**5. One-time expression with the new CRISPR/Cas9 system technology**

In recent years, several genetic tools have been elaborated and applied in various fungi and widely shared in different sectors of the economy. However, there is still a certain limitation in the studies of functional genomics for the production of recombinant cellulases in fungi. For this logic, emerging tools stand out, including CRISPR-Cas9-based genome editing (Clustered Regularly Interspaced Short Palindromic Repeats), i.e., Grouped and Regularly Interspaced Palindromic Repeats, as an agile tool for genome-specific gene edits [70]. The CRISPR-Cas9 system contains two components: the effector protein, which is Cas9 endonuclease, and a single chimeric guide RNA (sgRNA). This tool was involved to allow rapid editing of the genome of several organisms, among them, some varieties of filamentous fungi [71–75].

However, such approaches are not as useful as those available for yeasts and bacteria, considering the complexity of fungi, such as multicellular morphology, cell differentiation, thick chitinous cell walls and lack of adequate plasmids [76]. Composing the need to establish a genome editing system that can be used to develop a hyper cell factory for preparations of lignocellulolytic enzymes and other heterologous proteins, as well as to characterize the mechanisms that regulate protein induction, synthesis, and secretion [77].

In studies with *Myceliophthora thermophile* Li et al. [78] used the CRISPR/Cas9 technique in five highly expressed genes encoding extracellular proteases, degrade extracellular proteins and reduce cellulase yield. The results attest that Mtalp1 is a gene that degrades protease that inhibits cellulase production. To perform this study, five genes were selected and constructed using the CRISPR-Cas9 technique, resulting in a Mutant DMtalp1 that demonstrated protease activity substantially lower than 58.4%, and may be a good initial strain for additional metabolic engineering in order to produce cellulases and other proteins.

Liu et al. [74] used the CRISPR/Cas9 system for effective multiplexed genome engineering, successfully developed in thermophilic species *M. thermophila* and *M. heterothallica*. CRISPR/Cas9 can efficiently modify the imported a mdS gene into the genome through non-homologous nhej end-mediated events. As evidence of principle, the genes of the cellulase production pathway including cre-1, res-1, gh1–1 and alp-1, were chosen as editing targets. Simultaneous multigenic fissures of up to four of these different loci were prepared with the integration of the neomycin selection marker by means of a single transformation, using the CRISPR/Cas9 system.

This genome engineering tool gave rise to several strains that exhibit marked production of hypercellulase, among which extracellular secret activities of protein and lignocellulase increased substantially (up to 5 and 13 times, respectively), in analogy with the parent lineage. In their research, Salazar-Cerezo et al. [79], used *Penicillium subrubescens*, which is an ascomycete fungus with a robust content of families of active enzymes specific to carbohydrates involved in the degradation of ligonocellulosoic biomass. First, a method was developed for the engendering and transformation of protoplasts, using hygromycin as a selection marker. Subsequently, the CRISPR/Cas9 system was established in *P. subrubescens* by successfully excluding the KU70 gene, which was directly involved in the non-homologous end of the DNA repair mechanism. According to Salazar-Cerezo et al. [79], it was possible to consider the implementation of the CRISPR/Cas9 system in the filamentous fungus *P. subrubescens* and the effective protocols for generating and transforming protoplasts were optimized. In this way the MUTATED KU70 gene showed no discrepant phenotypic differences with the wild-type strains reported in the study, enabling the use of these mutants as parental strains for subsequent transformation events.

Rantasalo et al. [80] made use of CRISPR/Cas9 multiplexed in combination with System (S), classified as synthetic expression producing large amounts of the highly pure calB gene; this combination allowed the production of strains in a shorter time. Rantasalo et al. [80], when using the SES tool, it was used by the calB gene indices in an inducing medium, with highly constitutive expression provided by the SES, being possible to produce approximately 4 grams of glucose per liter of calB, in a cellulase inducing medium.

Zheng et al. [81], was able to create a CRISPR/Cas9 system in *Aspergillus niger* for sgRNA expression based on an endogenous U6 promoter and two heterologous promoters of U6. The three u6 promoters tested made feasible the transcription of sgRNA and the interruption of the gene of polyketide synthase albA gene in *A. niger*. In addition, this system allowed the insertion of highly efficient genes in the target genomic locus in *A. niger*, using DNAs from donors with homologous arms of up to 40 bp.

One of the alternatives for bioprospecting enzymes potentially for industry and the use of CRISPR/Cas9 with non-model species, such as the fungus *Huntiella omanensis*, a filamentous fungus ascomycete belonging to the family Ceratocystidaceae, will allow cutting in metabolic and genetic pathways that have not yet been studied in model species [82].

However, filamentous fungi are considered the largest producers of cellulases so far, since for genome editing systems using CRISPR-Cas9, it was stipulated in more than 40 different species of filamentous fungi and oomycetes, being therefore an important strategy regarding the production of potential strains for applications in the industry [83]. Cellulases and heterologous of fungi, with great industrial potential in the manufacture of bioethanol, using one of the most efficient techniques of recent times for genetic engineering, CRISPR/Cas9. It can be concluded that this technique, combined with biotechnological advances, will result in the improvement of fungal cells capable of producing biofuels economically and on an industrial scale, resulting in higher yield and quality of products.

*Recombinant Fungal Cellulases for the Saccharification of Sugarcane Bagasse DOI: http://dx.doi.org/10.5772/intechopen.98363*
