frequency in the absence of CRISPR/Cas9 elements. In addition to the afore-mentioned control transformation, strains were also transformed with the same deletion plasmid in addition to each of the CRIPSR/Cas9 plasmids described herein. Since a double stranded DNA break can increase the rate of homologous recombination for gene targeting (Rudin et al., 1989, Genetics 122(3): 519-534; Smith et al. , 1995, Nucleic Acids Res. 23(24): 5012-9), the more transformants obtained in a transformation containing the deletion construct and CRISPR/Cas9 plasmid is indicative of an increased rate of double stranded breaks. When transformed with a deletion construct and a CRIPSR/Cas9 construct, the latter is responsible for initiating the double stranded DNA break and the deletion construct provides a template DNA from which the cell's DNA repair machinery repair the break via homologous recombination between the flanking regions and the chromosome, a term called "homology- directed repair". Therefore, these studies identified elements that could increase the transformation efficiency using equal quantities of deletion plasmid, where transformations resulting in greater amount of transformants displaying the intended white phenotype indicated a more efficient CRISPR/Cas9/single guide RNA combination. In all cases the same Cas9 protein, gene optimized for expression in Aspergillus and under control of the Aspergillus nidulans tefl promoter (N0dvig et al., 2015, supra) was used as well as the same protospacer targeting a region located in the third exon of the wA gene. Each individual promoter tested as well as some additional modifications to the S. pyogenes single guide RNA are described in the following Examples.

The transformation efficiency of each batch of protoplasts can vary greatly, so in each example the pSMai271 fragment alone was included and transformation efficiency (and therefore CRIPSR/Cas9 efficiency) of each of the CRIPSR/Cas9 plasmids can be compared to the control within the same experiment. Due to the high variability of transformation efficiency between batches of protoplasts and subsequent transformations, transformant numbers or transformation efficiency cannot be compared between experiments/examples.

Example 19: Analysis of the Magnaporthe oryzae U6-2 promoter and Aspergillus fumigatus U6-1 promoter for single guide RNA expression

T. reesei strain JfyS 139/144-1 OB was transformed as described in Example 2 with the wA deletion plasmid pSMai271 by itself as well as in combination with plasmid pJfyS238 (Example 7) in which the S. pyogenes single guide RNA was driven by the A. fumigatus U6-1 promoter (Example 7), as well as in combination with pSMai274 (Example 5) in which the S. pyogenes single guide RNA was driven by the Magnaporthe oryzae U6-2 promoter (Example 5). For each construct combination, 8 transformations were performed. The results of the transformation are shown below in Table 1.

Table 1. Analysis of the Magnaporthe oryzae U6-2 promoter and Aspergillus fumigatus U6-1 promoters for wA single guide RNA expression in CRISPR/Cas9-based homology directed repair.

The A. fumigatus U6-1 promoter did not increase the transformation efficiency compared to the pSMai271 deletion plasmid alone, when used to control expression of the S. pyogenes single guide RNA sequence, suggesting that is was not effective in generating effective single guide RNAs. The M. oryzae U6-2 promoter, however, increased the transformation efficiency dramatically, where the white phenotype indicative of the correct targeting of the integration cassette was observed in 11 1 transformants compared to 2 white transformants obtained with the pSMai271 deletion plasmid alone. These results demonstrated that the M. oryzae U6-2 promoter is far superior to the A. fumigatus U6-1 promoter for S. pyogenes single guide RNA expression and demonstrated that the M. oryzae U6-2 promoter is an effective RNA polymerase III promoter for utilization of CRIPSR/Cas9 gene modification in T. reesei.

Example 20: Analysis of the Magnaporthe oryzae U6-2 promoter, Magnaporthe oryzae U6-2 promoter and terminator for single guide RNA expression

Since many U6 promoters contain transcriptional elements within the terminator region of the gene, the terminator for the M. oryzae U6-2 gene (Example 5) was identified from the genome sequence and added directly downstream of the S. pyogenes single guide RNA region in pSMai274 resulting in pSMai280 (Example 6). To analyze the plasmid's effectiveness in generating double stranded breaks, T. reesei strain JfyS139/144-1 OB was transformed according to the methods described in Example 2 with the wA deletion plasmid pSMai271 by itself as well as in combination with pSMai274 or pSMai280. For each construct combination 4 transformations reactions were performed. The results of the transformations are shown below in Table 2.

Table 2. Analysis of the Magnaporthe oryzae U6-2 promoter with and without terminator for wA single guide RNA expression in CRISPR/Cas9 initiated homology-directed repair.

The addition of the M. oryzae U6-2 terminator improved the function of the CRIPSR/Cas9 system dramatically, improving the transformation efficiency approximately 6- fold. Since many U6 promoters from other fungi contain transcriptional elements downstream of the transcript termination site (Marck et al., 2006, Nucleic Acids Res. 34 (6): 1816-35) this result provided an effective tool to increase single guide RNA expression.

Example 21 : Analysis of the Aspergillus oryzae U6-2 promoter and terminator, Aspergillus fumigatus U6-2 promoter and terminator, Aspergillus fumigatus U6-3 promoter and terminator, and Aspergillus fumigatus U6-1 promoter and A. fumigatus tRNAgly(GCC)1 -6 for single guide RNA expression In order to identify other U6 promoters from other species and compare their effectiveness, T. reesei strain JfyS139/144-10B was transformed as described in Example 2 with wA deletion plasmid pSMai271 by itself as well as in combination with CRISPR/Cas9 plasmids pJfyS244, pJfyS245, and pJfyS247 with the S. pyogenes single guide RNA driven by the A. fumigatus U6-2 promoter and terminator (pJfyS244), the A. fumigatus U6-3 promoter and terminator (pJfyS245), and the A. oryzae U6 promoter and terminator (pJfyS247). In addition to testing the other promoters to drive guide expression, tRNAgly(GCC)1-6 from A. fumigatus was added to the guide targeting the wA gene, upstream of the protospacer used previously, in an attempt to improve the transcription and consistency of RNA in the S. pyogenes single guide RNA sequence, facilitated by the endogenous RNase Z processing of the tRNA-single guide RNA fusion. Since RNAse Z recognizes the tRNA structure independent of sequence (Canino et al., 2009, Plant Physiol. 150(3): 1494-1502; Barbezier et ai, 2009, Plant Physiol. 150(3): 1598-1610) and the tRNA structure and processing are strictly conserved, the single guide RNA-tRNA fusion could be correctly processed by the endogenous T. reesei RNAse Z. The results of the analysis are shown below in Table 3.

Table 3. Analysis of the Aspergillus oryzae U6 promoter and terminator, Magnaporthe oryzae U6-2 promoter, Aspergillus fumigatus U6-2 promoter and terminator, Aspergillus fumigatus U6-3 promoter and terminator and A. fumigatus U6-1 with tRNAgly(GCC)1-6 for wA single guide RNA expression in CRISPR/Cas9 initiated homology-directed repair

The results indicated that the U6 promoters and terminator combinations tested in this experiment had similar efficiencies in generating double-stranded DNA breaks using the

CRIPSR/Cas9 system and all were effective in doing so. The addition of the A. fumigatus tRNAgly(GCC)1-6, however, was able to turn a previously non-functional promoter into one that worked far better than any other promoter and terminator alone. The presence of the A. fr m/gafi s tRNAgly(GCC)1-6 ensures accurate processing, and can solve problems in delayed transcription initiation, leading to an accurate 5' end of the protospacer as well as providing transcriptional enhancement. Sequences of tRNAs contain A and B box elements, which bind transcription factor IIIC, which then initiates the recruitment of the transcription complex containing transcription factor NIB and RNA polymerase III (White 201 1 , Nat. Rev. Genet. 12(7): 459-463; Died et al., 2007, Trends Genet. 23(12): 614-622). Addition of a tRNA in this fashion has also been seen to improve CRISPR/Cas9 efficiencies in the yeast Yarrowia lipolytica (Schwartz et al., 2016, ACS Synth. Biol. 5(4): 356-9) but effectiveness has not been shown in any studies to date in any filamentous fungus or other ascomycete and has not been shown with a U6 promoter.

Example 22: Analysis of the Trichoderma reesei U6 promoter and terminator, Trichoderma reesei U6 promoter and A. fumigatus tRNAgly and Trichoderma reesei U6 terminator, Aspergillus fumigatus U6-1 promoter and A. fumigatus tRNAgly(GCC)1 -6 for single guide RNA expression

In order to evaluate whether or not the T. reesei U6 promoter could be improved by the addition of a tRNA, T. reesei strain JfyS139/144-10B was transformed with the wA deletion plasmid pSMai271 alone as well as in combination with pSMai279 (Example 13), which contains the wA single guide RNA driven by the promoter and terminator of the T. reesei U6 gene, or pJfyS249 in which the same single guide RNA was driven by the T. reesei U6 promoter and terminator and in which the same A. fumigatus tRNAgly(GCC)1-6 described in Example 8 was added upstream of the protospacer. Plasmid pJfyS242 was also added as a control to compare the efficiencies between the A. fumigatus U6-1 and T. reesei U6 promoters when both contained tRNAgly(GCC)1-6. Four reactions were transformed for each plasmid combination using the methods described in Example 2.

Table 4. Analysis of the T. reesei U6 promoter and terminator with and without A. fumigatus tRNAgly addition, compared to A. fumigatus U6-1 with tRNAgly(GCC)1-6 for wA single guide

RNA expression in CRISPR/Cas9 initiated homology-directed repair

DNA Promoter for tRNA Terminator for Transformants

transformed per single guide single guide Total Total % white reaction RNA RNA white

5 μ9 pSMai271 NA NA NA 0 0 0

5 μ9 pSMai271 7. reesei U6 None 7 reesei U6 3 3 100

+ 3.8 ig

pSMai279

5 μg pSMai271 7. reesei U6 tRNAgly(GC 7 reesei U6 102 99 97.1 + 3.8 [}Q C)1-6

pJfyS249

5 μ9 pSMai271 A. fumigatus U6- tRNAgly(GC TTTTT 96 95 99 + 3.8 pJfyS242 1 C)1-6

The results of the transformation displayed a modest increase in transformation efficiencies when the T. reesei U6 promoter was used to control expression of the wA single guide RNA. This is likely due to the presence of an intron within the T. reesei U6 transcript, which contains the A box element necessary for maintaining normal transcript levels of the U6 gene (Canzler et al., 2016, RNA Biology 13: 1 19-127). Since the intron was not included in the transcript when the T. reesei U6 promoter was used to control expression of the wA single guide RNA, it is not surprising that the system did not function very well. tRNAgly(GCC)1-6 addition to that same construct resulting in plasmid pJfyS249, however, was able to dramatically improve transformation efficiencies more that 30-fold likely for the reasons stated above in Example 20.

