CRISPR-Cas9 system for fungi genome engineering toward industrial applications

Chapter 6

CRISPR-Cas9 system for fungi genome engineering toward industrial applications Lakkakula Satisha, b, Sasanala Shamilib, Balasubramanian C. Muthubharathic, Stanislaus Antony Ceasard, Ariel Kushmaroa, Vijai Singhe and Yaron Sitritb a

Department of Biotechnology Engineering, Ben-Gurion University of the Negev, Beer Sheva, Israel; bThe Jacob Blaustein Institutes for Desert

Research, Ben-Gurion University of the Negev, Bergman Campus, Beer Sheva, Israel; cDepartment of Biotechnology, Science Campus, Alagappa University, Karaikudi, Tamil Nadu, India; dFunctional Genomics and Molecular Imaging Lab, University of Liege, Liege, Belgium; eDepartment of Biosciences, School of Science, Indrashil University, Rajpur, Mehsana, Gujarat, India

6.1 Introduction The evolution of genome engineering approaches has made significant advancement in the past ten years (Glass et al., 2018). Formerly constrained to specific model organisms and ineffective at most, genome engineering started to develop into more predominantly useful through the exploration of various programmable DNA-binding proteins. Major among these tools are zinc finger nucleases (ZFNs) and transcription activator like effector nucleases (TALENs) (Chen et al., 2014). By combining such proteins to nucleases it is feasible to establish a double strand break (DSB) at a certain region in the genome sequence, that would again be repaired either by non-homologous end joining (NHEJ), frequently preeminent to a small insertions or deletions and thus gene knock-out, or homology directed recombination (HDR) with a desired gene, projecting to insertion of a desired DNA template (Miller et al., 2010; Urnov et al., 2005). Clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein 9 (Cas9) method was found in nature as a microbial adaptive host defense system, it has been remodeled into a key tool for genome engineering (Glass et al., 2018). CRISPRCas9 approach is balanced to transform developmental biology through an efficient and simple tool to modify the genome of virtually any developing organism precisely (Harrison et al., 2014; Komor et al., 2017). When DNA strand is cleaved using a Cas9 nuclease, it is feasible to be repaired through either NHEJ or HDR pathways. But, the exact mechanism underlying these repair pathways is unclear (Harrison et al., 2014). Traditional approaches for genome editing are however mostly inefficient. The CRISPR-Cas9 method has been extensively reported in bacteria, plants and mammals and has become a quick, simple and adaptable tool for systemic research on various fungi (Deng et al., 2017a). CRISPR-Cas9 method has empowered genome engineering in plentiful industrially significant organisms and allocated genetic systems that were inaccessible formerly (Donohoue et al., 2018). This chapter aims to focus on the current progresses of the CRISPR-Cas9 mediated genome engineering methods for targeted genome editing and its impending approaches in industrially important fungi.

6.2 Challenges in editing fungal genome Fungi affect many aspects of our life including health, food and livestock. Their benefits occur through various mechanisms such as improvement of plant nutrition and protection, production of enzymes, antibiotics and therapeutic molecules. Fungi are versatile organisms producing many valuable, natural compounds of industrial and pharmaceutical relevance such as alkaloids, polysaccharides, steroids and terpenes, some of which have antibacterial, anti-tumor activities (Deng et al., 2017b; Xiao and Zhong, 2016; Zhong and Xiao, 2009) (Fig. 6.1). The genetics unrevealed fungal behavior to

Genome Engineering via CRISPR-Cas9 System. https://doi.org/10.1016/B978-0-12-818140-9.00006-4 Copyright © 2020 Elsevier Inc. All rights reserved.

69

70

Genome Engineering via CRISPR-Cas9 System

FIG. 6.1 Specific produces synthesized in the industry using various fungal species as the main source.

environmental aspects, along with plant-fungal interactions, biological variations and the global carbon cycle have developed into typical aspects in molecular ecology (Wang et al., 2018). At present, the fungal cell factories are salient for large scale synthesis of secondary metabolites, proteins and organic acids. A number of issues must be addressed to enhance and improve the utilization of fungi as base originators for innovative molecules and microbial cell factories for wide-ranging production of cell-mediated products (Meyer et al., 2016) (Fig. 6.2). Synthesis of novel compounds using fungi will need effective genome editing methods to change the metabolism and to regulate the mechanisms (Pohl et al., 2016). Novel findings have indeed been made by various fungal genomics approaches (Ma and Fedorova, 2010). An example of how molecular intervention methodologies can affect the understanding of physiological processes in fungi includes the use of a traditional gene knock-out approach with 1 kb flanking region that usually forms heterokaryons and lead to multi cellular asexual spore development (Wenderoth et al., 2017). The lack of molecular approaches to study these organisms has not merely limited the insight of metabolic regulation of superior fungal species including mushrooms, as well as delayed other new strain development by genetic methods (Qin et al., 2017). Numerous genome editing methods has-been established with regard to obtain higher HDR rate in fungi (Weyda et al., 2017). However, the number of transformants with a gene knock-out in fungi was very limited including Pleurotus ostreatus (Salame et al., 2012) and Schizphylhls commne (Ohm et al., 2010). Breakdown of the NHEJ pathway by deleting the Ku protein complexes produced higher HDR rates of up to 98% with the use of protoplasts and a relatively

CRISPR-Cas9 system for fungi genome engineering toward industrial applications Chapter | 6

71

FIG. 6.2 Applications of various industrially important fungi.

small (less than 500 bp) homologous gene (Ninomiya et al., 2004). The CRISPR method is a substitute system to bypass these obstacles (Deng et al., 2017a). So far, this method has facilitated potent genome engineering and improvements in various model organisms. For genome engineering in fungi using CRISPR-Cas9, several approaches were employed for expressing sgRNA into the cells, i.e., expressing it by various fungal promoters or inclusion of in vitro transcribed sgRNA (Liu et al., 2015; Pohl et al., 2016). Since a decade, genome editing tools have been improved, and new developments in DNA sequencing methods has revealed several fungal species genome sequences, closely fitting many formerly unknown gene families of potential bioactive molecules (Gomez-Escribano et al., 2016). Details including genome size, karyotypes and ploidy for many species of fungi are however still unknown (Todd et al., 2017). In fungi, the poor competence of gene targeting, needed to accomplish the desired genetic modifications, hampers research on functional genomics (Shi et al., 2017; Weyda et al., 2017). Genome engineering methods should be assessed not only as a novel and modern system for studying the essential functions of fungi, but also as an advanced tool for enhancing and developing the useful traits as well (Poyedinok, 2018). Thus, the significance of CRISPR-Cas9 method in genome and epigenome editing emphasized importance in the last decade (Jeltsch, 2018). The applications of CRISPR-Cas9 genome editing in industrially important fungi have been depicted (Fig. 6.3; Table 6.1).

