Expression vectors and use thereof to enhance the efficiency of double-strand break induced mutagenesis

Abstract

This description relates to vectors for the genetic editing of filamentous fungi using the DNA double-strand break repair technique, capable of facilitating repair via homologous recombination, whether through the insertion, deletion or replacement of a DNA segment in the genome. These vectors preferably comprise the sequences defined in SEQ ID NO: 1 and SEQ ID NO: 23.

Description

Expression Vectors and Their Use to Increase the Efficiency of Double-Strand Break-Induced Mutagenesis (Description Field)

[0001] This description pertains to the field of compositions and methods for nucleotide editing or alteration of target sites in a cell's genome. FUNDAMENTALS OF THE DESCRIPTION

[0002] The CRISPR (clustered regularly interspaced short palindromic repeats) / Cas system has become an important genome editing tool (Sander & Young, 2014, doi.org / 10.1038 / nbt.2842). It introduces specific double-strand breaks in DNA for genetic recombination and relies on a Cas nuclease (associated with CRISPR), which uses a single chimeric guide RNA for targeting. However, the genetic engineering of filamentous fungal strains, such as species of great industrial interest, has faced significant challenges in achieving high gene editing efficiency through intrinsic recombination mechanisms (Bischof et al., 2016, doi.org / 10.1186 / s12934-016-0507-6).

[0003] The homologous recombination (HR) pathway is a natural DNA repair mechanism that plays a crucial role in facilitating precise and efficient genetic modifications, but it depends on the presence of a homologous DNA sequence for pairing and repairing DNA strands. The non-homologous end joining (NHEJ) pathway is also a natural DNA repair mechanism, but it acts differently from HR, inserting or deleting random nucleotides to rejoin two ends, without the need for homologous sequences as a guide (GUANGTAO et al., 2009, doi.org / 10.1016 / j.jbiotec.2008.10.007).

[0004] In the context of CRISPR / Cas9, HR is the most precise strategy organisms can employ to repair double-strand breaks in DNA made by the Cas9 enzyme and guide RNA (sqRNA) sequences. This strategy depends on the donor DNA strand carrying the desired information to be incorporated into the genome during editing. Thus, the process It allows the incorporation of desired genetic modifications into the genome, enabling precise editing of a specific locus in the genome (CAI et al., 2022, 10.1186 / sl 3068-022-02132-y).

[0005] Although the CRISPR / Cas technique is widely used in current genetic modifications of filamentous fungi, its efficiency is still far from ideal, since such fungi tend to naturally favor the non-homologous end joining (NHEJ) pathway (MACH-AIGNER; MARTZY, 2021, Trichoderma reesei: Methods and Protocols. New York, NY: Springer US, v. 2234).

[0006] In filamentous fungi, NHEJ naturally has a preference over HR, which impacts the efficiency of CRISPR / Cas-based genetic modifications with donor DNA, since the organism tends to repair its DNA without taking into account the template DNA that is provided (GUANGTAO et al., 2009, doi.org / 10.1016 / j.jbiotec.2008.10.007; MACH-AIGNER; MARTZY, 2021, Trichoderma reesei: Methods and Protocols. New York, NY: Springer US, v. 2234).

[0007] The literature presents strategies for suppressing NHEJ, which illustrate the challenges in achieving efficient gene editing. For example, Guangtao et al. (2009, doi.org / 10.1016 / j.jbiotec.2008.10.007) identified and deleted the tku70 gene, necessary for the NHEJ pathway, which promoted an increase in homologous recombination and greater efficiency in ectopic DNA integration in Trichoderma reesei, a filamentous fungus of great importance in industrial biotechnology. In this study, a 95% increase in the efficiency of donor DNA insertion by HR was observed in strains where the gene was deleted. However, this increase in efficiency may be accompanied by a lower growth rate and compromised stress resistance, especially in haploid T. strains.reesei as RUT-C30, which suggests that transient silencing strategies of the NHEJ pathway may be a more promising approach for industrial strains, which must withstand stress conditions during the bioprocess.

[0008] Another strategy to increase the efficiency of gene editing is the transcription of guide RNAs (gRNAs), such as small tRNAs or U6-type RNAs, driven by RNA polymerase III-dependent promoters (CHUTRAKUL et al., 2019, doi.org / 10.1007 / s00284-019-01770-0). In this context, the use of strong synthetic promoters dependent on RNA polymerase III to express guide RNAs (gRNAs), carrying cis-regulatory elements optimized for greater activation in transcription, proves to be an alternative to increase the efficiency of CRISPR / Cas-based gene editing in T. reesei.