Example 23: Comparison of different U6 promoters with enhancement by A. fumigatus tRNAgly(GCC)1 -6 upstream of the protospacer in the wA single guide RNA

In order to evaluate the ability of addition of a tRNA to a single guide RNA to increase transformation efficiency and to compare with many of the other promoters, tRNAgly(GCC)1- 6 tested above was added to plasmids containing Cas9, and wA single guide RNA driven by either the A fumigatus U6-3 promoter and terminator, M. oryzae U6-2 promoter, or A oryzae U6 promoter and terminator resulting in plasmids pJfyS253, pJfyS251 , pJfyS250, for the A fumigatus U6-3 promoter and terminator, M. oryzae U6-2 promoter, or A oryzae U6 promoter and terminator, respectively. The three plasmids were compared to the plasmids containing the M. oryzae U6-2 promoter (pSMai274), M. oryzae promoter and terminator (pSMai280), and T. reesei U6 promoter and A fumigatus tRNAgly(GCC)1-6. Three transformations for each plasmid combination were performed according to the procedure in Example 2 and the results are shown in Table 5 below.

After the addition of tRNA to each of the U6 promoters the T. reesei U6 promoter and terminator, the A fumigatus U6-1 promoter and polyT terminator, and M. oryzae U6 promoter each performed similarly resulting in approximately 100 transformants from the three reactions transformed. The A fumigatus U6-3 promoter and terminator coupled with the tRNAgly yielded approximately 130 transformants with the expected white phenotype representing the best combination tested. The A oryzae U6 promoter and terminator yielded slightly lower transformant numbers than the others when coupled to the tRNA demonstrating that addition of tRNA, while improving efficiency in all cases tested, did improve some promoters better than others. Table 5. Comparative analysis of five U6 promoters with A. fumigatus tRNAgly(GCC)1-6 addition compared to the M. oryzae U6-2 promoter and promoter and terminator without tRNAgly addition for wA single guide RNA expression in CRISPR/Cas9 initiated homology- directed repair.

Example 24: Analysis of the Aspergillus fumigatus U6-1 promoter and tRNAgly(GCC)1- 6, and A. fumigatus tRNAhis(GTG)1-2 promoter and tRNAhis(GTG)1-2 for single guide RNA expression

This study evaluated whether a tRNA promoter would have comparable efficiency to the U6 promoters coupled with tRNA. The tRNAhis(GTG)1-2 promoter was identified from the Aspergillus fumigatus strain 293 genome and the promoter and corresponding tRNA were used to drive expression of the wA single guide RNA resulting in plasmid pjfy254. The plasmid was used in four transformations: T. reesei JfyS139/144-1 OB with the wA deletion plasmid pSMai271 and transformed alongside plasmid pJfyS242 containing the A. fumigatus U6-1 promoter coupled with tRNAgly as well as pSMai274 and pSMai280 which contain the M. oryzae U6-2 promoter and M. oryzae U6-2 promoter and terminator without tRNA for pSMai274 and pSMai280, respectively. Table 6. Comparative analysis of A. ft/m/gafivs tRNAhis(GTG)1-2 promoter and corresponding tRNA compared to A. fumigatus U6-1 promoter with A. fumigatus tRNAgly(GCC)1-6 addition compared to the M. oryzae U6-2 promoter and promoter and terminator without tRNAgly addition for wA single guide RNA expression in CRISPR/Cas9 initiated homology-directed DNA repair

The tRNAhis promoter + tRNAhis(GTG)1-2 dramatically increased the efficiency of wA gene deletion compared to the wA deletion plasmid (pSMai271) only control, especially since this transformation yielded no transformants with pSMai271 plasmid alone. With 106 transformants with the expected phenotype obtained the efficiency was greater than 100X improved compared to the deletion fragment alone. Compared to the A. fumigatus U6-1 promoter with the A. fumigatus tRNAgly(GCC)1-6, the tRNAhis(GTG)1-2 promoter and corresponding tRNA was not as effective as the A. fumigatus U6-1 promoter with the A. fumigatus tRNAgly(GCC)1-6, which resulted in 142 transformants compared to the tRNA promoter. Both promoters coupled with tRNAs, however, were better than the M. oryzae U6- 2 promoter and terminator as well as the M. oryzae U6-2 promoter alone. These results further demonstrated the effectiveness of the tRNA in improving the CRIPSR/Cas9 system functionality in T. reesei.

Example 25: Construction of plasmid pAT1153 [A. oryzae U6 promoter)

Two plasmids, pFC330 and pFC336, 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), were employed below, except the plasmids carry different antibiotic selection markers: plasmid pFC330 carries an A. fumigatus pyrG selection marker, the Streptomyces pyogenes Cas9 endonuclease expression cassette, and the AMA1 sequence for autonomous replication in Aspergillus, and plasmid pFC336 carries an A. fumigatus pyrG selection marker, the Streptomyces pyogenes Cas9 endonuclease expression cassette, an AMA1 sequence for autonomous replication in Aspergillus, and a single guide RNA expression cassette containing a wA protospacer. Plasmid pAT1153 (Figure 16) was constructed to contain the A. oryzae U6 small nuclear RNA promoter, A. fumigatus tRNAgly(GCC)1-6 sequence, wA protospacer, Streptomyces pyogenes single guide RNA sequence, A. oryzae U6 small nuclear RNA terminator, Streptomyces pyogenes Cas9 gene and the A. fumigatus pyrG gene.

Construction was by PCR of the A. oryzae U6 promoter single guide RNA expression cassette containing the wA protospacer (5'-AGTGGGATCTCAAGAACTAC-3'; SEQ ID NO: 63) found in plasmid pFC336. Two fragments were amplified by PCR with one primer set of primers oAT1 142 and oAT1 143 and a second primer set of primers oAT1 144 and oAT1145 shown below using plasmid pJfyS250 as template generating PCR fragments of 639 bp and 256 bp, respectively. The 639 bp fragment contains the A. oryzae U6 promoter, A. fumigatus tRNAgly(GCC)1-6 sequence, and the A. oryzae wA protospacer. The 256 bp fragment contains the Streptomyces pyogenes single guide RNA sequence, and the A. oryzae U6 terminator.

Primer oAT1142:

5'-TCCGCTGAGGGTTTAATTAATGGTTCACTTCTCTTTAG-3' (SEQ ID NO: 64)

Primer oAT1143:

5'-GTAGTTCTTGAGATCCCACTTGCATCATCCGTGAATC-3' (SEQ ID NO: 65)

Primer oAT1144:

5'-TCGGCTGAGGTCTTAATTAAAGCAGCTCTATATCACG-3' (SEQ ID NO: 66)

Primer oAT1145:

5'- AGTGGGATCTCAAGAACTACGTTTTAGAGCTAGAAATAGC-3' (SEQ ID NO: 67)

The PCRs were performed in a volume of 100 μΙ containing 2.5 units of Taq DNA polymerase (Invitrogen), 100 ng of plasmid pJfyS250, 250 nM of each dNTP, and 10 pmol of each of the two primer sets described above in a reaction buffer of 50 mM KCI, 10 mM Tris- HCI pH 8.0, 1.5 mM MgC . Amplification was carried out in a thermocycler programmed for one cycle at 94°C for 3 minutes, followed by 25 cycles each at 94°C for 1 minute, 55°C for 30 seconds, and 72°C for 1 minute.

The two PCR fragments were cloned together with a 15,764 bp Pac I fragment of plasmid pFC330 using an IN-FUSION® HD ECODRY™ Cloning Kit (Takara Bio USA, Inc.) according to the manufacturer to produce plasmid pAT1 153. The Pac I fragment contains the

A. fumigatus pyrG selection marker, the Streptomyces pyogenes Cas9 endonuclease expression cassette, and the AMA1 sequence for autonomous replication in Aspergillus. The plasmid was verified by restriction digestion and sequencing of the inserted PCR fragments. Example 26: Construction of plasmid pAT1154 (A. fumigatus U6-3 promoter) Plasmid pAT1 154 (Figure 17) was constructed to contain the A. fumigatus U6-3 small nuclear RNA promoter, Aspergillus fumigatus tRNAgly(GCC)1-6 sequence, wA protospacer, Streptomyces pyogenes single guide RNA sequence, A. fumigatus U6-3 small nuclear RNA terminator, Streptomyces pyogenes Cas9 gene, and the A. fumigatus pyrG gene. Construction was by PCR of the A. fumigatus U6-3 promoter single guide RNA expression cassette containing the wA protospacer (5'-AGTGGGATCTCAAGAACTAC-3'; SEQ ID NO: 63) found in plasmid pFC336. Two fragments were amplified by PCR with one primer set of primers oAT1 146 and oAT1147 and a second primer set of primers oAT1 145 and oAT1148 shown below using plasmid pJfyS253 as template generating PCR fragments of 631 bp and 328 bp, respectively. The 631 bp fragment contains the A. fumigatus U6-3 promoter, A. fumigatus tRNAgly(GCC)1-6 sequence, and the A. oryzae wA protospacer. The 328 bp fragment contains the Streptomyces pyogenes single guide RNA sequence and the A. fumigatus U6-3 terminator.

Primer oAT1146:

5'-TCCGCTGAGGGTTTAATTAAAACCGGAGAGTACTATCAAC-3' (SEQ ID NO: 68) Primer oAT1147:

5'-GTAGTTCTTGAGATCCCACTTGCATCATCCGTGAATCG-3' (SEQ ID NO: 69)

Primer oAT1145:

5'-AGTGGGATCTCAAGAACTACGTTTTAGAGCTAGAAATAGC-3' (SEQ ID NO: 70) Primer oAT1148:

5'- TCGGCTGAGGTCTTAATTAATTTTTTTCTGGATCCTACAG-3' (SEQ ID NO: 71)

The PCRs were performed in a volume of 100 μΙ containing 2.5 units of Taq DNA polymerase, 100 ng of plasmid pJfyS253, 250 nM of each dNTP, and 10 pmol of each of the two primer sets described above in a reaction buffer of 50 mM KCI, 10 mM Tris-HCI pH 8.0, 1.5 mM MgC . Amplification was carried out in a thermocycler programmed for one cycle at

94°C for 3 minutes, followed by 25 cycles each at 94°C for 1 minute, 55°C for 30 seconds, and 72°C for 1 minute.

The two PCR fragments were cloned together with a 15,764 bp Pac I fragment of plasmid pFC330 using an IN-FUSION® HD ECODRY™ Cloning Kit according to the manufacturer to produce plasmid pAT1 154. Plasmid were verified by restriction digestion and sequencing of the inserted PCR fragments.