72

Genome Engineering via CRISPR-Cas9 System

FIG. 6.3 Applications of CRISPR-Cas9 system genome engineering of in industrially important fungi.

6.3 Industrial applications of CRISPR-Cas9 methods in fungi genome editing The existing drug discovery system is highly expensive, lengthy and originates through a host of barriers. Genome editing is one of the promising approaches amid industrial and clinical applications considering the fast growing facilities for genome engineering toward fruitful applications (Singh et al., 2017). Researchers involved in drug detection can exploit CRISPR system to knock out the target sequence to understand the mechanism of specific gene function in the organism. Research on fungal natural compound identification and synthesis is at an emphasizing phase, as number of existing industrial advances are effective to elevate the growth in the arena in many ways (van der Lee and Medema, 2016). Fungi tend to have several beneficial applications as industrially attractive strains with their higher relative complexity. Majority of the synthetic biology systems have been industrialized in Saccharomyces cerevisiae since it is a well-established model species and for its well-annotated genome and genetic tractability (Nielsen et al., 2013). Type-II CRISPR-Cas9 approach is well categorized after discovery of single protein Cas9 (Gasiunas et al., 2012; Jinek et al., 2012), and has observed the comprehensive adoption for programmable targeting in industrially essential genus of yeast and fungi (DiCarlo et al., 2013). CRISPR-Cas9 mechanism allows genome organization of various industrially important strains which are highly beneficial if engineered genetically since the lack of selection markers and also the complications in transforming multiple alleles (Le Borgne, 2012; Stovicek et al., 2017). S. cerevisiae is a model organism which is widely utilizesd as an industrial host for the invention of recombinant proteins, food products, beverages, analyticals and fuels (Borodina and Nielsen, 2014; Li and Borodina, 2015; Stovicek et al., 2017). Multiplex CRISPR system was executed for evolution experimentations in S. cerevisiae through the direct modifications by inserting/deleting specific nucleotides in a DNA strand which code for functional proteins (Ryan et al., 2014). A collective DNA cleaves were created in the genome and barcoded DNA sequences joined at the cleaved regions to complete the gaps and it was tested and proved to produce improved proteins (Ryan et al., 2014). CRISPR-Cas9 mechanism was also being used to produce auxotrophic markers by gene disruption and developed S. cerevisiae mutants’ resistant to heat stress. This was successfully used as a sustainable fermentation inhibitor which is extremely applicable in the alcoholic beverages (wine and beer) and fermentation (organic acids, glucose and glycerol etc.) industries (Zhang et al., 2014). The simultaneous functional DNA knock-in and a gene disruption system was developed successfully in lactic acid producing industrial fungal strain (Stovicek et al., 2015). Multiple (three) gene targeting and genome editing in Yarrowia lipolytica through CRISPR system resulted in elevated lipid synthesis with modest feedstock utilization (Friedlander et al., 2016).

6.4 Implementations of the CRISPR-Cas9 in fungi The budding yeast has used for since more than a millennium for making of bread and alcoholic beverage. Although, early 1980s, various molecular biology tools have been developed and used to introduce rDNA concerning fungal genome

TABLE 6.1 Summary of CRISPR-Cas9-mediated genome editing reported in various fungal species. Transformation method

Target gene

Type of promoter Molecular function

Cas9

gRNA

Trait

Phenotype

Mutation frequency (%)

C. militaris

Agrobacterium and PEGmediated

cmcas9

Gene disruption

Pcmlsm3

Ura3

e

Conidia

64

Chen et al. (2018a)

Aspergillus niger

PEG-CaCl2

albA

Polyketide synthase

PglaA

PhU6

Black spores

Albino colonies

15

Zheng et al. (2018)

Saccharomyces cerevisiae

e

kanMX natMX hphMX

Marker-free genetic manipulations

CLN2

Ura3

Yeast

e

90

Soreanu et al. (2018)

A. carbonarius

Protoplast and Agrobacteriummediated

Ayg1

1,3,6,8tetrahydroxynaphthalene; conidia-enriched protein

Ptef

PgpdA

Black conidia

Yellow/black conidia

27

Weyda et al. (2017)

A. fumigatus

PEG-mediated

PksP

Dihydroxy napthalene (DHN)-melanin

e

gpdA

Yellow conidia

White conidia

40e74

Al Abdallah et al. (2017)

Protoplastmediated

tynC

PKS coding gene

gpdA

tet

e

Trypacidin production

8/10

Weber et al. (2017)

Protoplastmediated

GFP

Fluorescence

e

T7

e

Presence of fluorescence

95e100

Zhang and Lu (2017)

Protoplastmediated

mttA

e

PcoxA

PmbfA

e

Aconitic acid production

100

Sarkari et al. (2017)

e

GluD GluF

2-Keto-L-gulonate reductase and L-idonate 5dehydrogenase

Tef1

In vitro synthesis

e

Catabolization of D-GlcUA

e

Kuivanen et al. (2016)

Alternaria alternata

Protoplastmediated

brm2

THN reductase encoding gene

Tef1

e

Brown colonies

Uracil auxotrophic mutants

9/14

pksA

Polyketide synthase

Wenderoth et al. (2017)

Nodulisporium sp.