[0009] The literature also discusses the repression of the NHEJ pathway with stimulation of HR through the overexpression of proteins involved in HR repair, such as the Rad52 protein. Rad52 can compete with the complex formed by Ku70-Ku80 proteins that participate in NHEJ repair and promote the initiation of repair by homologous recombination (CAI et al., 2022, 10.1186 / s13068-022-02132-y). STATE OF THE ART

[0010] The state of the art comprises gene editing strategies for unconventional and filamentous unicellular fungi using the CRISPR / Cas system.

[0011] Patent document EP3387134 discloses expression cassettes for nucleotide editing or alteration of target sites in the genome of a cell, more specifically a yeast of the genus Yarrowia. The methods and compositions employ a system containing a guide RNA and a Cas endonuclease and a known inhibitor of the DNA Ligase IV (LIG4) / XRCC4 complex of the classical NHEJ pathway, which is the synthetic molecule Scr7.

[0012] Document EP3180425 discloses yeast strains that received a construct containing a Cas endonuclease and DNA encoding an RNA complementary to a target site on a chromosome or episome. The Cas endonuclease and RNA together form an RNA-guided endonuclease (RGEN) capable of binding to the target site to be cleaved. Means of repression are not disclosed. from the NHEJ pathway. Conversely, it is noted that, for yeasts such as those of the genus Yarrowia, the frequency of HR at a Cas9 / sgRNA cleavage site, using sgRNA expressed from the cassette disclosed in the invention, was only about 2.25%, with mutation rates of about 15%.

[0013] US patent document US9044492 describes a method for enhancing DNA double-strand break-induced mutagenesis involving the transformation of a cell with small synthetic interfering RNAs to modulate the NHEJ pathway. Such interfering RNAs include siRNA, miRNA, and shRNA. In this disclosure, endonucleases may also be interfering agents for inducing mutagenesis targeted to a site in the genome.

[0014] Document EP3234160 discloses a method for silencing the NHEJ pathway to facilitate the integration of DNA cassettes into the genome by homologous recombination (HR), in which the fungal cell is fused with a cell from an auxiliary fungal lineage in which the NHEJ pathway is already silenced.

[0015] Document EP3362560 describes a strategy to increase homologous recombination in microbial cells in which the template polynucleotide for modification is protected by a phosphorothioate linkage at the 5' end of the chain.

[0016] Therefore, vectors for fungal cell transformation that simultaneously promote the homologous recombination (HR) pathway to increase the efficiency of gene-editing tools such as the CRISPR / Cas system are not disclosed in the state of the art. BRIEF DESCRIPTION OF THE INVENTION

[0017] One of the objectives of this description is to reveal expression vectors developed to more efficiently transform filamentous fungi, containing components that facilitate the proper incorporation of donor DNA into the microorganism's genome by favoring the homologous recombination pathway, and that are capable of promoting gene editing in T. reesei with an efficiency of at least 25%. These vectors find application both in research on gene function and in other environments. production methods to increase the efficiency of producing molecules of commercial interest.

[0018] The objectives of this description are achieved by vectors for gene editing of filamentous fungi using the technique of repairing double-strand breaks in DNA, capable of promoting repair by homologous recombination. BRIEF DESCRIPTION OF THE FIGURES

[0019] The present invention is illustrated in the embodiments shown in the figures, as briefly described below.

[0020] Figure 1 is a diagram showing the expression cassettes of guide RNA (gRNA) containing synthetic polymerase III-dependent promoters and the terminator region in the cassettes (T), formed by the combination of at least two sequences defined in SEQ ID NO: 08, SEQ ID NO: 09, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13.

[0021] Figure 2 is a bar graph showing the percentage of colonies grown in geneticin as a function of the number of colonies subcultured on the plate with PDA medium, after transformation with the pTRED-Si vector along with donor DNA corresponding to a geneticin resistance cassette, which was inserted into the genome via HR. NC: negative control (transformation with the pTRED-Si vector without a gRNA cassette, but with donor DNA containing the geneticin resistance cassette).

[0022] Figure 3 is a bar graph showing the percentage of positive T. reesei RUT-C30 transforms confirmed by amplification of the geneticin cassette inserted into the genome. Statistical analysis of all data was conducted using one-way ANOVA and Tukey's multiple comparisons test (p-value < 0.05). The analyzed constructs that did not show a significant difference are indicated in the graph (ns).

[0023] Figure 4 is a bar graph showing the percentage of gene editing efficiency of the pTRED and pTRED-Rad vectors compared to the pTRED-Si vector.

[0024] Figure 5 is a bar graph showing the percentage of positive pTred-Si colonies evaluated by PCR. For the PCR analysis, three independent experiments were performed. Statistical analysis of all data was conducted using one-way ANOVA and Tukey's multiple comparisons test (p-value < 0.05). The analyzed constructs that did not show a significant difference are indicated in the graph (ns).