Example 27: Preparation of donor DNA for deletion of Aspergillus oryzae wA gene

An A. oryzae wA donor fragment for making a deletion in the A. oryzae wA gene (SEQ ID NO: 72 of the DNA sequence and SEQ ID NO: 73 for the deduced amino acid sequence) was constructed by overlap PCR amplification of two PCR products with one primer set of primers oAT916 and oAT917 and a second primer set of primers oAT918 and oAT919 shown below using genomic DNA of A oryzae strain Jal_355 (WO 2005/070962 Example 10) as template generating PCR fragments of 619 bp and 636 bp, respectively. The two PCR products were mixed and amplified by PCR with primers oAT916 and oAT919 generating a fragment on 1218 bp. The 1218 bp fragment was used as a donor DNA for generating a 65 bp deletion in the A oryzae wA gene.

Primer oAT916:

5'-CATTCTGGTGAAGACTGTCG-3' (SEQ ID NO: 74)

Primer oAT917:

5'-CTTCAGCTCCAGCCACCAGCTGCCTTACGAAGGGTGC-3' (SEQ ID NO: 75)

Primer oAT918:

5'-GCACCCTTCGTAAGGCAGCTGGTGGCTGGAGCTGAAG-3' (SEQ I D NO: 76)

Primer oAT919:

5'-CTGGCTGTCAAGGCTTCC-3' (SEQ ID NO: 77)

The PCRs were performed in a volume of 100 μΙ containing 2.5 units of Taq DNA polymerase, 100 ng of A oryzae genomic DNA or 619 bp and 636 bp fragments, 250 nM of each dNTP, and 10 pmol of each of the two primer sets described above in a reaction buffer of 50 mM KCI, 10 mM Tris-HCI pH 8.0, 1.5 mM MgCI₂. Amplification was carried out in a themocycler programmed for one cycle at 94°C for 3 minutes, followed by 25 cycles each at 94°C for 1 minute, 55°C for 30 seconds, and 72°C for 1 minute.

Example 28: Expression of single guide RNA in A. oryzae strain Jal_355 from A. oryzae and A. fumigatus U6 promoters

The purpose of the experiment was to test editing efficiency by expression of single guide RNA in Aspergillus oryzae with U6 promoters from A oryzae and A fumigatus using plasmids pAT1153 and pAT1 154 where in both constructs the single guide RNA was processed by the A fumigatus tRNAgly(GCC)1-6. The editing efficiency of single guide RNA expression from the U6 promoter was measured by editing of the A oryzae wA gene AO090102000545 with and without donor DNA. These was compared to the use of the CRISPR/Cas9 system described by N0dvig et ai, 2015, supra.

Plasmids pAT1 153, pAT1154 and pFC336 were transformed into A oryzae Jal_355 (pyrG^~) according to Table 7. The number of transformants were then counted after 4 days of incubation at 30°C.

Aspergillus transformation was performed according to Christensen et ai, 1988, Biotechnolog 6: 1419-1422. In short, A oryzae mycelia were grown in a rich nutrient broth. The mycelia were separated from the broth by filtration. The enzyme preparation GLUCANEX® (Novozymes A/S) was added to the mycelia in an osmotically stabilizing buffer such as 1.2 M MgS0₄ buffered to pH 5.0 with sodium phosphate. The suspension was incubated for 60 minutes at 37°C with agitation. The protoplasts were filtered through MIRACLOTh® (Calbiochem Inc.) to remove mycelial debris. The protoplasts were harvested and washed twice with STC. The protoplasts were then resuspended in 200-1000 μΙ of STC.

For transformation, 10 μg of plasmid pAT1153 or pAT1 154 and 1 μg of donor DNA were added to 100 μΙ of protoplast suspension and then 200 μΙ of PEG buffer were added. The mixture was incubated for 20 minutes at room temperature. The protoplasts were harvested, washed twice with 1.2 M sorbitol, and resuspended in 200 μΙ of 1.2 M sorbitol. Transformants containing the amdS gene were selected for their ability to used acetamide as the sole source for nitrogen on minimal plates (Cove, 1966, Biochem. Biophys. Acta. 113: 51- 56) containing 1.0 M sucrose as carbon source and 10 mM NaN0₄ as nitrogen source. After 5-7 days of growth at 37°C, stable transformants appeared as vigorously growing and sporulating colonies. Transformants were purified once through conidiospores.

The results shown in Table 7 demonstrated that both the A. fumigatus U6-3 promoter and the A. oryzae U6 promoter drove expression of the single guide RNA in A. oryzae and that both promoters yielded a higher mutation frequency with and without donor DNA compared to plasmid pFC336. Table 7. Comparative analysis of gRNA expression from A. oryzae U6 promoter with A. fumigatus tRNAgly(GCC)1-6 addition, and A. fumigatus U6-3 promoter with A. fumigatus tRNAgly(GCC)1-6 addition compared to the A. nidulans gpdA promoter for wA single guide RNA expression in CRISPR/Cas9 initiated homology-directed DNA repair and non- homologous-end-joining repair

Example 29: Construction of plasmid pJfyS259 containing the Magnaporthe oryzae U6- 2 promoter, A. fumigatus tRNAgly(GCC)1 -6 sequence, wA protospacer, Streptomyces pyogenes single guide RNA sequence, and Magnaporthe oryzae U6-2 terminator

A synthetic DNA sequence containing 500 bp of the Magnaporthe oryzae U6-2 promoter (Example 5), the A. fumigatus tRNAgly(GCC)1-6 sequence with the 3' leader sequence removed, the 20 bp protospacer region targeting the T. reesei wA gene, the S. pyogenes single guide RNA sequence, 5 bp of the poly (T) terminator, and flanking homologous sequences for insertion into plasmid pSMai268 was synthesized as a STRING™ DNA fragment (SEQ ID NO: 78) by GENEART® cloned into a commercially supplied GENEART® standard cloning vector. The lyophilized plasmid DNA supplied by GENEART® containing the fragment was resuspended in water at a concentration of 30 ng/μΙ. The sequence containing the elements above was PCR amplified using PHUSION® Hot Start High Fidelity DNA Polymerase and the forward and reverse primers shown below.

Forward Primer:

5' - AG ATG A ATATTG CCTG C AG G- 3' (SEQ ID NO: 48)

Reverse Primer:

5'-TGATTCTGCTGTCTCGAAGC-3' (SEQ ID NO: 49)

The fragment was inserted into Hind Ill-digested pSMai268 using an IN-FUSION® HD Cloning Kit. The reaction was composed of 125 ng of Hind III digested pSMai268, 60 ng of the PCR fragment, and 1X IN-FUSION® HD Premix in a 10 μΙ reaction volume. After incubating the mixture for 15 minutes at 50°C, 2 μΙ of the reaction were transformed into 50 μΙ of E. coli STELLAR™ competent cells. The cells were heat shocked at 42°C for 45 seconds after which 100 μΙ of SOC medium were added and the total volume was spread onto a 150 mm 2XYT + Amp plate and incubated at 37°C overnight. The resulting E. coli transformants were individually inoculated into 3 ml of LB + Amp medium in 14 ml round-bottom polypropylene tubes and incubated at 37°C overnight with shaking at 200 rpm. Plasmid DNA was isolated using a BIOROBOT® 9600. The insert was confirmed by DNA sequencing with a Model 377 XL Automated DNA Sequencer using dye-terminator chemistry (Giesecke et al., 1992, supra). One plasmid designated pJfyS259 (Figure 18) was selected for analyzing the Magnaporthe oryzae U6-2 promoter and terminator for single guide RNA with tRNAgly(GCC)1-6 expression in T. reesei. Example 30: Comparison of Magnaporthe oryzae U6-2 promoter and terminator with enhancement by A. u/77/^'gafus tRNAgly(GCC)1 -6 upstream of the protospacer in the wA single guide RNA

This study evaluated whether a tRNA would increase efficiency of the Magnaporthe oryzae U6-2 promoter and terminator. T. reesei HgD- strain JfyS154-78-3C was transformed with the wA deletion plasmid pSMai271 and transformed together with plasmid pJfyS259 containing the Magnaporthe oryzae U6-2 promoter coupled with tRNAgly as well as pSMai274 and pSMai280 which contain the M. oryzae U6-2 promoter and M. oryzae U6-2 promoter and terminator without tRNA for pSMai274 and pSMai280, respectively.

The results shown in Table 8 demonstrated that addition of the tRNAgly(GCC)1-6 directly upstream of the protospacer dramatically improves CRISPR/Cas9-based gene deletion efficiency when the M. oryzae U6-2 promoter is used to drive sgRNA expression.

Table 8. Comparative analysis of M. oryzae U6-2 promoter and terminator with A. fumigatus tRNAgly(GCC)1-6 addition compared to the M. oryzae U6-2 promoter and promoter and terminator without tRNAgly addition for wA single guide RNA expression in CRISPR/Cas9 initiated homology-directed repair.

Example 31 : Construction of pCHSN2

Plasmid pCHSN2 is a derivative of pAT1153 (Example 25) and was constructed in a two-step cloning process. First, a general purpose AMA-pyrG vector (with a Pac I insertion site, described in WO 2005/121351A2, pCHSNI , was constructed as a three-fragment fusion using an NEBUILDER® HiFi DNA Assembly Cloning Kit (New England Biolabs), using the PCR products from Table 9. Cloning and transformation was done as per manufacturer's protocol, using E. coli STELLAR™ competent cells (ClonTech) while subsequent purification of plasmid DNA was done using a QIAprep Spin Miniprep kit, following the included manual. Secondly pCHSN2, was constructed that contains the gene encoding the Mad7 endonuclease under control of the A. nidulans TEF1 promoter and terminator in pCHSNI . The pCHSNI vector was digested with Pac I and used as vector backbone for pCHSN2, in which four fragments were inserted using an NEBUILDER® HiFi DNA Assembly Cloning Kit, assembling Mad7 (from two fragments) with promoter and terminator. The PCR product for pCHSN2 is defined in Table 10. The DNA sequences of the primers used for construction of pCHSN 1 and pCHSN2 can be found in Table 11. PCR products for both pCHSNI and pCHSN2 were made with PHUSION® Hot Start II High Fidelity DNA polymerase (New England Biolabs), following the manufacturer's protocol. Both pCHSNI and pCHSN2 vectors were verified by Bsp El digestion (following manufactures protocol), and the insert in pCHSN2 sequenced by Sanger sequencing.