Protoplastmediated

neo

Neomycin phosphotransferase II (neomycin resistance)

trpC

U6

e

G418 resistant colonies

11/12 mutants

Zheng et al. (2017)

Ganoderma lucidum

PEG-mediated

Ura3

e

gpdA

T7

e

5-FOA resistant mutant

8/9 colonies

Qin et al. (2017)

Species

White colonies

73

Continued

CRISPR-Cas9 system for fungi genome engineering toward industrial applications Chapter | 6

A. niger

Reference

74

Type of promoter

Mutation frequency (%)

Species

Transformation method

Target gene

Molecular function

Cas9

gRNA

Trait

Phenotype

Talaromyces atroroseus

hph SM based plasmid-PEG

albA

Naphthagpyrone

e

e

Green colonies

White phenotype

10 fold

Nielsen et al. (2017)

Coprinopsis cinerea

e

CC1G

e

CcDED1

U6

e

e

21

Sugano et al. (2017)

Beauveria bassiana

PEG-mediated

Bar

Phosphinothricin acetyl transferase gene

gpdA

In vitro synthesis

e

5-FOA resistant colonies

>5

Chen et al. (2017)

Shiraia bambusicola

PEG-mediated

MFS

Transporter

Tef1

U6

Hypocrellin production

Able to cause spores on bamboo leaves

90

Deng et al. (2017a)

Candida albicans

Lithium acetate

RFP

Red fluorescence

ADH1

SNR52

White colonies

Red colonies under fluorescence microscope

10 fold

Ng and Dean (2017)

Myceliophthora thermophila

Agrobacteriummediated

amdS cre-1

Hyper-cellulase production

Ptef1

U6

e

e

95

Liu et al. (2017)

wA

Polyketide Synthase

amyB

U6

White conidia

e

10e20

yA

Conidial laccase

Yellow conidia

100

Katayama et al. (2016)

pyrG

c

e

10

A. oryzae

A. niger

AMA1 plasmid

GaaA

Galactaric acid production

Ptef1

gpdA

Galactaric acid catabolism

A. fumigatus

AMA1 plasmid

PksP

Melanin

pgdA

U6

cnaA

Calcineurin and catalytic subunit of Ca2þ

Reference

Hygromycin resistant colonies

28

Kuivanen et al. (2016)

e

Pigment less Albino

95e100

Zhang et al. (2016)

Penicillium chrysogenum

Protoplastmediated

pks17

Polyketide synthase

lysY

T7

Green colonies

White colonies

60

Pohl et al. (2016)

Ustilago maydis

e

bE1, bW2

Homeodomain protein

Otef

U6

Loss of filament formation in charcoal containing agar

Carboxin resistant colonies

50e90

Schuster et al. (2016)

Genome Engineering via CRISPR-Cas9 System

TABLE 6.1 Summary of CRISPR-Cas9-mediated genome editing reported in various fungal species.dcont’d

Protoplastmediated

Avr4/6

Infect soybean cultivars

Ham34

U6

e

G418 resistant

100

Fang and Tyler (2016)

A. fumigatus

PEG-mediated

abr2

Polyketide synthase

Ptef1

SNR52

Colourless mutants

e

25e53

Fuller et al. (2015)

A. nidulans

Protoplastation

yA

Conidial laccase

Ptef1

PgpdA

Green conidia

Yellow conidia

20e30

albA

Black spore pigment

Black colonies

No colony formed in 5-FOA (confirmed)

Nødvig et al. (2015)

A. aculeatus

0

pyrG

Orotidine 5 -phosphate decarboxylase

Trichoderma reesei

Protoplastmediated

URA5, clr2

(Uridine dependent Cas9 expression cassette)

Ppdc & Pcbh1

In vitro synthesis

e

5FOA resistant

100

Liu et al. (2015)

Neurospora crassa

e

Clr2 & csr1

Cellulose regulator & cyclosporin A-binding protein

trpC

SNR52

e

Cellulase production

200 fold

Matsu-ura et al. (2015)

Pyricularia oryzae

PEG-mediated

SDH

Scytalone dehydratase

Tef1

U6

e

White colonies

36.1e80.5

Arazoe et al. (2015)

Details on transformation method, name of the target gene, observed phenotype, types of promoters used for the expression of Cas9 and gRNA and percentage mutations observed are presented with respective references. abr2, Brown pigmentation; ADH1, Alcohol dehydrogenase 1; albA, Polyketide synthase; amdS, Acetamidase; amyB, a amylase; avr4/6, Avirulence protein precursor; Ayg1, Aspergillus yellow green; Bar, Herbicide bialaphos gene; bE1, B mating type protein; brm2, 1,3,8 Trihydroxy naphthalene reductase; bW2, Homeodomain transcription factor; CCD1, Carotenoid cleavage dioxygenase 1; CcDED1, Coprinopsis cinerea DED1 gene; clr2, Cryptic loci regulator; cnaA, Calcineurin-encoding gene; coxA, Cytochrome C oxidase; csr1, Cyclosporine A binding protein; gaaA, Gene responsible for conversion of D-Galacturonic acid to L-Galactonic acid; GFP, Green fluorescence protein; GluD & GluF, NAD and FAD specific glutamate dehydrogenease; lysY, Promoter from lysine Y gene of bacteria; mbfA, Ferrous iron exporter; MFS, Major facilated superfamily (Multi gene tgransporter); mttA, Mitochondrial transporter; neo, neomycin; pks17, Probable polyketide synthase 17; pksA, Polyketide synthase; pyrG, Orotidine 5’-phosphate decarboxylase; PEG, Poly ethylene glycol; PglaA, Promoter of glucoamylase; PgpdA, Promoter of glyceraldehyde 3-phosphate dehydrogenase; PhU6, Initial identity of a snRNA; PksP, Promoter of polyketide synthase; RFP, Red Fluorescence protein; SDH, Scytalone dehydratase; SNR52, Small nucleolar RNA; T7, promoter from T7 bacteriophage; Tef, Translation elongation factor; trpC, Tryptophan biosynthesis; tynC, tet- tetracycline; Ura3, URAcil-3 requiring; URA5, URAcil-5 requiring; wA and yA, Conidia colour pigmentation genes in Aspergillus.