[0025] Figure 6 is an image of an agarose gel with 1200 bp amplicons confirming the insertion of the geneticin cassette into the T. reesei RUT-C30 genome. The arrow indicates the 1200 bp amplicon that confirms the insertion of the geneticin cassette into the genome. An untransformed T. reesei RUT-C30 strain was used as a negative control. M: 1kb plus DNA marker (Thermo).

[0026] Figure 7 is a map of the pTRED expression vector according to one embodiment in the present description.

[0027] Figure 8 is a map of the pTRED-Si expression vector according to one embodiment in the present description.

[0028] Figure 9 is a map of the pTRED-Rad expression vector according to one embodiment in the present description. DETAILED DESCRIPTION OF THE INVENTION

[0029] This description refers to vectors specifically designed to contain gene sequences that promote the success of gene editing techniques in filamentous fungi using the CRISPR / Cas system, resulting in enhanced repair of DNA double-strand breaks through homologous recombination.

[0030] In one embodiment of the present description, the gene expression vector comprises:

[0031] (i) an origin of replication for filamentous fungi;

[0032] (ii) a source of bacterial replication;

[0033] (iii) a selection cassette for filamentous fungi, in which a gene terminator is a terminator originating from yeasts;

[0034] (iv) a cassette containing a Cas endonuclease in which a gene terminator is a terminator originating from yeast;

[0035] (v) a cassette for expression of at least one guide RNA.

[0036] In one embodiment, an origin of replication for filamentous fungi is an origin of replication as defined in SEQ ID NO: 01.

[0037] In one embodiment, a bacterial replication origin is as defined in SEQ ID NO: 18.

[0038] In one embodiment, a gene terminator that is usually suitable for yeast is employed in a selection cassette for filamentous fungi and consists of the yeast ADH1 terminator.

[0039] In one embodiment, a gene terminator that is usually suitable for yeast is employed in a cassette containing a Cas endonuclease and consisting of the yeast CYC1 terminator.

[0040] In one embodiment, an expression cassette for a guide RNA comprises at least one sequence as defined in SEQ ID NO: 07, and may be multiple consecutive guide RNA sequences, in multiplex, of the sequence as defined in SEQ ID NO: 07.

[0041] In one embodiment, a modified origin of replication for filamentous fungi, as defined in SEQ ID NO: 01, is an origin of replication derived from AMA (referred to as AMA-half or AMAh), where AMAh comprises the functional half of the AMA sequence.

[0042] In one embodiment, the vector comprises an origin of E. coli replication as per SEQ ID NO: 18.

[0043] In one embodiment, the AMAh fragment is inserted into an EcoRI site.

[0044] In one embodiment, the cassette containing a Cas endonuclease enzyme comprises a sequence containing codons optimized for expression in the filamentous fungus T. reesei or phylogenetically related fungi, preferably Cas9, Cas12, or Casphi.

[0045] In one embodiment, the cassette containing a Cas endonuclease contains a sequence that encodes a Cas9 enzyme, as defined by SEQ ID NO: 02.

[0046] In one embodiment, the cassette containing a Cas endonuclease contains a sequence encoding a Cas12 enzyme, as defined by SEQ ID NO: 03.

[0047] In one embodiment, the cassette containing a Cas endonuclease contains a sequence encoding a CasPhi enzyme, as defined by SEQ ID NO: 04.

[0048] In one embodiment, the cassette containing a Cas endonuclease contains a sequence encoding a Cas12 enzyme, as defined by SEQ ID NO: 05.

[0049] In one embodiment, the cassette containing a Cas endonuclease is inserted into a Bglll site.

[0050] In one embodiment, any of the sequences SEQ ID NO: 02, SEQ ID NO: 03, SEQ ID NO: 04, and SEQ ID NO: 05 possess a targeting signal sequence for the nucleus.

[0051] In one embodiment, an expression cassette containing any of the sequences SEQ ID NO: 02, SEQ ID NO: 03, SEQ ID NO: 04, or SEQ ID NO: 05 has a promoter derived from the PDC1 gene of T. reesei and the CYC1 terminator from yeast (S. cerevisiae).

[0052] In an exemplary embodiment of the present description, the cassette containing a Cas endonuclease has SEQ ID NO: 02 and is as defined in SEQ ID NO: 06.

[0053] The Cas endonuclease sequences as defined in SEQ ID NO: 02, SEQ ID NO: 03, SEQ ID NO: 04, and SEQ ID NO: 05 are also suitable for species phylogenetically close to T. reesei.

[0054] In one embodiment, a guide RNA (gRNA) expression cassette contains a synthetic promoter sequence that is dependent on polymerase III.

[0055] Figure 1 is a schematic representation of the sequences. promoters for cloning gRNA expression cassettes into vectors as described herein.

[0056] In one embodiment, a promoter sequence of a gRNA expression cassette may be one of the sequence SEQ ID NO: 08, designated 6U-TATA.