Table 9. PCR products for creating of general purpose AMA1- pyrG vector, pChSNI , with Pac\ for insertion

Table 10. PCR products for insertion of CRISPR-Mad7 expression cassette into pChSNI to create pChSN2

Table 11. Primers used to PCR amplify DNA fragments to construct pChSNI and pChSN2

ChSN-P20 caacgatcaaggcgagttacatgat (SEQ ID NO: 86)

ChSN-P21 cggaaacagctatgaccatgagatc (SEQ ID NO: 87)

ChSN-P22 gatctcatggtcatagctgtttccgTTAATTAAtcctcgtgtactgtgtaagcgccc (SEQ ID NO:

88)

ChSN-P23 gcggacattcgatttatgccg (SEQ ID NO: 89)

ChSN-P24 ggtgaaggttgtgttatgttttgtgg (SEQ ID NO: 90)

ChSN-P25 gatctcatggtcatagctgtttccgTTAATTAAcgagacagcagaatcaccgc (SEQ ID NO:

91)

ChSN-P26 gggcgcttacacagtacacgaggagtattgggatgaattttgtatgcacg (SEQ ID NO: 92)

Example 32: Construction of pSMai322a containing CRISPR-Mad7 and wd-sgRNA expression cassettes

Plasmid pSMai322a (Figure 20) is a CRISPR-Mad7 expression plasmid containing an Eubacterium rectale Mad7 protein coding sequence [SEQ ID NO: 93 for the DNA sequence, SEQ I D NO: 94 for the deduced amino acid sequence, and SEQ ID NO: 95 for the codon- optimized DNA sequence with 3' extension sequence encoding a SV40 nuclear localization signal for use in Aspergillus oryzae, under control of the Aspergillus nidulans tefl promoter (N0dvig et ai, 2015, supra). In addition, it has a i½A-sgRNA expression cassette, comprising a Magnaporthe oryzae U6-2 promoter, Aspergillus fumigatus tRNAgly(GCC)1-6 sequence, wA protospacer, Eubacterium rectale single guide RNA sequence, and Magnaporthe oryzae U6- 2 terminator.

A PCR fragment (PCR fragment 1) containing the Eubacterium rectale Mad7 gene codon optimized for A. oryzae with a SV40 NLS sequence under the transcriptional control of the A. nidulans tefl promoter and terminator was generated using the following primers: Forward Primer 1226078:

TAGTTTCTGCCATTTGCAATCGAGACAGCAGAATCACCG (SEQ ID NO: 96)

Reverse Primer 1226079:

GCTATGACCATGATTACGCCATCCTGCCCTCTTGCTCAAT (SEQ ID NO: 97)

The PCR was composed of 10 ng of pCHSN2 (Example 31j, 10 mM dNTPs, 50 pmol of forward primer 1226078, 50 pmol of reverse primer 1226079, 1X PHUSION® HF buffer, and 2 units of PHUSION® Hot Start DNA polymerase in a final volume of 50 μΙ. The reaction was incubated in a thermocycler programmed for 1 cycle at 98°C for 3 minutes; 30 cycles each at 98°C for 10 seconds, 58°C for 30 seconds, and 72°C for 3 minutes; 1 cycle at 72°C for 10 minutes; and a 10°C hold. The resulting 5147 bp PCR fragment was purified by 0.9% agarose gel electrophoresis using TAE buffer, excised from the gel, and extracted using a NUCLEOSPIN® Gel and PCR Clean-up Kit. A PCR fragment (PCR fragment 2) containing the Magna port he oryzae U6-2 promoter and the Aspergillus fumigatus the tRNAgly(GCC)1-6 sequence with the 3' trailer sequence removed was generated using the following primers:

Forward Primer 1226073:

GAGTCGACCTGCAGGCATGCAAGCTTTCTGCTCGAGGCCATCTGGCTT (SEQ ID NO: 98)

Reverse Primer 1226074:

AATTAAAAAGGTCTTTTGACTGCATCATCCGTGAATCGAACACGG (SEQ ID NO: 99)

The PCR was composed of 10 ng of pJfyS259 (Example 29;, 10 mM dNTPs, 50 pmol of forward primer 1226073, 50 pmol of reverse primer 1226074, 1X PHUSION® HF buffer, and 2 units of PHUSION® Hot Start DNA polymerase in a final volume of 50 μΙ. The reaction was incubated in a thermocycler programmed for 1 cycle at 98°C for 3 minutes; 30 cycles each at 98°C for 10 seconds, 58°C for 30 seconds, and 72°C for 3 minutes; 1 cycle at 72°C for 10 minutes; and a 10°C hold. The resulting 637 bp PCR fragment was purified by 0.9% agarose gel electrophoresis using TAE buffer, excised from the gel, and extracted using a NUCLEOSPIN® Gel and PCR Clean-up Kit.

A 311 bp synthetic DNA fragment containing the Eubacterium rectale single guide RNA sequence, the 21 bp protospacer region targeting the T. reesei wA gene, the Magnaporthe oryzae U6-2 terminator, and flanking homologous sequences for insertion into plasmid pUC19 was synthesized as a STRING™ DNA fragment (SEQ ID NO: 100) by GENEART®. The DNA fragment was resuspended in 10 mM Tris pH 8 at a concentration of 10 ng/μΙ.

The two PCR fragments (PCR fragments 1 and 2) described above and the 311 bp synthetic DNA fragment were inserted into Hind Ill-digested pUC19 using an NEBUILDER® HiFi DNA Assembly Kit. The reaction was composed of 100 ng of Hind Ill-digested pUC19, 191 ng of PCR fragment 1 , 24 ng of PCR fragment 2, 15 ng of the 311 bp DNA fragment, and 1X HiFi master mix in total volume of 20 μΙ. After incubating the mixture for 1 hour at 50°C, 2 μΙ of the reaction were transformed into 50 μΙ of E. coli STELLAR™ competent cells. The cells were heat shocked at 42°C for 45 seconds after which 450 μΙ of SOC medium were added and the total volume was spread onto a 150 mm 2XYT + Amp plate and incubated at 37°C overnight. The resulting E. coli transformants were individually inoculated into 3 ml of LB + Amp medium in 14 ml round-bottom polypropylene tubes and incubated at 37°C overnight with shaking at 200 rpm. Plasmid DNA was isolated using a BIOROBOT® 9600 and sequenced with a Model 377 XL Automated DNA Sequencer using dye-terminator chemistry (Giesecke et al., 1992, supra). DNA sequencing identified three mutations in the Eubacterium rectale Mad7 gene. These mutations were corrected using a QUIKCHANGE® Multi Site- Directed Mutagenesis Kit (Agilent Technologies) with the mutagenic insertion primers shown below.

Primer 1226359:

GCCACATCGTTCAAAGACTATTTTAAGAATCGTGC (SEQ ID NO: 101)

Primer 1226360:

ATTTGTGGTAAAGTCAACTCGTTCATGAACCTC (SEQ ID NO: 102)

Primer 1226361 :

AAAACTGGAAGGAGGACGGCAAATTCTCGAGG (SEQ ID NO: 103)

The mutagenic PCR contained 100 ng of pSMai322, 1 μΙ of dNTP mix, 10 pmol of each primer, 1X QUIKCHANGE® Multi Reaction Buffer (Agilent Technologies), 0.5 μΙ of QUIKSOLUTION® reagent, and 1 μΙ of QUI KCHANGE® Multi Enzyme Blend (Agilent Technologies) in a final volume of 25 μΙ. The PCR was performed in a thermocycler programmed for 1 cycle at 95°C for 1 minute; 30 cycles each at 95°C for 1 minute, 55°C for 1 minute, and 65°C for 18 minutes; and 1 cycle at 65°C for 5 minutes. Following temperature cycling, the reaction was cooled and 1 μΙ of Dpn I restriction enzyme was added directly to amplification reaction and incubated for 1 hour at 37°C. Two μΙ of the Dpn l-treated reaction was transformed into 50 μΙ of E. coli STELLAR™ competent cells. The cells were heat shocked at 42°C for 45 seconds after which 450 μΙ of SOC medium were added and the total volume was spread onto a 150 mm 2XYT + Amp plate and incubated at 37°C overnight. The resulting E. coli transformants were individually inoculated into 3 ml of LB + Amp medium in 14 ml round-bottom polypropylene tubes and incubated at 37°C overnight with shaking at 200 rpm. Plasmid DNA was isolated using a BIOROBOT® 9600 and sequenced with a Model 377 XL Automated DNA Sequencer using dye-terminator chemistry (Giesecke et al., 1992, supra). One plasmid was identified as containing the desired sequence and designated pSMai322a (Figure 20).

Example 33: Analysis of CRISPR-Mad7 genome editing in Trichoderma reesei

The purpose of the experiment was to test editing efficiency of the T. reesei wA gene with and without repair DNA by RNA-guided CRISPR-Mad7 genome editing system in T. reesei. The Mad7-CRISPR plasmid, pSMai322a (Example 32), was transformed into T. reesei GMer62-1A9 strain with the wA deletion plasmid pSMai271 (repair DNA) as described in Example 2. From three independent transformation reactions, transformation efficiency was dramatically increased with CRISPR-Mad7 compared to transformation with repair DNA alone (Table 12). Most of the obtained transformants (98-100%) had the expected white phenotype, indicating CRISPR-Mad7 system can function in T. reesei with high efficiency. Table 12. Analysis of homology directed CRISPR-Mad7 genome editing of the wA gene in Trichoderma reesei

The present disclosure is further described by the following numbered paragraphs: Paragraph 1. A method for modifying the genome of a fungal cell, the method comprising the steps of:

(A) introducing into the fungal cell:

(i) a first nucleic acid construct comprising a polynucleotide encoding an RNA-guided DNA endonuclease for introducing a double-strand break at a target site in the genome of the fungal cell, wherein the fungal cell comprises a protospacer adjacent motif (PAM) sequence for the RNA-guided DNA endonuclease immediately before the 5' end or immediately following the 3' end of the target site;

(iii) a third nucleic acid construct comprising a donor DNA comprising a nucleotide sequence of interest for modifying the target site, wherein the third nucleic acid construct is incorporated into the target site by endogenous DNA repair; and

(B) selecting a transformant of the fungal cell, wherein the target site is modified with the nucleotide sequence of interest.

Paragraph 2. The method of paragraph 1 , wherein the RNA-guided DNA endonuclease is a Cas9 endonuclease.

Paragraph 3. The method of paragraph 2, wherein the Cas9 endonuclease is a

Bordetella pseudohinzii, Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus mutans, Campylobacter jejuni, Neisseria meningitidis, Francisella novicida, or Pasteurella multocida Cas9 endonuclease, or a homologue thereof.

Paragraph 4. The method of paragraph 2, wherein the Cas9 endonuclease comprises a sequence selected from the group consisting of SEQ ID NO: 11 , SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, and SEQ ID NO: 62; and a homologue thereof.

Paragraph 5. The method of paragraph 2, wherein the Cas9 endonuclease is a variant of a Class-ll Cas9 endonuclease having only one active nuclease domain.