CRISPR-Cas9 system for fungi genome engineering toward industrial applications Chapter | 6

Phytophthora sojae

75

76

Genome Engineering via CRISPR-Cas9 System

FIG. 6.4 Overall view of CRISPR-Cas9 genome editing in fungi for enzyme production.

modification (Orr-Weaver et al., 1981). To gain an insight into the cell-biology of various fungi cannot be only substantially influence the industrial production besides that have applicability to human health (Fraczek et al., 2018). Current developments in genetic engineering, sequencing approaches, and optimization of gene transfection methods on industrially essential fungal species has become useful for mycologists to precisely use engineered cells for creating a novel freeliving fungi traits (Shanmugam et al., 2019). CRISPR-Cas9 genome engineering was efficiently used in Aspergillus fumigatus and polyketide enzyme (crucial for virulence) was biosynthesized after identifying the polyketide synthase gene that is responsible for pigment bio-synthesis (Langfelder et al., 1998). Later on several types of metabolites/enzymes have been produced in various industries by filamentous fungi at distinctive environments (Liu et al., 2015) (Fig. 6.4). Filamentous fungi constitute an important provenance of active pharmaceutical compounds including the most extensively used anti-bacterial (cephalosporin and penicillin), the anti-fungals (echinocandin and griseofulvin), and statins such as cholesterol lowering agents (Hoffmeister and Keller, 2007). Improvement of novel and versatile systems for genome editing of fungi with considerable benefit in synthesizing the poor yields of complex bioactive compounds (Zheng et al.,

CRISPR-Cas9 system for fungi genome engineering toward industrial applications Chapter | 6

77

2017). These results proven that, the CRISPR-Cas9 mechanism works in Trichoderma reesei like an effective and amenable genome editing approach in to sequence specific mode. Further, the Cas9 nuclease expression in specific promoters helps in precise genome modifications of fungi. The CRISPR-Cas9 mutagenesis will enable significantly more rapid generation of intended mutants, that will permit it simpler to associate molecules to precise biosynthetic gene assembly (van der Lee and Medema, 2016). A marker-free CRISPR mediated genome editing system was reported in S. cerevisiae with complete gene and new promoter changes and with high throughput operative array combination (Soreanu et al., 2018). Another marker-free CRISPR system was developed in Penicillium chrysogenum which is very flexible and can be used for genome targeting with least cloning efforts (Pohl et al., 2016). Marker-free genetic engineering is a novel idea and highly required for desired gene replacement and several other practices in different organisms including fungi (Soreanu et al., 2018).

6.5 CRISPR-based gene regulation in fungi CRISPR-Cas9 system opens the path for gene regulation and enables genome editing and offers the possibility to use of this method in various areas of the biological and industrial research. After the primary characterization of CRISPR-Cas9 genome editing system, researchers perceived how it could be applicable for defined genome engineering in fungi (Pineda et al., 2019; Thurtle-Schmidt and Lo, 2018). CRISPR-based genetic modification systems have advanced genetic studies in numerous widespread organisms (Vyas et al., 2018). Defined targeting, insertions or removal of the target gene for affirmative preference are the two significant applications of CRISPRCas9 system in fungi genome editing (Stovicek et al., 2017). Recently, a CRISPR mechanism was employed to organize the gene expression and also to engineer a varied virulence pathways in Candida albicans (Román et al., 2019). Improvement of CRISPR-Cas9 genome engineering procedure in C. albicans is essential for improvement of novel antifungal agents and also for understanding the regulation of several key genes. This group revealed an exact activation or repression of catalase gene that encode the cytosolic catalase (Román et al., 2019). Further improvement of CRISPR-Cas9 mechanism was shown not to let any selection marker is intended to be left in the genetically modified strains (Stovicek et al., 2015).

6.6 CRISPR-Cas9 a novel approach for biological control Most of the diseases in various plant species are caused by fungal pathogens which are liable for the significant decline in growth and productivity of the crops with massive economic losses (Giraud et al., 2010). Genome engineering methods have been improved quickly and become one of the most significant genetic tools in the development of resistant crops to a number of pathogens. Also, an understanding of communications among the plants and fungi, bacterial communities is an important research interest for several years (Borrelli et al., 2018). Developments in genome engineering approaches have shown new techniques to accomplish the biotic stress tolerance in crop plants. After the CRISPR-Cas9 mechanism has been started using to unravel wide range of agricultural challenges, biotic stress tolerance crops also improved successfully (Arora and Narula, 2017). Many feasible candidate genes and its derivative products responsible for fungal disease resistance in plants were illustrated, and currently which are the major objectives for genome engineering through CRISPR-Cas9 method. It is clear that various fungi cause huge complications in the plant production industry and that impotent controller that can start to many severe problems in food production (Shuping and Eloff, 2017). After establishment of the CRISPR-Cas9 approach as a way for intended genome alteration, it has been rapidly developed as a key resource for crop improvement (Jinek et al., 2012). The CRISPR-Cas9 mechanism was exploited to produce transgenic Solanum lycopersicum (Malnoy et al., 2016), Triticum aestivum (Nekrasov et al., 2017), and Vitis vinifera (Wei et al., 2018) plants by targeting Mildew Locus O (MLO) susceptible gene which confers heritable resistance to Blumeria graminis fungus causing powdery mildew. The Natriuretic Peptide Receptor 3 (NPR3) gene responsible for regulating the immune system in Theobroma cacao plants was targeted and mutants resistant to black pod disease caused by Phytophthora tropicalis were generated (Fister et al., 2018). The rice blast disease in Oryza sativa caused by Magnaporthe oryzae was significantly controlled by targeting a transcription factor Ethylene response factor (ERF) implicated in multiple stress responses (Wang et al., 2016), and a subunit of the Exocyst complex component 3A (SEC3A) gene (Ma et al., 2018). Altogether, these summarized results validate the prevailing and beneficial applications of the CRISPR-Cas9 genome engineering for the progression of some plants resistant to fungal diseases.