[0057] In one embodiment, a promoter sequence of a gRNA expression cassette can be a tRNA, for example, a tRNAglyl as defined in SEQ ID NO: 13.

[0058] In one embodiment, a promoter sequence of a gRNA expression cassette may comprise the sequence SEQ ID NO: 08 followed by a sequence SEQ ID NO: 09 or SEQ ID NO: 10. Optionally, the promoter sequence may also comprise a third sequence, which is a sequence encoding a tRNA, as defined in SEQ ID NO: 13.

[0059] In one embodiment, a promoter sequence of a gRNA expression cassette can be a sequence SEQ ID NO: 11, designated 5S1, or a sequence SEQ ID NO: 12, designated 5S2.

[0060] In one embodiment, a promoter sequence of a gRNA expression cassette may comprise a sequence SEQ ID NO: 11 or a sequence SEQ ID NO: 12 followed by a second sequence that is a tRNA-encoding sequence, as defined in SEQ ID NO: 13.

[0061] Other tRNAs may be more suitable for use with Cas12 and LbCpfl endonucleases. A specialist in the field will know how to select the most appropriate tRNA.

[0062] In one embodiment of the present description, a gRNA expression cassette contains a terminator sequence represented by the nucleotide sequence TTTTTT.

[0063] A gRNA expression cassette conforming to the embodiments of the present description is suitable for T. reesei and other filamentous fungi. phylogenetically close.

[0064] In one embodiment, the expression vector also includes a bacterial selection cassette.

[0065] In an exemplary embodiment of the present description, a resistance cassette for bacterial selection is a cassette containing a gene that confers resistance to ampicillin under the control of the AmpR promoter and the T7 Terminator of E. coli, as defined in SEQ ID NO: 19.

[0066] An expert in the field will know how to use a more suitable bacterial screening cassette.

[0067] In one embodiment, a selection cassette for filamentous fungi is a cassette that confers resistance to hygromycin B containing the hph gene (Hygromycin B phosphotransferase) under the control of the GDPA promoter of Aspergillus nidulans and the ADH1 terminator of yeast, referred to as Hygr in the present description and defined in SEQ ID NO: 20.

[0068] In one embodiment, the Hygr gene cassette is inserted into the vector at a Pstl site.

[0069] The Hygr gene cassette could be replaced by another gene cassette, for example, by a resistance selection marker such as geneticin or ptrA (pyritiamine) or by another auxotrophic selection such as pyr4 (OMP decarboxylase). A person skilled in the art will know how to choose an alternative gene cassette for the selection of a specific filamentous fungus that is most suitable for the experimental work.

[0070] In one embodiment, a vector comprising a modified origin of replication for filamentous fungi, a site-directed mutation-inactivated Bsal site, a selection cassette for filamentous fungi, and a cassette containing a Cas endonuclease is termed the pTRED vector and is defined as per SEQ ID NO: 21.

[0071] In one embodiment, a vector comprising a modified origin of replication for filamentous fungi, a site-directed mutation-inactivated Bsal site, a selection cassette for filamentous fungi, and a A cassette containing a Cas endonuclease further comprises a cassette for silencing the NHEJ pathway targeting the mus53 gene, which encodes the ligase IV enzyme of the NHEJ pathway of T. reesei, and is named the pTRED-Si vector in this description and defined by the sequence SEQ ID NO: 22.

[0072] In one embodiment, the pTRED-Si vector carries an NHEJ pathway silencing cassette encoding a double-stranded antisense RNA of approximately 500 nucleotides, as defined by SEQ ID NO: 14, to silence ligase IV enzyme (mus53). In the silencing cassette, two 500 bp fragments of the mus53 gene are inverted and separated by intron 2 of the cutinase (CUT) gene from the fungus Magnaporthe oryzae.

[0073] In one embodiment, a cassette for silencing the NHEJ pathway targeting the mus53 gene comprises the sequence SEQ ID NO: 14 flanked by a promoter of the T. reesei cDNA1 constitutive expression gene, as defined in SEQ ID NO: 16, and a yeast PGK1 terminator, as defined by the sequence SEQ ID NO: 17.

[0074] In one embodiment, the inverted mus53 sequences in the cassette for silencing the NHEJ pathway can be replaced by another mus53 gene sequence encoded in the genome of other filamentous fungi, when the pTRED vector is used to transform other species of filamentous fungi.

[0075] In one embodiment, for constructing the pTRED-Si vector, an NHEJ pathway silencing cassette is inserted into the Hindlll site of the pTRED vector, as described herein.