Paragraph 6. The method of paragraph 5, wherein the Cas9 endonuclease variant comprises a substitution of aspartic acid for alanine in the amino acid position corresponding to position 10 in the Streptococcus pyogenes Cas9 endonuclease amino acid sequence of SEQ ID NO: 1 1.

Paragraph 7. The method of paragraph 1 , wherein the RNA-guided DNA endonuclease is a Mad7 endonuclease.

Paragraph 8. The method of paragraph 7, wherein the Mad7 endonuclease is an

Eubacterium rectale Mad7 endonuclease, or a homologue thereof.

Paragraph 9. The method of paragraph 7, wherein the Mad7 endonuclease comprises the sequence of SEQ ID NO: 94 or a homologue thereof.

Paragraph 10. The method of paragraph 1 , wherein the RNA-guided DNA endonuclease is a Cpf1 endonuclease.

Paragraph 11. The method of paragraph 10, wherein the Cpf1 endonuclease is an Acidaminococcus sp., Lachnospiraceae sp., or Francisella novicide Cpf1 endonuclease.

Paragraph 12. The method of paragraph 10, wherein the Cpf1 endonuclease comprises a sequence selected from the group consisting of SEQ ID NO: 105, SEQ ID NO: 107, and SEQ ID NO: 109; and a homologue thereof.

Paragraph 13. The method of any one of paragraphs 1-12, wherein the first nucleic acid construct comprising a polynucleotide encoding an RNA-guided DNA endonuclease further comprises a sequence encoding a nuclear localization signal (NLS).

Paragraph 14. The method of any one of paragraphs 1-13, wherein the protospacer adjacent motif (PAM) sequence is a 2-6 bp DNA sequence located immediately before the 5' end or immediately following the 3' end of the target site.

Paragraph 15. The method of any one of paragraphs 1-14, wherein the U6 promoter is an Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Magnaporthe oryzae, Neurospora crassa, or Trichoderma reesei U6 promoter.

Paragraph 16. The method of paragraph 15, wherein the U6 promoter comprises a sequence selected from the group consisting of SEQ I D NO: 13; SEQ I D NO: 20, SEQ I D NO: 27, SEQ ID NO: 30, SEQ ID NO: 33, and SEQ ID NO: 37.

Paragraph 17. The method of any one of paragraphs 1-16, wherein the U6 transcriptional terminator is some Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Magnaporthe oryzae, Neurospora crassa, or Trichoderma reesei U6 terminator.

Paragraph 18. The method of paragraph 17, wherein the U6 transcriptional terminator comprises a sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO: 31 , SEQ ID NO: 34, and SEQ ID NO: 44.

Paragraph 19. The method of any one of paragraphs 1-18, wherein the transfer RNA gene comprises the sequence of SEQ ID NO: 22 or SEQ ID NO: 25.

Paragraph 20. The method of any one of paragraphs 1-19, wherein the single guide RNA comprises (1) a crRNA sequence or (2) a crRNA sequence and a tracer sequence in the form of a single polynucleotide open reading frame, and wherein the crRNA sequence and the tracrRNA sequence of the single guide RNA form a stem-loop structure when hybridized with each other.

Paragraph 21. The method of paragraph 20, wherein the crRNA sequence comprises at least 20 nucleotides that are at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% complementary to and capable of hybridizing to the target site.

Paragraph 22. The method of any one of paragraphs 1-21 , wherein the target site to be modified comprises at least 20 nucleotides.

Paragraph 23. The method of any one of paragraphs 1-21 , wherein the target site to be modified is a gene.

Paragraph 24. The method of any one of paragraphs 1-23, wherein the nucleotide modification is an insertion, a deletion and/or a substitution of one or more nucleotides, codons, coding sequences, expression constructs, or regulatory sequences.

Paragraph 25. The method of any one of paragraphs 1-24, wherein the nucleotide sequence of interest for modifying the target site comprises at least 1 , 5, 10, 20, 40, 60, 100, 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 10,000 nucleotides.

Paragraph 26. The method of any one of paragraphs 1-25, wherein the first nucleic acid construct and the second nucleic acid construct are on a single DNA fragment or a single vector.

Paragraph 27. The method of any one of paragraphs 1-25, wherein the first nucleic acid construct and the third nucleic acid construct are on a single DNA fragment or a single vector.

Paragraph 28. The method of any one of paragraphs 1-25, wherein the second nucleic acid construct and the third nucleic acid construct are on a single DNA fragment or a single vector.

Paragraph 29. The method of any one of paragraphs 1-25, wherein the first nucleic acid construct, the second nucleic acid construct, and the third nucleic acid construct are on a single DNA fragment or a single vector.

Paragraph 30. The method of any one of paragraphs 1-25, wherein the first nucleic acid construct, the second nucleic acid construct, and the third nucleic acid construct are on separate DNA fragments or vectors.

Paragraph 31. The method of any one of paragraphs 1-30, wherein the fungal cell is a filamentous fungal cell or a yeast cell.

Paragraph 32. The method of paragraph 31 , wherein the filamentous fungal 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.

Paragraph 33. The method of paragraph 31 , wherein the filamentous fungal 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, Talaromyces emersonii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cell.

Paragraph 34. The method of paragraph 31 , wherein the yeast cell is a Candida,

Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell. Paragraph 35. The method of paragraph 31 , wherein the yeast cell is a Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica cell.

Paragraph 36. The method of any one of paragraphs 1-35, wherein the endogenous

DNA repair of the target site is by homologous recombination.

Paragraph 37. The method of paragraph 36, wherein the donor DNA further comprises a 5' homology sequence and a 3' homology sequence flanking the nucleotide sequence of interest, wherein the 5' homology sequence and the 3' homology sequence correspond to an upstream region and a downstream region, respectively, of the target site for incorporation of the donor DNA into the double-strand break by homologous recombination.

Paragraph 38. The method of paragraph 37, wherein the flanking 5' homology sequence and the flanking 3' homology sequence each comprise at least 5, 10, 20, 40, 60, 100, 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 10,000 nucleotides.

Paragraph 39. The method of any one of paragraphs 36-38, wherein the fungal cell comprises an inactivated non-homologous end joining (NHEJ) system.

Paragraph 40. The method of paragraph 39, wherein the fungal cell comprises an inactivated DNA Ligase D (LigD) and/or DNA-end-binding protein Ku.

Paragraph 41. The method of paragraph 40, wherein the fungal cell comprises an inactivated HgD gene, kulO gene, and/or ku80 gene, or homologue(s) thereof.

Paragraph 42. The method of any one of paragraphs 1-35, wherein the endogenous DNA repair is by non-homologous end joining.

Paragraph 43. The method of paragraph 42, wherein the fungal cell comprises an active non-homologous end joining (NHEJ) system.

Paragraph 44. The method of any one of paragraphs 1-43, wherein the selecting of the transformant of the fungal cell is accomplished with a selectable marker or counterselection.

Paragraph 45. A fungal host cell transformed with one or more plasmids or DNA fragments, comprising:

(i) a first nucleic acid construct comprising a polynucleotide encoding an RNA- guided DNA endonuclease for introducing a double-strand break at a target site in the genome of the fungal cell, wherein the fungal cell comprises a protospacer adjacent motif (PAM) sequence for the RNA-guided DNA endonuclease immediately before the 5' end or immediately following the 3' end of the target site; (ii) a second nucleic acid construct comprising (a) a U6 promoter sequence operably linked at the 5' end of (1) a sequence encoding a transfer RNA and (2) a sequence encoding a single guide RNA at the 3' end of the transfer RNA sequence, and (b) a U6 transcriptional terminator sequence operably linked at the 3' end of the sequence encoding the single guide RNA, wherein the single guide RNA directs the RNA-guided DNA endonuclease to the target site to introduce the double-strand break, and wherein the second nucleic acid construct increases the frequency of the RNA-guided DNA endonuclease in producing the double-strand break at the target site; and

Paragraph 46. The fungal host cell of paragraph 45, wherein the RNA-guided DNA endonuclease is a Cas9 endonuclease.

Paragraph 47. The fungal host cell of paragraph 46, wherein the Cas9 endonuclease is a Bordetella pseudohinzii, Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus mutans, Campylobacter jejuni, Neisseria meningitidis, Francisella novicida, or Pasteurella multocida Cas9 endonuclease, or a homologue thereof.

Paragraph 48. The fungal host cell of paragraph 46, wherein the Cas9 endonuclease comprises a sequence selected from the group consisting of SEQ ID NO: 1 1 , SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, and SEQ ID NO: 62; and a homologue thereof.

Paragraph 49. The fungal host cell of paragraph 46, wherein the Cas9 endonuclease is a variant of a Class-ll Cas9 endonuclease having only one active nuclease domain.

Paragraph 50. The fungal host cell of paragraph 49, wherein the Cas9 endonuclease variant comprises a substitution of aspartic acid for alanine in the amino acid position corresponding to position 10 in the Streptococcus pyogenes Cas9 endonuclease amino acid sequence of SEQ I D NO: 1 1.

Paragraph 51. The fungal host cell of paragraph 45, wherein the RNA-guided DNA endonuclease is a Mad7 endonuclease.

Paragraph 52. The fungal host cell of paragraph 51 , wherein the Mad7 endonuclease is an Eubacterium rectale Mad7 endonuclease, or a homologue thereof.

Paragraph 53. The fungal host cell of paragraph 51 , wherein the Mad7 endonuclease comprises the sequence of SEQ ID NO: 94 or a homologue thereof.

Paragraph 54. The fungal host cell of paragraph 45, wherein the RNA-guided DNA endonuclease is a Cpf1 endonuclease.

Paragraph 55. The fungal host cell of paragraph 54, wherein the Cpf1 endonuclease is an Acidaminococcus sp., Lachnospiraceae sp., or Francisella novicide Cpf1 endonuclease.

Paragraph 56. The fungal host cell of paragraph 54, wherein the Cpf1 endonuclease comprises a sequence selected from the group consisting of SEQ ID NO: 105, SEQ ID NO: 107, and SEQ ID NO: 109; and a homologue thereof.

Paragraph 57. The fungal host cell of any one of paragraphs 45-56, wherein the first nucleic acid construct comprising a polynucleotide encoding an RNA-guided DNA endonuclease further comprises a sequence encoding a nuclear localization signal (NLS).

Paragraph 58. The fungal host cell of any one of paragraphs 45-57, wherein the protospacer adjacent motif (PAM) sequence is a 2-6 bp DNA sequence immediately before the 5' end or immediately following the 3' end of the target site.

Paragraph 59. The fungal host cell of any one of paragraphs 45-58, wherein the U6 promoter is an Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Magnaporthe oryzae, Neurospora crassa, or Trichoderma reesei U6 promoter.