78

Genome Engineering via CRISPR-Cas9 System

6.7 Further developments required for fungi genome editing CRISPR-Cas9 mechanism is a novel and proficient way in order to gene modification, however it is additionally an alluring method to determine the gene expression (Román et al., 2019). Recent advances in genome engineering of various fungal species have untied a different frontline through CRISPR-Cas9 system (Krappmann, 2016). There is an enormous challenge to advance the third generation bio-refineries that incorporate energy generation with the invention of valuable and complex chemicals from renewable materials. For this, various stress resistant and robust industrial fungal strains are need to be developed through the genome engineering approaches (Stovicek et al., 2015). Nutrient absorption is the outset that consents biological systems to thrive and produce invaluable products. Synthesis of sustainable chemicals, pharmaceuticals and biofuels must be cost effective, ecofriendly, and to be usually accepted by arcades and society. In this regard, genetically modified yeast/fungi and other organisms will play a significant role in the production of eco-products with the applicability at low cost feedstocks such as starch, sucrose, xylose, cellobiose and lignocellulosic biomass, etc. (Endalur Gopinarayanan and Nair, 2018). Very limited studies were reported on CRISPR-Cas9-mediated marker-free gene modification system in fungi including Ustilago trichophora (Huck et al., 2018), M. oryzae (Foster et al., 2018), S. cerevisiae (Soreanu et al., 2018; Jessop-Fabre et al., 2016) and Mucor circinelloides (Nagy et al., 2017) and many more investigations have to be done in model organisms. Similarly, marker recycling system was reported C. albicans through CRISPR induced marker excision which allows recycling of a selection marker for repeated use (Huang and Mitchell, 2017). Bioremediation is a vital approach in the reduction of plastic and other waste which relies on bio-reduction methods as certain microorganisms and many enzymes are believed to take place in break down different kinds of pollutants. To discover the diversity of genes involved in the plastic bio-reduction, several endophytic fungi were tested for their capability in plastic degradation (Russell et al., 2011). Scientists believe that bioremediation might be done at enormous levels with CRISPR engineered fungi. Insufficiency of capable methods to image nonrepetitive DNA sequences in living organisms has pinned down our competence to discover the functional impact of novel genes (Chen et al., 2018b).

6.8 Conclusion and future prospects CRISPR-Cas9 gene editing mechanism has empowered the genetic engineering in wide variety of industrially adequate fungi species and delivered various genetic methods that were inaccessible earlier. Over the past decade, after the CRISPRCas9 mechanism was introduced for genome editing in various model organisms especially as regards fungi, this tool has been repurposed to accomplish several strains of genome alterations in a different group of species concerned to industrial biotechnology. As a result of its apt iterative policy, ease of application and numerous ways to use, the CRISPR-Cas9 mechanism is certainly used in broad areas of research. The novel findings of CRISPR-Cas9 applications in fungi genome editing with unique and novel competencies and latent applications in industrial biotechnology demonstrates the existence of other molecular approaches covert in the genomes of uncultured fungi. Applications of CRISPR-Cas9 genome engineering mechanism in fungi will undergo to effect the industrial biotechnology, where the consistent methods for use athwart non model organisms are required and explored.

Acknowledgment The financial support of the Planning and Budgeting Committee Post-Doctoral fellowship, Israel to LS is kindly acknowledged. SS acknowledges the Kreitman School of Advanced Graduate Studies, Israel for financial support.

References Al Abdallah, Q., Ge, W., Fortwendel, J.R., 2017. A simple and universal system for gene manipulation in Aspergillus fumigatus: in vitro-assembled Cas9guide RNA ribonucleoproteins coupled with microhomology repair templates. mSphere 2 (6) pp e00446-17. Arazoe, T., Miyoshi, K., Yamato, T., Ogawa, T., Ohsato, S., Arie, T., Kuwata, S., 2015. Tailor-made CRISPR/Cas system for highly efficient targeted gene replacement in the rice blast fungus. Biotechnol. Bioeng. 112 (12), 2543e2549. Arora, L., Narula, A., 2017. Gene editing and crop improvement using CRISPR-Cas9 system. Front. Plant Sci. 8, 1932. Borodina, I., Nielsen, J., 2014. Advances in metabolic engineering of yeast Saccharomyces cerevisiae for production of chemicals. Biotechnol. J. 9 (5), 609e620. Borrelli, V.M.G., Brambilla, V., Rogowsky, P., Marocco, A., Lanubile, A., 2018. The enhancement of plant disease resistance using CRISPR/Cas9 technology. Front. Plant Sci. 9, 1245.

CRISPR-Cas9 system for fungi genome engineering toward industrial applications Chapter | 6