[0076] In one embodiment, a vector comprising a modified origin of replication for filamentous fungi, a site-directed mutation-inactivated Bsal site, a selection cassette for filamentous fungi, and a cassette containing a Cas endonuclease further comprises a cassette carrying the rad52 gene, which participates in the HR pathway, and is named pTRED-Rad vector in the present description and defined by the sequence SEQ ID NO: 23.

[0077] In one embodiment, a Bsal site is inactivated by a site-directed mutation mediated by the oligonucleotides SEQ ID NO: 45 and SEQ ID NO: 46. The mutation results in the modification of the 5'-GGTCTC-3' site to 5'-GCAGCC-3', making it unrecognizable by the Bsal enzyme. In this way, the ampicillin resistance cassette present in the scaffold that gives rise to the vectors of the present description becomes inactive.

[0078] In one embodiment, there are specific sites for type II restriction enzymes (endonucleases) such as Kpnl and Aflll in the pTRED vectors where additional gene cassettes can be inserted, for example, expression cassettes for guide RNA(s). These sites are indicated in the maps of the pTRED vectors (as shown in Figure 7), pTRED-Si (as shown in Figure 8) and pTRED-Rad (as shown in Figure 9).

[0079] In one embodiment, for the multiplex system, an expression cassette carrying more than one sequence encoding a guide RNA is inserted into the Kpnl and Aflll sites in any of the pTRED, pTRED-Si, and TRED-Rad type vectors.

[0080] In one embodiment, sites for type II restriction enzymes can be EcoRI, Pstl, Bglll, Hindlll, Kpnl, Aflll, Saci, BamHI, SnaBI, Acc65l, among others.

[0081] In one embodiment, a Hindlll site can be preferentially used for insertion of a Rad52 expression cassette or an NHEJ pathway silencing cassette.

[0082] In one embodiment, an expression cassette of at least one guide RNA is inserted and flanked, preferably, by the Kpnl and Aflll sites.

[0083] In one embodiment, for constructing the pTRED-Rad vector, a cassette carrying the rad52 gene from T. reesei is inserted into the Hindlll site of the pTRED vector, as described herein.

[0084] In one embodiment, a cassette carrying the rad52 gene comprises the SEQ ID sequence NO: 15 flanked by a promoter of the T. reesei constitutive expression gene CDNA1, as defined in SEQ ID NO: 16 and a PGK1 yeast terminator, as defined by the sequence SEQ ID NO: 17.

[0085] A subject matter expert could replace the CDNA1 gene promoter or the PGK1 terminator in yeast with another promoter or another constitutively expressed gene terminator.

[0086] In one embodiment, an expression vector as described herein is used to insert, replace, or delete a portion of DNA in the genome of filamentous fungi, preferably T. reesei, to promote increased efficiency of double-strand break-induced mutagenesis in filamentous fungi.

[0087] Table 1 provides a list with the names of all sequences mentioned in this description, along with their respective reference numbers.

[0088] Table 1: List of sequences: EXAMPLES OF IMPROVEMENT OF THE INVENTION

[0089] What follows presents exemplary, non-restrictive examples of the object described herein, illustrating the results and advantages achieved.

[0090] 1. Growth and maintenance of fungal strains:

[0091] The T. reesei RUT-C30 strain (IHEM_5652 / ATCC_56765) was cultivated on Potato Dextrose Agar (PDA) plates (Merck, Darmstadt, Germany) at 28 °C for 16 h and then transferred to a bench for growth at room temperature and with light exposure until the formation of Spores (5-10 days). Spores were suspended in a 20% (v / v) glycerol solution and stored at -80 °C.

[0092] 2. Assembly and confirmation of the pTRED, pTRED-Si, and pTRED-Rad vectors:

[0093] Plasmids were propagated in the E. coli DH5a strain and purified using the QIAprep Miniprep kit (Qiagen, Hilden, Germany). Polymerase chain reaction (PCR) amplifications were performed using the high-fidelity DNA polymerase Phusion, following the manufacturer's instructions (NEB, Ipswich, USA). PCR products and restriction DNA fragments were purified using QIAquick kits (Qiagen). Sanger DNA sequencing was conducted using a 3500xL Genetic Analyzer from Applied Biosystems (Foster City, USA). The primers used in this work are listed in Table 2, in Item 3 of this section.

[0094] The pTRED vector was assembled by adding parts to the pTRED vector scaffold, which was artificially designed and synthesized (Genscript, Piscataway, USA) with specific sites for subsequent modifications. The pTRED-Si and pTRED-Rad vectors were assembled by adding parts to the already constructed pTRED vector. First, for the assembly of the pTRED vector, a directed mutation was performed on the Bsal site, aiming to make this Bsal site suitable for later use in assembling gene cassettes in the vector. Subsequently, the origin of replication for the filamentous fungus AMAh, the hygromycin resistance cassette (Hygr), and a cassette containing Cas9 were inserted. For the construction of the pTRED-Si vector, a cassette for silencing the NHEJ pathway targeting the mus53 gene was cloned into the pTRED vector. Finally, for the construction of the pTRED-Rad vector, a cassette carrying the rad52 gene, which participates in the HR pathway, was cloned into the pTRED vector.