Paragraph 60. The fungal host cell of paragraph 59, wherein the U6 promoter comprises a sequence selected from the group consisting of SEQ ID NO: 13; SEQ ID NO: 20, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 33, and SEQ ID NO: 37.

Paragraph 61. The fungal host cell of any one of paragraphs 45-60, wherein the U6 transcriptional terminator is an Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Magnaporthe oryzae, Neurospora crassa, or Trichoderma reesei U6 terminator.

Paragraph 62. The fungal host cell of paragraph 61 , wherein the U6 transcriptional terminator comprises a sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO: 31 , SEQ ID NO: 34, and SEQ ID NO: 44.

Paragraph 63. The fungal host cell of any one of paragraphs 45-62, wherein the transfer RNA gene comprises the sequence of SEQ ID NO: 22 or SEQ ID NO: 25.

Paragraph 64. The fungal host cell of any one of paragraphs 45-63, wherein the single guide RNA comprises (1) a crRNA sequence or (2) a crRNA sequence and a tracrRNA sequence in the form of a single polynucleotide open reading frame, and wherein the crRNA sequence and the tracrRNA sequence of the single guide RNA form a stem-loop structure when hybridized with each other.

Paragraph 65. The fungal host cell of paragraph 64, wherein the crRNA sequence comprises at least 20 nucleotides that are at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% complementary to and capable of hybridizing to the target site.

Paragraph 66. The fungal host cell of any one of paragraphs 45-65, wherein the target site to be modified comprises at least 20 nucleotides. Paragraph 67. The fungal host cell of any one of paragraphs 45-65, wherein the target site to be modified is a gene.

Paragraph 68. The fungal host cell of any one of paragraphs 45-67, wherein the nucleotide modification is an insertion, a deletion and/or a substitution of one or more nucleotides, codons, coding sequences, expression constructs, or regulatory sequences.

Paragraph 69. The fungal host cell of any one of paragraphs 45-68, wherein the nucleotide sequence of interest for modifying the target site comprises at least 1 , 5, 10, 20, 40, 60, 100, 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 10,000 nucleotides.

Paragraph 70. The fungal host cell of any one of paragraphs 45-69, wherein the first nucleic acid construct and the second nucleic acid construct are on a single DNA fragment or a single vector.

Paragraph 71. The fungal host cell of any one of paragraphs 45-69, wherein the first nucleic acid construct and the third nucleic acid construct are on a single DNA fragment or a single vector.

Paragraph 72. The fungal host cell of any one of paragraphs 45-69, wherein the second nucleic acid construct and the third nucleic acid construct are on a single DNA fragment or a single vector.

Paragraph 73. The fungal host cell of any one of paragraphs 45-69, wherein the first nucleic acid construct, the second nucleic acid construct, and the third nucleic acid construct are on a single DNA fragment or a single vector.

Paragraph 74. The fungal host cell of any one of paragraphs 45-69, wherein the first nucleic acid construct, the second nucleic acid construct, and the third nucleic acid construct are on separate DNA fragments or vectors.

Paragraph 75. The fungal host cell of any one of paragraphs 45-74, wherein the fungal cell is a filamentous fungal cell or a yeast cell.

Paragraph 76. The fungal host cell of paragraph 75, wherein the filamentous fungal 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.

Paragraph 77. The fungal host cell of paragraph 75, wherein the filamentous fungal 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, Talaromyces emersonii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cell.

Paragraph 78. The fungal host cell of paragraph 75, wherein the yeast cell is a

Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell.

Paragraph 79. The fungal host cell of paragraph 75, wherein the yeast cell is a Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica cell.

Paragraph 80. The fungal host cell of any one of paragraphs 45-79, wherein the endogenous DNA repair of the target site is by homologous recombination.

Paragraph 81. The fungal host cell of paragraph 80, wherein the donor DNA further comprises a 5' homology sequence and a 3' homology sequence flanking the nucleotide sequence of interest, wherein the 5' homology sequence and the 3' homology sequence correspond to an upstream region and a downstream region, respectively, of the target site for incorporation of the donor DNA into the double-strand break by homologous recombination.

Paragraph 82. The fungal host cell of paragraph 81 , wherein the flanking 5' homology sequence and the flanking 3' homology sequence each comprise at least 5, 10, 20, 40, 60, 100, 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 10,000 nucleotides.

Paragraph 83. The fungal host cell of any one of paragraphs 80-82, wherein the fungal cell comprises an inactivated non-homologous end joining (NHEJ) system.

Paragraph 84. The fungal host cell of paragraph 83, wherein the fungal cell comprises an inactivated DNA Ligase D (LigD) and/or DNA-end-binding protein Ku. Paragraph 85. The fungal host cell of paragraph 84, wherein the fungal cell comprises an inactivated HgD gene, kulO gene and/or ku80 gene, or homologue(s) thereof.

Paragraph 86. The fungal host cell of any one of paragraphs 45-79, wherein the endogenous DNA repair is by non-homologous end joining.

Paragraph 87. The fungal host cell of paragraph 86, wherein the fungal cell comprises an active non-homologous end joining (NHEJ) system.

Paragraph 88. A method for modifying the genome of a fungal cell, the method comprising the steps of:

(A) introducing into the fungal cell:

(i) a first nucleic acid construct comprising a polynucleotide encoding an RNA-guided DNA endonuclease for introducing a double-strand break at a target site in the genome of the fungal cell, wherein the fungal cell comprises a protospacer adjacent motif (PAM) sequence for the Cas9 endonuclease immediately following the 3' end of the target site;

(ii) a second nucleic acid construct comprising (a) a U6 promoter sequence operably linked at the 5' end of (1) a sequence encoding a transfer RNA and (2) a sequence encoding a single guide RNA at the 3' end of the transfer RNA sequence, and (b) a U6 transcriptional terminator sequence operably linked at the 3' end of the sequence encoding the single guide RNA, wherein the single guide RNA directs the RNA-guided DNA endonuclease to the target site to introduce the double-strand break, and wherein the second nucleic acid construct increases the frequency of the RNA- guided DNA endonuclease in producing the double-strand break at the target site; wherein the double-strand break is repaired by endogenous DNA repair; and

(B) selecting a transformant of the fungal cell, wherein the target site is modified.

Paragraph 89. The method of paragraph 88, wherein the RNA-guided DNA endonuclease is a Cas9 endonuclease.

Paragraph 90. The method of paragraph 89, wherein the Cas9 endonuclease is a Bordetella pseudohinzii, Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus mutans, Campylobacter jejuni, Neisseria meningitidis, Francisella novicida, or Pasteurella multocida Cas9 endonuclease, or a homologue thereof.

Paragraph 91. The method of paragraph 89, wherein the Cas9 endonuclease comprises a sequence selected from the group consisting of SEQ ID NO: 1 1 , SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, and SEQ ID NO: 62; and a homologue thereof.

Paragraph 92. The method of paragraph 89, wherein the Cas9 endonuclease is a variant of a Class-ll Cas9 endonuclease having only one active nuclease domain. Paragraph 93. The method of paragraph 92, wherein the Cas9 endonuclease variant comprises a substitution of aspartic acid for alanine in the amino acid position corresponding to position 10 in the Streptococcus pyogenes Cas9 endonuclease amino acid sequence of SEQ ID NO: 1 1.

Paragraph 94. The method of paragraph 88, wherein the RNA-guided DNA endonuclease is a Mad7 endonuclease.

Paragraph 95. The method of paragraph 94, wherein the Mad7 endonuclease is an Eubacterium rectale Mad7 endonuclease, or a homologue thereof.

Paragraph 96. The method of paragraph 94, wherein the Mad7 endonuclease comprises the sequence of SEQ ID NO: 94 or a homologue thereof.

Paragraph 97. The method of paragraph 88, wherein the RNA-guided DNA endonuclease is a Cpf1 endonuclease.

Paragraph 98. The method of paragraph 97, wherein the Cpf1 endonuclease is an Acidaminococcus sp., Lachnospiraceae sp., or Francisella novicide Cpf1 endonuclease.

Paragraph 99. The method of paragraph 97, wherein the Cpf1 endonuclease comprises a sequence selected from the group consisting of SEQ ID NO: 105, SEQ ID NO: 107, and SEQ ID NO: 109; and a homologue thereof.

Paragraph 100. The method of any one of paragraphs 88-99, wherein the first nucleic acid construct comprising a polynucleotide encoding an RNA-guided DNA endonuclease further comprises a sequence encoding a nuclear localization signal (NLS).

Paragraph 101. The method of any one of paragraphs 88-100, wherein the protospacer adjacent motif (PAM) sequence is a 2-6 bp DNA sequence located immediately before the 5' end or immediately following the 3' end of the target site.

Paragraph 102. The method of any one of paragraphs 88-101 , wherein the U6 promoter is an Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Magnaporthe oryzae, Neurospora crassa, or Trichoderma reesei U6 promoter.

Paragraph 103. The method of paragraph 102, wherein the U6 promoter comprises a sequence selected from the group consisting of SEQ I D NO: 13; SEQ I D NO: 20, SEQ I D NO: 27, SEQ ID NO: 30, SEQ ID NO: 33, and SEQ ID NO: 37.

Paragraph 104. The method of any one of paragraphs 88-103, wherein the U6 transcriptional terminator is an Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Magnaporthe oryzae, Neurospora crassa, or Trichoderma reesei U6 terminator.

Paragraph 105. The method of paragraph 104, wherein the U6 transcriptional terminator comprises a sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO: 31 , SEQ ID NO: 34, and SEQ ID NO: 44. Paragraph 106. The method of any one of paragraphs 88-105, wherein the transfer RNA gene comprises the sequence of SEQ ID NO: 22 or SEQ ID NO: 25.

Paragraph 107. The method of any one of paragraphs 88-106, wherein the single guide RNA comprises (1) a crRNA sequence or (2) a crRNA sequence and a tracrRNA sequence in the form of a single polynucleotide open reading frame, and wherein the crRNA sequence and the tracrRNA sequence of the single guide RNA form a stem-loop structure when hybridized with each other.

Paragraph 108. The method of paragraph 107, wherein the crRNA sequence comprises at least 20 nucleotides that are at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% complementary to and capable of hybridizing to the target site.

Paragraph 109. The method of any one of paragraphs 88-108, wherein the target site to be modified comprises at least 20 nucleotides.

Paragraph 110. The method of any one of paragraphs 88-108, wherein the target site to be modified is a gene.

Paragraph 1 11. The method of any one of paragraphs 88-110, wherein the first nucleic acid construct and the second nucleic acid construct are on a single DNA fragment or a single vector.