79

Chen, B.-X., Wei, T., Ye, Z.-W., Yun, F., Kang, L.-Z., Tang, H.-B., Guo, L.-Q., Lin, J.-F., 2018a. Efficient CRISPR-Cas9 gene disruption system in edible-medicinal mushroom Cordyceps militaris. Front. Microbiol. 9, 1157. Chen, B., Zou, W., Xu, H., Liang, Y., Huang, B., 2018b. Efficient labeling and imaging of protein-coding genes in living cells using CRISPR-Tag. Nat. Commun. 9 (1), 5065. Chen, J., Lai, Y., Wang, L., Zhai, S., Zou, G., Zhou, Z., Cui, C., Wang, S., 2017. CRISPR/Cas9-mediated efficient genome editing via blastospore-based transformation in entomopathogenic fungus Beauveria bassiana. Sci. Rep. 7, 45763. Chen, L., Tang, L., Xiang, H., Jin, L., Li, Q., Dong, Y., Wang, W., Zhang, G., 2014. Advances in genome editing technology and its promising application in evolutionary and ecological studies. GigaScience 3 (1), 24. Deng, H., Gao, R., Liao, X., Cai, Y., 2017a. Genome editing in Shiraia bambusicola using CRISPR-Cas9 system. J. Biotechnol. 259, 228e234. Deng, H., Gao, R., Liao, X., Cai, Y., 2017b. CRISPR system in filamentous fungi: current achievements and future directions. Gene 627, 212e221. DiCarlo, J.E., Norville, J.E., Mali, P., Rios, X., Aach, J., Church, G.M., 2013. Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Res. 41 (7), 4336e4343. Donohoue, P.D., Barrangou, R., May, A.P., 2018. Advances in industrial biotechnology using CRISPR-Cas systems. Trends Biotechnol. 36 (2), 134e146. Endalur Gopinarayanan, V., Nair, N.U., 2018. A semi-synthetic regulon enables rapid growth of yeast on xylose. Nat. Commun. 9 (1), 1233. Fang, Y., Tyler, B.M., 2016. Efficient disruption and replacement of an effector gene in the oomycete Phytophthora sojae using CRISPR/Cas9. Mol. Plant Pathol. 17 (1), 127e139. Fister, A.S., Landherr, L., Maximova, S.N., Guiltinan, M.J., 2018. Transient expression of CRISPR/Cas9 machinery targeting TcNPR3 enhances defense response in Theobroma cacao. Front. Plant Sci. 9, 268. Foster, A.J., Martin-Urdiroz, M., Yan, X., Wright, H.S., Soanes, D.M., Talbot, N.J., 2018. CRISPR-Cas9 ribonucleoprotein-mediated co-editing and counterselection in the rice blast fungus. Sci. Rep. 8 (1), 14355. Fraczek, M.G., Naseeb, S., Delneri, D., 2018. History of genome editing in yeast. Yeast 35 (5), 361e368. Friedlander, J., Tsakraklides, V., Kamineni, A., Greenhagen, E.H., Consiglio, A.L., MacEwen, K., Crabtree, D.V., Afshar, J., Nugent, R.L., Hamilton, M.A., Joe Shaw, A., South, C.R., Stephanopoulos, G., Brevnova, E.E., 2016. Engineering of a high lipid producing Yarrowia lipolytica strain. Biotechnol. Biofuels 9 (1), 77. Fuller, K.K., Chen, S., Loros, J.J., Dunlap, J.C., 2015. Development of the CRISPR/Cas9 system for targeted gene disruption in Aspergillus fumigatus. Eukaryot. Cell 14 (11), 1073e1080. Gasiunas, G., Barrangou, R., Horvath, P., Siksnys, V., 2012. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc. Natl. Acad. Sci. U.S.A. 109 (39), E2579eE2586. Giraud, T., Gladieux, P., Gavrilets, S., 2010. Linking the emergence of fungal plant diseases with ecological speciation. Trends Ecol. Evol. 25 (7), 387e395. Glass, Z., Lee, M., Li, Y., Xu, Q., 2018. Engineering the delivery system for CRISPR-based genome editing. Trends Biotechnol. 36 (2), 173e185. Gomez-Escribano, J.P., Alt, S., Bibb, M.J., 2016. Next generation sequencing of actinobacteria for the discovery of novel natural products. Mar. Drugs 14 (4), 78. Harrison, M.M., Jenkins, B.V., O’Connor-Giles, K.M., Wildonger, J., 2014. A CRISPR view of development. Genes Dev. 28 (17), 1859e1872. Hoffmeister, D., Keller, N.P., 2007. Natural products of filamentous fungi: enzymes, genes, and their regulation. Nat. Prod. Rep. 24 (2), 393e416. Huang, M.Y., Mitchell, A.P., 2017. Marker recycling in Candida albicans through CRISPR-Cas9-induced marker excision. mSphere 2 (2) e00050-17. Huck, S., Bock, J., Girardello, J., Gauert, M., Pul, Ü., 2018. Marker-free genome editing in Ustilago trichophora with the CRISPR-Cas9 technology. RNA Biol. 1e7. Jeltsch, A., 2018. From bioengineering to CRISPR/Cas9 e a personal retrospective of 20 Years of research in programmable genome targeting. Front. Genet. 9, 5. Jessop-Fabre, M.M., Jakociunas, T., Stovicek, V., Dai, Z., Jensen, M.K., Keasling, J.D., Borodina, I., 2016. EasyClone-MarkerFree: a vector toolkit for marker-less integration of genes into Saccharomyces cerevisiae via CRISPR-Cas9. Biotechnol. J. 11 (8), 1110e1117. Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J.A., Charpentier, E., 2012. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337 (6096), 816e821. Katayama, T., Tanaka, Y., Okabe, T., Nakamura, H., Fujii, W., Kitamoto, K., Maruyama, J.-I., 2016. Development of a genome editing technique using the CRISPR/Cas9 system in the industrial filamentous fungus Aspergillus oryzae. Biotechnol. Lett. 38 (4), 637e642. Komor, A.C., Badran, A.H., Liu, D.R., 2017. CRISPR-based technologies for the manipulation of eukaryotic genomes. Cell 168 (1e2), 20e36. Krappmann, S., 2016. CRISPR-Cas9, the new kid on the block of fungal molecular biology. Med. Mycol. 55 (1), 16e23. Kuivanen, J., Wang, Y.-M.J., Richard, P., 2016. Engineering Aspergillus Niger for galactaric acid production: elimination of galactaric acid catabolism by using RNA sequencing and CRISPR/Cas9. Microb. Cell Factories 15 (1), 210. Langfelder, K., Jahn, B., Gehringer, H., Schmidt, A., Wanner, G., Brakhage, A.A., 1998. Identification of a polyketide synthase gene (pksP) of Aspergillus fumigatus involved in conidial pigment biosynthesis and virulence. Med. Microbiol. Immunol. 187 (2), 79e89. Le Borgne, S., 2012. Genetic engineering of industrial strains of Saccharomyces cerevisiae. In: Lorence, A. (Ed.), Recombinant Gene Expression. Humana Press, Totowa, NJ. Li, M., Borodina, I., 2015. Application of synthetic biology for production of chemicals in yeast Saccharomyces cerevisiae. FEMS Yeast Res. 15 (1), 1e12. Liu, Q., Gao, R., Li, J., Lin, L., Zhao, J., Sun, W., Tian, C., 2017. Development of a genome-editing CRISPR/Cas9 system in thermophilic fungal Myceliophthora species and its application to hyper-cellulase production strain engineering. Biotechnol. Biofuels 10 (1), 1.