[0095] Fragments carrying gene cassettes for hygromycin resistance (Hygr) and Cas9, as well as the origin of replication for filamentous fungi AMAh, were amplified from the pTrCas9gRNA1 vector. (FONSECA et al, 2020, doi.org / 10.1186 / s13068-020-01732-w). These amplified fragments were inserted into the initial scaffold of the designed and synthesized starter vector (Genscript, Piscataway, USA) with specific sites for subsequent modifications and mutated at the Bsal site within the ampicillin resistance cassette. The AMAh fragment was inserted into the EcoRI site, followed by the Hygr and Cas9 cassettes, which were cloned into the Pstl and Bglll sites, respectively. For the construction of the pTRED-Si vector, an additional step consisted of inserting the NHEJ pathway silencing cassette into the Hindi 11 site. The insertion of the fragments into the pTRED and pTRED-Si vectors was confirmed by PCR and 1% agarose gel electrophoresis (Merck, Darmstadt, Germany) for each region added to the pTRED vector.Cloning of the fragment corresponding to the NHEJ pathway silencing cassette, targeting the mus53 gene, was also confirmed by digestion with the Hindi 11 enzyme and release of a fragment approximately 2200 bp in size. Furthermore, for the pTRED-Si and pTRED-Rad vectors, digestion at the Hindlll sites flanking the inserted cassettes for NHEJ pathway silencing or Rad52 protein production, respectively, was conducted, followed by agarose gel electrophoresis to confirm the insert size.

[0096] For the construction of the pTRED-Rad vector, the rad52 gene, which participates in the HR pathway, was amplified from a T. reesei RUT-C30 cDNA library using specific primers. The approximately 1500 bp rad52 cDNA was amplified, purified, and cloned into the Saci and BamHI sites of the cassette with the T. reesei cDNA1 constitutive promoter and the Saccharomyces cerevisiae PGK1 terminator. The complete rad52 cassette was amplified and cloned into the Hindlll site of the pTRED vector to produce the pTRED-Rad vector. Confirmation of the pTRED-Rad vector was performed with the Hindlll restriction enzyme through the release of the approximately 2300 bp inserted cassette for the production of the Rad52 protein. The result of this reaction was observed by agarose gel electrophoresis to confirm the size of the insert.

[0097] 3. Cloning and confirmation of the cloning of cassettes with gRNA in the pTRED, pTRED-Si and pTRED-Rad vectors:

[0098] Nine different cassettes containing different synthetic promoters dependent on RNA Pol III, a guide RNA, and a terminator were synthesized to be tested in T. reesei. For each vector, the nine expression cassettes carrying the guide RNA (gRNA) with different synthetic promoters and terminator (as shown in Figure 1) were cloned into the Kpnl and Aflll sites of the pTRED, pTRED-Si, and pTRED-Rad vectors, totaling 27 constructs.

[0099] Successful promoter cloning was confirmed using PCR with the primers Forwardcf-Kpnl and Reverse-Aflll. The generated amplicon was subsequently subjected to 1% agarose gel electrophoresis, amplifying the region corresponding to the size of the expression cassette, including the synthetic promoter, gRNA, and terminator. All constructs were confirmed by sequencing using the Forward-CYC primer.

[0100] Table 2 presents the oligonucleotide sequences revealed in this description.

[0101] Table 2: Oligonucleotides:

[0102] 4. Transformation by protoplasts:

[0103] T. reesei protoplasts were generated and transformed as described by Fonseca, Parreiras, and Murakami (2020). For each transformation event, approximately 5 pg of the pTRED-Si vector and about 5 pg of the linear DNA cassette carrying a geneticin resistance cassette for genomic integration were used. The transformations were... selected by plating on a minimum medium containing 15.0 g L 1 of KH2PO4, 5.0 g L 1 of (NH4)2SO4, 0.59 g L 1 of MgSO4, 0.45 g L 1 of CaCl2, 5.0 mg L 1 of FeSO4-7H2O, 2.0 mg L 1 of CoCl2-2H2O, 1.6 mg L 1 of MnSO4-4H2O, 1.4 mg L -1of ZnSO47H2O, 1 M sorbitol, 20.0 g L -1 of agar and 200 pg mL 1 hygromycin B (Merck) was added. The pH of the medium was adjusted to 5.5 with 3 M KOH. The transformation plates were incubated at 28 °C for approximately 5 days until colonies were observed.