Paragraph 1 12. The method of any one of paragraphs 88-110, wherein the first nucleic acid construct and the second nucleic acid construct are on separate DNA fragments or vectors.

Paragraph 1 13. The method of any one of paragraphs 88-112, wherein the fungal cell is a filamentous fungal cell or a yeast cell.

Paragraph 114. The method of paragraph 1 13, wherein the filamentous fungal 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.

Paragraph 115. The method of paragraph 1 13, wherein the filamentous fungal 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, Talaromyces emersonii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cell.

Paragraph 1 16. The method of paragraph 1 13, wherein the yeast cell is a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell.

Paragraph 1 17. The method of paragraph 1 13, wherein the yeast cell is a Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica cell.

Paragraph 118. The method of any one of paragraphs 88-117, wherein the endogenous DNA repair is by non-homologous end joining.

Paragraph 1 19. The method of paragraph 1 18, wherein the fungal cell comprises an active non-homologous end joining (NHEJ) system.

Paragraph 120. A fungal host cell transformed with one or more plasmids or DNA fragments, comprising:

(i) a first nucleic acid construct comprising a polynucleotide encoding an RNA- guided DNA endonuclease for introducing a double-strand break at a target site in the genome of the fungal cell, wherein the fungal cell comprises a protospacer adjacent motif (PAM) sequence for the RNA-guided DNA endonuclease immediately before the 5' end or immediately following the 3' end of the target site; and

(ii) a second nucleic acid construct comprising (a) a U6 promoter sequence operably linked at the 5' end of (1) a sequence encoding a transfer RNA and (2) a sequence encoding a single guide RNA at the 3' end of the transfer RNA sequence, and (b) a U6 transcriptional terminator sequence operably linked at the 3' end of the sequence encoding the single guide RNA, wherein the single guide RNA directs the RNA-guided DNA endonuclease to the target site to introduce the double-strand break, and wherein the second nucleic acid construct increases the frequency of the RNA-guided DNA endonuclease in producing the double-strand break at the target site; wherein the double-strand break is repaired by endogenous DNA repair. Paragraph 121. The fungal host cell of paragraph 120, wherein the RNA-guided DNA endonuclease is a Cas9 endonuclease.

Paragraph 122. The fungal host cell of paragraph 121 , wherein the Cas9 endonuclease is a Bordetella pseudohinzii, Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus mutans, Campylobacter jejuni, Neisseria meningitidis, Francisella novicida, or Pasteurella multocida Cas9 endonuclease, or a homologue thereof.

Paragraph 123. The fungal host cell of paragraph 121 , wherein the Cas9 endonuclease comprises a sequence selected from the group consisting of SEQ ID NO: 11 , SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, and SEQ ID NO: 62; and a homologue thereof.

Paragraph 124. The fungal host cell of paragraph 121 , wherein the Cas9 endonuclease is a variant of a Class-ll Cas9 endonuclease having only one active nuclease domain.

Paragraph 125. The fungal host cell of paragraph 124, wherein the Cas9 endonuclease variant comprises a substitution of aspartic acid for alanine in the amino acid position corresponding to position 10 in the Streptococcus pyogenes Cas9 endonuclease amino acid sequence of SEQ ID NO: 11.

Paragraph 126. The fungal host cell d of paragraph 120, wherein the RNA-guided DNA endonuclease is a Mad7 endonuclease.

Paragraph 127. The fungal host cell of paragraph 126, wherein the Mad7 endonuclease is an Eubacterium rectale Mad7 endonuclease, or a homologue thereof.

Paragraph 128. The fungal host cell of paragraph 126, wherein the Mad7 endonuclease comprises the sequence of SEQ ID NO: 94 or a homologue thereof.

Paragraph 129. The fungal host cell of paragraph 120, wherein the RNA-guided DNA endonuclease is a Cpf1 endonuclease.

Paragraph 130. The fungal host cell of paragraph 129, wherein the Cpf1 endonuclease is an Acidaminococcus sp., Lachnospiraceae sp., or Francisella novicide Cpf1 endonuclease.

Paragraph 131. The fungal host cell of paragraph 129, wherein the Cpf1 endonuclease comprises a sequence selected from the group consisting of SEQ ID NO: 105, SEQ ID NO: 107, and SEQ ID NO: 109; and a homologue thereof.

Paragraph 132. The fungal host cell of any one of paragraphs 120-131 , wherein the first nucleic acid construct comprising a polynucleotide encoding an RNA-guided DNA endonuclease further comprises a sequence encoding a nuclear localization signal (NLS).

Paragraph 133. The fungal host cell of any one of paragraphs 120-132, wherein the protospacer adjacent motif (PAM) sequence is a 2-6 bp DNA sequence located immediately before the 5' end or immediately following the 3' end of the target site.

Paragraph 134. The fungal host cell of any one of paragraphs 120-133, wherein the U6 promoter is an Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Magnaporthe oryzae, Neurospora crassa, or Trichoderma reesei U6 promoter.

Paragraph 135. The fungal host cell of paragraph 134, wherein the U6 promoter comprises a sequence selected from the group consisting of SEQ ID NO: 13; SEQ ID NO: 20, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 33, and SEQ ID NO: 37.

Paragraph 136. The fungal host cell of any one of paragraphs 120-135, wherein the U6 transcriptional terminator is an Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Magnaporthe oryzae, Neurospora crassa, or Trichoderma reesei U6 terminator.

Paragraph 137. The fungal host cell of paragraph 136, wherein the U6 transcriptional terminator comprises a sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO: 31 , SEQ ID NO: 34, and SEQ ID NO: 44.

Paragraph 138. The fungal host cell of any one of paragraphs 120-137, wherein the transfer RNA gene comprises the sequence of SEQ ID NO: 22 or SEQ ID NO: 25.

Paragraph 139. The fungal host cell of any one of paragraphs 120-138, wherein the single guide RNA comprises (1) a crRNA sequence or (2) a crRNA sequence and a tracrRNA sequence in the form of a single polynucleotide open reading frame, and wherein the crRNA sequence and the tracrRNA sequence of the single guide RNA form a stem-loop structure when hybridized with each other.

Paragraph 140. The fungal host cell of paragraph 139, wherein the crRNA sequence comprises at least 20 nucleotides that are at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% complementary to and capable of hybridizing to the target site.

Paragraph 141. The fungal host cell of any one of paragraphs 120-140, wherein the target site to be modified comprises at least 20 nucleotides.

Paragraph 142. The fungal host cell of any one of paragraphs 120-140, wherein the target site to be modified is a gene.

Paragraph 143. The fungal host cell of any one of paragraphs 120-142, wherein the first nucleic acid construct and the second nucleic acid construct are on a single DNA fragment or a single vector.

Paragraph 144. The fungal host cell of any one of paragraphs 120-142, wherein the first nucleic acid construct and the second nucleic acid construct are on separate DNA fragments or vectors. Paragraph 145. The fungal host cell of any one of paragraphs 120-144, wherein the fungal cell is a filamentous fungal cell or a yeast cell.

Paragraph 146. The fungal host cell of paragraph 145, wherein the filamentous fungal 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.

Paragraph 147. The fungal host cell of paragraph 145, wherein the filamentous fungal 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, Talaromyces emersonii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cell.

Paragraph 148. The fungal host cell of paragraph 145, wherein the yeast cell is a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell.

Paragraph 149. The fungal host cell of paragraph 145, wherein the yeast cell is a

Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica cell.

Paragraph 150. The fungal host cell of any one of paragraphs 120-149, wherein the endogenous DNA repair is by non-homologous end joining.

Paragraph 151. The fungal host cell of paragraph 150, wherein the fungal cell comprises an active non-homologous end joining (NHEJ) system.

Paragraph 152. A nucleic acid construct comprising (a) a U6 promoter sequence operably linked at the 5' end of (1) a sequence encoding a transfer RNA and (2) a sequence encoding a single guide RNA at the 3' end of the transfer RNA sequence, and (b) a U6 transcriptional terminator sequence operably linked at the 3' end of the sequence encoding the single guide RNA, wherein the single guide RNA directs an RNA-guided DNA endonuclease to a target site in the genome of a fungal cell to introduce a double-strand break, wherein the fungal cell comprises a protospacer adjacent motif (PAM) sequence for the RNA- guided DNA endonuclease immediately before the 5' end or immediately following the 3' end of the target site, and wherein the second nucleic acid construct increases the frequency of the RNA-guided DNA endonuclease in producing the double-strand break at the target site.

Paragraph 153. The nucleic acid construct of paragraph 152, wherein the RNA- guided DNA endonuclease is a Cas9 endonuclease.

Paragraph 154. The nucleic acid construct of paragraph 153, wherein the Cas9 endonuclease is a Bordetella pseudohinzii, Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus mutans, Campylobacter jejuni, Neisseria meningitidis, Francisella novicida, or Pasteurella multocida Cas9 endonuclease, or a homologue thereof.

Paragraph 155. The nucleic acid construct of paragraph 153, wherein the Cas9 endonuclease comprises a sequence selected from the group consisting of SEQ ID NO: 11 , SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, and SEQ ID NO: 62; and a homologue thereof.

Paragraph 156. The nucleic acid construct of paragraph 153, wherein the Cas9 endonuclease is a variant of a Class-ll Cas9 endonuclease having only one active nuclease domain.

Paragraph 157. The nucleic acid construct of paragraph 156, wherein the Cas9 endonuclease variant comprises a substitution of aspartic acid for alanine in the amino acid position corresponding to position 10 in the Streptococcus pyogenes Cas9 endonuclease amino acid sequence of SEQ ID NO: 11.

Paragraph 158. The nucleic acid construct of paragraph 152, wherein the RNA- guided DNA endonuclease is a Mad7 endonuclease.

Paragraph 159. The nucleic acid construct of paragraph 158, wherein the Mad7 endonuclease is an Eubacterium rectale Mad7 endonuclease, or a homologue thereof.

Paragraph 160. The nucleic acid construct of paragraph 158, wherein the Mad7 endonuclease comprises the sequence of SEQ ID NO: 94 or a homologue thereof.

Paragraph 161. The nucleic acid construct of paragraph 152, wherein the RNA- guided DNA endonuclease is a Cpf1 endonuclease. Paragraph 162. The nucleic acid construct of paragraph 161 , wherein the Cpfl endonuclease is an Acidaminococcus sp., Lachnospiraceae sp., or Francisella novicide Cpf1 endonuclease.

Paragraph 163. The nucleic acid construct of paragraph 161 , wherein the Cpfl endonuclease comprises a sequence selected from the group consisting of SEQ ID NO: 105, SEQ ID NO: 107, and SEQ ID NO: 109; and a homologue thereof.