80

Genome Engineering via CRISPR-Cas9 System

Liu, R., Chen, L., Jiang, Y., Zhou, Z., Zou, G., 2015. Efficient genome editing in filamentous fungus Trichoderma reesei using the CRISPR/Cas9 system. Cell Discov. 1, 15007. Ma, J., Chen, J., Wang, M., Ren, Y., Wang, S., Lei, C., Cheng, Z., Sodmergen, 2018. Disruption of OsSEC3A increases the content of salicylic acid and induces plant defense responses in rice. J. Exp. Bot. 69 (5), 1051e1064. Ma, L.-J., Fedorova, N.D., 2010. A practical guide to fungal genome projects: strategy, technology, cost and completion. Mycology 1 (1), 9e24. Malnoy, M., Viola, R., Jung, M.-H., Koo, O.-J., Kim, S., Kim, J.-S., Velasco, R., Nagamangala Kanchiswamy, C., 2016. DNA-free genetically edited grapevine and apple protoplast using CRISPR/Cas9 ribonucleoproteins. Front. Plant Sci. 7, 1904. Matsu-ura, T., Baek, M., Kwon, J., Hong, C., 2015. Efficient gene editing in Neurospora crassa with CRISPR technology. Fungal Biol. Biotechnol. 2 (1), 4. Meyer, V., Andersen, M.R., Brakhage, A.A., Braus, G.H., Caddick, M.X., Cairns, T.C., de Vries, R.P., Haarmann, T., Hansen, K., Hertz-Fowler, C., 2016. Current challenges of research on filamentous fungi in relation to human welfare and a sustainable bio-economy: a white paper. Fungal Biol. Biotechnol. 3 (1), 6. Miller, J.C., Tan, S., Qiao, G., Barlow, K.A., Wang, J., Xia, D.F., Meng, X., Paschon, D.E., Leung, E., Hinkley, S.J., 2010. A TALE nuclease architecture for efficient genome editing. Nat. Biotechnol. 29 (2), 143. Nagy, G., Szebenyi, C., Csernetics, Á., Vaz, A.G., Tóth, E.J., Vágvölgyi, C., Papp, T., 2017. Development of a plasmid free CRISPR-Cas9 system for the genetic modification of Mucor circinelloides. Sci. Rep. 7 (1), 16800. Nekrasov, V., Wang, C., Win, J., Lanz, C., Weigel, D., Kamoun, S., 2017. Rapid generation of a transgene-free powdery mildew resistant tomato by genome deletion. Sci. Rep. 7 (1), 482. Ng, H., Dean, N., 2017. Dramatic improvement of CRISPR/Cas9 editing in Candida albicans by increased single guide RNA expression. mSphere 2 (2) e00385-16. Nielsen, J., Larsson, C., van Maris, A., Pronk, J., 2013. Metabolic engineering of yeast for production of fuels and chemicals. Curr. Opin. Biotechnol. 24 (3), 398e404. Nielsen, M.L., Isbrandt, T., Rasmussen, K.B., Thrane, U., Hoof, J.B., Larsen, T.O., Mortensen, U.H., 2017. Genes linked to production of secondary metabolites in Talaromyces atroroseus revealed using CRISPR-Cas9. PLoS One 12 (1), e0169712. Ninomiya, Y., Suzuki, K., Ishii, C., Inoue, H., 2004. Highly efficient gene replacements in Neurospora strains deficient for nonhomologous end-joining. Proc. Natl. Acad. Sci. U.S.A. 101 (33), 12248e12253. Nødvig, C.S., Nielsen, J.B., Kogle, M.E., Mortensen, U.H., 2015. A CRISPR-Cas9 system for genetic engineering of filamentous fungi. PLoS One 10 (7), e0133085. Ohm, R.A., de Jong, J.F., Berends, E., Wang, F., Wösten, H.A.B., Lugones, L.G., 2010. An efficient gene deletion procedure for the mushroom-forming basidiomycete Schizophyllum commune. World J. Microbiol. Biotechnol. 26 (10), 1919e1923. Orr-Weaver, T.L., Szostak, J.W., Rothstein, R.J., 1981. Yeast transformation: a model system for the study of recombination. Proc. Natl. Acad. Sci. U.S.A. 78 (10), 6354e6358. Pineda, M., Lear, A., Collins, J.P., Kiani, S., 2019. Safe CRISPR: challenges and possible solutions. Trends Biotechnol. 37, 389e401. Pohl, C., Kiel, J., Driessen, A., Bovenberg, R., Nygard, Y., 2016. CRISPR/Cas9 based genome editing of Penicillium chrysogenum. ACS Synth. Biol. 5 (7), 754e764. Poyedinok, N.L., 2018. Advances, problems, and prospects of genetic transformation of fungi. Cytol. Genet. 52, 154e2018. Qin, H., Xiao, H., Zou, G., Zhou, Z., Zhong, J.-J., 2017. CRISPR-Cas9 assisted gene disruption in the higher fungus Ganoderma species. Process Biochem. 56, 57e61. Román, E., Coman, I., Prieto, D., Alonso-Monge, R., Pla, J., 2019. Implementation of a CRISPR-based system for gene regulation in Candida albicans. mSphere 4, e00001e19. Russell, J.R., Huang, J., Anand, P., Kucera, K., Sandoval, A.G., Dantzler, K.W., Hickman, D., Jee, J., Kimovec, F.M., Koppstein, D., Marks, D.H., Mittermiller, P.A., Núñez, S.J., Santiago, M., Townes, M.A., Vishnevetsky, M., Williams, N.E., Vargas, M.P.N., Boulanger, L.-A., BascomSlack, C., Strobel, S.A., 2011. Biodegradation of polyester polyurethane by endophytic fungi. Appl. Environ. Microbiol. 77 (17), 6076e6084. Ryan, O.W., Skerker, J.M., Maurer, M.J., Li, X., Tsai, J.C., Poddar, S., Lee, M.E., DeLoache, W., Dueber, J.E., Arkin, A.P., Cate, J.H.D., 2014. Selection of chromosomal DNA libraries using a multiplex CRISPR system. eLife 3, e03703. Salame, T.M., Knop, D., Tal, D., Levinson, D., Yarden, O., Hadar, Y., 2012. Predominance of a Versatile-Peroxidase-Encoding gene, mnp4, as Demonstrated by Gene Replacement via a gene targeting system for Pleurotus ostreatus. Mycology 78 (15), 5341e5352. Sarkari, P., Marx, H., Blumhoff, M.L., Mattanovich, D., Sauer, M., Steiger, M.G., 2017. An efficient tool for metabolic pathway construction and gene integration for Aspergillus niger. Bioresour. Technol. 245, 1327e1333. Schuster, M., Schweizer, G., Reissmann, S., Kahmann, R., 2016. Genome editing in Ustilago maydis using the CRISPReCas system. Fungal Genet. Biol. 89, 3e9. Shanmugam, K., Ramalingam, S., Venkataraman, G., Hariharan, G.N., 2019. The CRISPR/Cas9 system for targeted genome engineering in free-living fungi: advances and opportunities for lichenized fungi. Front. Microbiol. 10, 62. Shi, T.-Q., Liu, G.-N., Ji, R.-Y., Shi, K., Song, P., Ren, L.-J., Huang, H., Ji, X.-J., 2017. CRISPR/Cas9-based genome editing of the filamentous fungi: the state of the art. Appl. Microbiol. Biotechnol. 101 (20), 7435e7443. Shuping, D.S.S., Eloff, J.N., 2017. The use of plants to protect plants and food against fungal pathogens: a review. AJTCAM 14 (4), 120e127. Singh, V., Braddick, D., Dhar, P.K., 2017. Exploring the potential of genome editing CRISPR-Cas9 technology. Gene 599, 1e18.