[0104] 5. Screening of transformants on geneticin-based selection plates and extraction of genomic DNA and PCR of potential transformants:

[0105] Forty colonies from each transformation were transferred to two PDA plates, which were supplemented with the antibiotic geneticin (400 pg mL). -1Each plate contained 20 colonies and was left to grow for 3 days at 28 °C. After this period, it was possible to perform the first screening, identifying the positive colonies, i.e., those resistant to geneticin. Pieces of the 8 positive colonies that showed the greatest growth in each transformation were transferred to liquid potato dextrose medium (Sigma, St. Louis, USA) and incubated at 30 °C at 200 rpm for 72 hours to generate a sufficient quantity of hyphae for genomic DNA extraction. Genomic DNA extraction was performed using the phenol:chloroform:isoamyl alcohol method, using glass beads (Sigma, St. Louis, USA) in a sample homogenizer, such as a "beadbeate" homogenizer model Geno Grinder 2010 (Spex Sample Prep, Metuchen, USA).

[0106] Colony PCR was performed using DNA extracted from selected colonies and the F-upGen and R-Gen primers, which anneal to the genome region upstream of the inserted geneticin cassette and within the geneticin cassette, respectively. The presence of an amplicon of approximately 1200 base pairs (bp) in the transformants, but not in the parental strain RUT-C30, indicates that the cassette was inserted into the T. reesei genome, confirming gene editing and insertion at the target locus.

[0107] Figure 2 shows the results obtained for the selection. The geneticin from the pTRED-Si vector constructs carrying the different synthetic promoters of RNA Pol III and the gRNA that directs Cas9 cleavage in the upstream region of the pap1 gene promoter (TRIREDRAFT_74156) of T. reesei RUT-C30 was used. The pTRED-Si vector constructs that showed a transformation percentage greater than 20% were selected for confirmation of geneticin cassette insertion via polymerase chain reaction with specific primers that anneal upstream at the insertion locus in the genome and within the insert. An amplicon of approximately 1200 bp is expected for positive transformants. Amplification results showed gene editing efficiency of over 50% for four pTRED-Si constructs carrying the 5S1, 5S1-tRNA, 5S2, and 5S2-tRNA promoters (as shown in Figure 3). The 5S1 and 5S1-tRNA promoters resulted in a reduced number of false positives and an editing efficiency greater than 80%.

[0108] The promoter that showed the highest efficiency in gene editing along with the pTRED-Si vector was the 5S1-tRNA promoter. Therefore, this promoter was used to evaluate whether the other vectors pTRED and pTRED-Rad also showed the same effectiveness in promoting gene editing with CRISPR / Cas9. The results indicate that the three vectors showed efficiency above 20% for the geneticin selection plate test (as shown in Figure 4), with the pTRED-Si vector standing out, showing a greater number of possible transformants. For the PCR test, which was used to confirm the insertion of the geneticin cassette into the genome of the transformants, all vectors showed results above 50% efficiency in editing, with pTRED and pTRED-Rad showing a greater number of false-positive clones, that is, clones that grew with geneticin but do not have the resistance cassette inserted into the genome, as shown in Figures 5 and 6.These results highlight the pTRED-Si vector, which showed a reduced number of false-positive colonies and a greater number of gene-edited clones.

[0109] These findings reinforce not only the excellence of the 5S1-tRNA promoter during evaluation on selection plates, but also demonstrate that the colonies from these tests were, for the most part, truly positive, that is, gene editing actually occurred in these colonies, which therefore carry the geneticin cassette inserted into the genome.

[0110] 6. Evaluation of pTRED vector loss by T. reesei colonies:

[0111] After subculturing in PDA medium without hygromycin, confirmation of pTRED vector loss during conidiogenesis was obtained by cooling the isolates on PDA plates without hygromycin and with 200 pg ml⁻¹. 1Hygromycin B was used for comparison of colony growth. Cured T. reesei colonies, i.e., those that had lost the pTRED vector, only grew in PDA medium without antibiotic.

[0112] To evaluate the loss of the pTRED-Si vector in T. reesei-positive colonies, different colonies that were grown on PDA were subcultured again on PDA and PDA-Hygromycin media. Approximately 66% of T. reesei colonies lost the ability to grow on hygromycin-containing medium after subculture, indicating that the pTRED-Si vector was lost after passage on PDA without the antibiotic.

[0113] Although exemplary embodiments of the processes and products described have been presented in this report, the scope of protection is not intended to be limited to the literal wording of those embodiments. Therefore, the description should be interpreted not as restrictive, but merely as examples of particular embodiments that embody the inventive concept presented herein. A person skilled in the art may readily apply the teachings presented herein to analogous solutions arising from the same, limited only by the scope of the claims in this application.