Paragraph 164. The nucleic acid construct of any one of paragraphs 152-163, wherein the protospacer adjacent motif (PAM) sequence is a 2-6 bp DNA sequence located immediately before the 5' end or immediately following the 3' end of the target site.

Paragraph 165. The nucleic acid construct of any one of paragraphs 152-164, wherein the U6 promoter is an Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Magnaporthe oryzae, Neurospora crassa, or Trichoderma reesei U6 promoter.

Paragraph 166. The nucleic acid construct of paragraph 165, wherein the U6 promoter comprises a sequence selected from the group consisting of SEQ ID NO: 13; SEQ ID NO: 20, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 33, and SEQ ID NO: 37.

Paragraph 167. The nucleic acid construct of any one of paragraphs 152-166, wherein the U6 transcriptional terminator is an Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Magnaporthe oryzae, Neurospora crassa, or Trichoderma reesei U6 terminator.

Paragraph 168. The nucleic acid construct of paragraph 152, wherein the U6 transcriptional terminator comprises a sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO: 31 , SEQ ID NO: 34, and SEQ ID NO: 44.

Paragraph 169. The nucleic acid construct of any one of paragraphs 152-168, wherein the transfer RNA gene comprises the sequence of SEQ ID NO: 22 or SEQ ID NO: 25.

Paragraph 170. The nucleic acid construct of any one of paragraphs 152-169, wherein the single guide RNA comprises (1) a crRNA sequence or (2) a crRNA sequence and a tracrRNA sequence in the form of a single polynucleotide open reading frame, and wherein the crRNA sequence and the tracrRNA sequence of the single guide RNA form a stem-loop structure when hybridized with each other.

Paragraph 171. The nucleic acid construct of paragraph 170, wherein the crRNA sequence comprises at least 20 nucleotides that are at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% complementary to and capable of hybridizing to the target site. Paragraph 172. The nucleic acid construct of any one of paragraphs 152-171 , wherein the target site to be modified comprises at least 20 nucleotides.

Paragraph 173. The nucleic acid construct of any one of paragraphs 152-171 , wherein the target site to be modified is a gene.

The invention described and claimed herein is not to be limited in scope by the specific aspects herein disclosed, since these aspects are intended as illustrations of several aspects of the invention. Any equivalent aspects are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In the case of conflict, the present disclosure including definitions will control.

Claims

What is claimed is: 1. A method for modifying the genome of a fungal cell, the method comprising the steps of:

(A) introducing into the fungal cell:

2. The method of claim 1 , wherein the RNA-guided DNA endonuclease is a Cas9 endonuclease, a Mad7 endonuclease, or a Cpf 1 endonuclease.

3. The method of claim 1 or 2, wherein the U6 promoter is an Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Magnaporthe oryzae, Neurospora crassa, or Trichoderma reesei U6 promoter, e.g., SEQ ID NO: 13; SEQ ID NO: 20, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 33, and SEQ ID NO: 37.

4. The method of any one of claims 1-3, wherein the U6 transcriptional terminator is an Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Magna port he oryzae, Neurospora crassa, or Trichoderma reesei U6 terminator, e.g., SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO: 31 , SEQ ID NO: 34, and SEQ ID NO: 44.

5. The method of any one of claims 1-4, wherein the transfer RNA gene comprises the sequence of SEQ ID NO: 22 or SEQ ID NO: 25.

6. The method of any one of claims 1-5, wherein the single guide RNA comprises (1) a crRNA sequence or (2) a crRNA sequence and a tracrRNA sequence in the form of a single polynucleotide open reading frame, and wherein the crRNA sequence and the tracrRNA sequence of the single guide RNA form a stem-loop structure when hybridized with each other.

7. The method of any one of claims 1-6, wherein the donor DNA further comprises a 5' homology sequence and a 3' homology sequence flanking the nucleotide sequence of interest, wherein the 5' homology sequence and the 3' homology sequence correspond to an upstream region and a downstream region, respectively, of the target site for incorporation of the donor DNA into the double-strand break by homologous recombination.

8. A fungal host cell transformed with one or more plasmids or DNA fragments, comprising:

(i) a first nucleic acid construct comprising a polynucleotide encoding an RNA- guided DNA endonuclease for introducing a double-strand break at a target site in the genome of the fungal cell, wherein the fungal cell comprises a protospacer adjacent motif (PAM) sequence for the RNA-guided DNA endonuclease immediately before the 5' end or immediately following the 3' end of the target site;

9. The fungal host cell of claim 8, wherein the RNA-guided DNA endonuclease is a Cas9 endonuclease, a Mad7 endonuclease, or a Cpf 1 endonuclease.

10. The fungal host cell of claim 8 or 9, wherein the U6 promoter is an Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Magnaporthe oryzae, Neurospora crassa, or Trichoderma reesei U6 promoter, e.g., SEQ ID NO: 13; SEQ ID NO: 20, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 33, and SEQ ID NO: 37.

11. The fungal host cell of any one of claims 8-10, wherein the U6 transcriptional terminator is an Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Magnaporthe oryzae, Neurospora crassa, or Trichoderma reesei U6 terminator, e.g., SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO: 31 , SEQ ID NO: 34, and SEQ ID NO: 44.

12. The fungal host cell of any one of claims 8-11 , wherein the transfer RNA gene comprises the sequence of SEQ ID NO: 22 or SEQ ID NO: 25.

13. The fungal host cell of any one of claims 8-12, wherein the single guide RNA comprises (1) a crRNA sequence or (2) a crRNA sequence and a tracrRNA sequence in the form of a single polynucleotide open reading frame, and wherein the crRNA sequence and the tracrRNA sequence of the single guide RNA form a stem-loop structure when hybridized with each other.

14. The fungal host cell of any one of claims 8-13, wherein the donor DNA further comprises a 5' homology sequence and a 3' homology sequence flanking the nucleotide sequence of interest, wherein the 5' homology sequence and the 3' homology sequence correspond to an upstream region and a downstream region, respectively, of the target site for incorporation of the donor DNA into the double-strand break by homologous recombination.

15. A method for modifying the genome of a fungal cell, the method comprising the steps of:

(A) introducing into the fungal cell:

(ii) a second nucleic acid construct comprising (a) a U6 promoter sequence operably linked at the 5' end of (1) a sequence encoding a transfer RNA and (2) a sequence encoding a single guide RNA at the 3' end of the transfer RNA sequence, and (b) a U6 transcriptional terminator sequence operably linked at the 3' end of the sequence encoding the single guide RNA, wherein the single guide RNA directs the RNA-guided DNA endonuclease to the target site to introduce the double-strand break, and wherein the second nucleic acid construct increases the frequency of the RNA- guided DNA endonuclease in producing the double-strand break at the target site; wherein the double-strand break is repaired by endogenous DNA repair; and (B) selecting a transformant of the fungal cell, wherein the target site is modified.

16. The method of claim 15, wherein the RNA-guided DNA endonuclease is a Cas9 endonuclease, a Mad7 endonuclease, or a Cpf1 endonuclease.

17. The method of claim 15 or 16, wherein the U6 promoter is an Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Magnaporthe oryzae, Neurospora crassa, or Trichoderma reesei U6 promoter, e.g., SEQ ID NO: 13; SEQ ID NO: 20, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 33, and SEQ ID NO: 37.

18. The method of any one of claims 15-17, wherein the U6 transcriptional terminator is an Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Magnaporthe oryzae, Neurospora crassa, or Trichoderma reesei U6 terminator, e.g., SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO: 31 , SEQ ID NO: 34, and SEQ ID NO: 44.

19. The method of any one of claims 15-18, wherein the transfer RNA gene comprises the sequence of SEQ ID NO: 22 or SEQ ID NO: 25.

20. The method of any one of claims 15-19, wherein the single guide RNA comprises (1) a crRNA sequence or (2) a crRNA sequence and a tracrRNA sequence in the form of a single polynucleotide open reading frame, and wherein the crRNA sequence and the tracrRNA sequence of the single guide RNA form a stem-loop structure when hybridized with each other.

21. A fungal host cell transformed with one or more plasmids or DNA fragments, comprising:

(ii) a second nucleic acid construct comprising (a) a U6 promoter sequence operably linked at the 5' end of (1) a sequence encoding a transfer RNA and (2) a sequence encoding a single guide RNA at the 3' end of the transfer RNA sequence, and (b) a U6 transcriptional terminator sequence operably linked at the 3' end of the sequence encoding the single guide RNA, wherein the single guide RNA directs the RNA-guided DNA endonuclease to the target site to introduce the double-strand break, and wherein the second nucleic acid construct increases the frequency of the RNA-guided DNA endonuclease in producing the double-strand break at the target site;

wherein the double-strand break is repaired by endogenous DNA repair.

22. The fungal host cell of claim 21 , wherein the RNA-guided DNA endonuclease is a Cas9 endonuclease, a Mad7 endonuclease, or a Cpf 1 endonuclease.

23. The fungal host cell of claim 21 or 22, wherein the U6 promoter is an Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Magnaporthe oryzae, Neurospora crassa, or Trichoderma reesei U6 promoter, e.g., SEQ ID NO: 13; SEQ ID NO: 20, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 33, and SEQ ID NO: 37.

24. The fungal host cell of any one of claims 21-23, wherein the U6 transcriptional terminator is an Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Magnaporthe oryzae, Neurospora crassa, or Trichoderma reesei U6 terminator, e.g., SEQ ID NO: 16, SEQ ID NO: 18, SEQ I D NO: 28, SEQ ID NO: 31 , SEQ ID NO: 34, and SEQ ID NO: 44.

25. The fungal host cell of any one of claims 21-24, wherein the transfer RNA gene comprises the sequence of SEQ ID NO: 22 or SEQ ID NO: 25.

26. The fungal host cell of any one of claims 21-25, wherein the single guide RNA comprises (1) a crRNA sequence or (2) a crRNA sequence and a tracrRNA sequence in the form of a single polynucleotide open reading frame, and wherein the crRNA sequence and the tracrRNA sequence of the single guide RNA form a stem-loop structure when hybridized with each other.

27. A nucleic acid construct comprising (a) a U6 promoter sequence operably linked at the 5' end of (1) a sequence encoding a transfer RNA and (2) a sequence encoding a single guide RNA at the 3' end of the transfer RNA sequence, and (b) a U6 transcriptional terminator sequence operably linked at the 3' end of the sequence encoding the single guide RNA, wherein the single guide RNA directs an RNA-guided DNA endonuclease to a target site in the genome of a fungal cell to introduce a double-strand break, wherein the fungal cell comprises a protospacer adjacent motif (PAM) sequence for the RNA-guided DNA endonuclease immediately before the 5' end or immediately following the 3' end of the target site, and wherein the second nucleic acid construct increases the frequency of the RNA- guided DNA endonuclease in producing the double-strand break at the target site.