Claims

CLAIMS 1. Gene expression vector characterized by comprising: (i) an origin of replication for filamentous fungi; (ii) a source of bacterial replication; (iii) a selection cassette for filamentous fungi, in which a gene terminator is a terminator originating from yeasts; (iv) a cassette containing a Cas endonuclease, wherein a gene terminator is a terminator originating from yeast; (v) a cassette for expression of at least one guide RNA.

2. Vector, according to claim 1, characterized in that an origin of replication for filamentous fungi is as defined in SEQ ID NO:

01.

3. Vector, according to any one of claims 1 to 2, characterized in that a bacterial replication origin is as defined in SEQ ID NO:

18.

4. Vector, according to any one of claims 1 to 3, characterized in that a terminator of the selection cassette for filamentous fungi is the ADH1 terminator of yeasts.

5. Vector, according to any one of claims 1 to 4, characterized in that a cassette terminator containing a Cas endonuclease is the yeast CYC1 terminator.

6. Vector, according to any one of claims 1 to 5, characterized in that it further comprises a bacterial resistance cassette containing a Bsal site or having a site-directed mutation-inactivated form.

7. Vector, according to any one of claims 1 to 6, characterized by further comprising a gene cassette for selection for filamentous fungi that is preferably a cassette for selection for antibiotic resistance or a cassette for auxotrophic selection.

8. Vector, according to any one of claims 1 to 7, characterized by the fact that the nucleotide sequence encoding a Cas endonuclease enzyme is selected from the group comprising SEQ ID NO: 02, SEQ ID NO: 03, SEQ ID NO: 04 and SEQ ID NO:

05.

9. Vector, according to any one of claims 1 to 8, characterized in that the expression cassette of a Cas endonuclease enzyme is as defined in SEQ ID NO:

06.

10. Vector, according to any one of claims 1 to 9, characterized in that the nucleotide sequence encoding a guide RNA for Cas9 endonuclease is preferably as defined in SEQ ID NO:

07.

11. Vector, according to any one of claims 1 to 10, characterized in that the promoter of the expression cassette of at least one guide RNA is selected from the group comprising SEQ ID NO: 08, SEQ ID NO: 09, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO:

13.

12. Vector, according to any one of claims 1 to 11, characterized in that the promoter of the expression cassette of at least one guide RNA is optionally combined with a sequence selected from the group comprising SEQ ID NO: 08, SEQ ID NO: 09, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO:

12.

13. Vector, according to any one of claims 1 to 12, characterized in that the promoter of the expression cassette of at least one guide RNA is optionally combined with SEQ ID NO: 09 or SEQ ID NO:

10.

14. Vector, according to claim 13, characterized in that the promoter of the expression cassette of at least one guide RNA further comprises SEQ ID NO:

13.

15. Vector, according to any one of claims 1 to 14, characterized in that the expression cassette of at least one RNA comprises more than one SEQ ID NO: 07 in a multiplex system.

16. Vector, according to any one of claims 1 to 15, characterized by further comprising a mus53 gene silencing cassette.

17. Vector, according to any one of claims 1 to 16, characterized in that the nucleotide sequence that promotes the silencing of the mus53 gene is as defined in SEQ ID NO:

14.

18. Vector, according to any one of claims 1 to 17, characterized by further comprising a rad52 gene expression cassette.

19. Vector, according to any one of claims 1 to 18, characterized in that the nucleotide sequence encoding the rad52 gene is as defined in SEQ ID NO:

15.

20. Vector, according to any one of claims 1 to 19, characterized in that the rad52 gene expression cassette and the mus53 gene silencing cassette contain a promoter, which is preferably as defined in SEQ ID NO: 16, and a terminator, which is preferably as defined in SEQ ID NO:

17.

21. Vector, according to any one of claims 1 to 20, characterized in that a resistance cassette for bacterial selection is preferably an ampicillin resistance cassette, as defined in SEQ ID NO:

19.

22. Vector, according to any one of claims 1 to 21, characterized in that a selection gene cassette for filamentous fungi is preferably a cassette containing the hph gene under the control of the GDPA promoter of Aspergillus nidulans and the ADH1 terminator of yeasts, to confer resistance to hygromycin B, as defined in SEQ ID NO:

20.

23. Vector, according to any one of claims 1 to 22, characterized by further comprising at least four specific cloning sites for restriction enzymes, preferably Kpnl, Aflll, SnaBI and Acc65l.

24. Vector, according to any one of claims 1 to 23, characterized by being as defined in SEQ ID NO:

21.

25. Vector, according to any one of claims 1 to 23, characterized by being as defined in SEQ ID NO:

22.

26. Vector, according to any one of claims 1 to 23, characterized by being as defined in SEQ ID NO:

23.

27. Use of the expression vector as defined in claims 1 to 26, characterized by being for insertion, substitution or deletion of a portion of DNA in the genome of filamentous fungi, increasing the efficiency of mutagenesis induced by double-strand breaks.

Images