CRISPR-Cas9 system for fungi genome engineering toward industrial applications Chapter | 6

81

Soreanu, I., Hendler, A., Dahan, D., Dovrat, D., Aharoni, A., 2018. Marker-free genetic manipulations in yeast using CRISPR/CAS9 system. Curr. Genet. 64 (5), 1129e1139. Stovicek, V., Borodina, I., Forster, J., 2015. CRISPReCas system enables fast and simple genome editing of industrial Saccharomyces cerevisiae strains. Metab. Eng. Commun. 2, 13e22. Stovicek, V., Holkenbrink, C., Borodina, I., 2017. CRISPR/Cas system for yeast genome engineering: advances and applications. FEMS Yeast Res. 17 (5) fox030. Sugano, S.S., Suzuki, H., Shimokita, E., Chiba, H., Noji, S., Osakabe, Y., Osakabe, K., 2017. Genome editing in the mushroom-forming basidiomycete Coprinopsis cinerea, optimized by a high-throughput transformation system. Sci. Rep. 7 (1), 1260. Thurtle-Schmidt, D.M., Lo, T.-W., 2018. Molecular biology at the cutting edge: a review on CRISPR/CAS9 gene editing for undergraduates. Biochem. Mol. Biol. Educ. 46 (2), 195e205. Todd, R.T., Forche, A., Selmecki, A., 2017. Ploidy variation in fungi: polyploidy, aneuploidy, and genome evolution. Microbiol. Spectr. 5 (4) https:// doi.org/10.1128/microbiolspec.FUNK-0051-2016. Urnov, F.D., Miller, J.C., Lee, Y.-L., Beausejour, C.M., Rock, J.M., Augustus, S., Jamieson, A.C., Porteus, M.H., Gregory, P.D., Holmes, M.C., 2005. Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature 435 (7042), 646. van der Lee, T.A.J., Medema, M.H., 2016. Computational strategies for genome-based natural product discovery and engineering in fungi. Fungal Genet. Biol. 89, 29e36. Vyas, V.K., Bushkin, G.G., Bernstein, D.A., Getz, M.A., Sewastianik, M., Barrasa, M.I., Bartel, D.P., Fink, G.R., 2018. New CRISPR mutagenesis strategies reveal variation in repair mechanisms among fungi. mSphere 3 (2) e00154-18. Wang, F., Wang, C., Liu, P., Lei, C., Hao, W., Gao, Y., Liu, Y.-G., Zhao, K., 2016. Enhanced rice blast resistance by CRISPR/Cas9-Targeted mutagenesis of the ERF transcription factor gene OsERF922. PLoS One 11 (4), e0154027. Wang, Z., Wang, J., Li, N., Li, J., Trail, F., Dunlap, J.C., Townsend, J.P., 2018. Light sensing by opsins and fungal ecology: NOP-1 modulates entry into sexual reproduction in response to environmental cues. Mol. Ecol. 27 (1), 216e232. Weber, J., Valiante, V., Nødvig, C.S., Mattern, D.J., Slotkowski, R.A., Mortensen, U.H., Brakhage, A.A., 2017. Functional reconstitution of a fungal natural product gene cluster by advanced genome editing. ACS Synth. Biol. 6 (1), 62e68. Wei, W., Qianli, P., Fei, H., Alina, A., Shiaoman, C., Harold, T., Eduard, A., 2018. Transgenerational CRISPR-Cas9 activity facilitates multiplex gene editing in allopolyploid wheat. CRISPR J. 1 (1), 65e74. Wenderoth, M., Pinecker, C., Voß, B., Fischer, R.J., 2017. Establishment of CRISPR/Cas9 in Alternaria alternata. Fungal Genet. Biol. 101, 55e60. Weyda, I., Yang, L., Vang, J., Ahring, B.K., Lübeck, M., Lübeck, P.S., 2017. A comparison of Agrobacterium-mediated transformation and protoplastmediated transformation with CRISPR-Cas9 and bipartite gene targeting substrates, as effective gene targeting tools for Aspergillus carbonarius. J. Microbiol. Methods 135, 26e34. Xiao, H., Zhong, J.-J., 2016. Production of useful terpenoids by higher-fungus cell factory and synthetic biology approaches. Trends Biotechnol. 34 (3), 242e255. Zhang, C., Lu, L., 2017. Precise and efficient in-frame integration of an exogenous GFP tag in Aspergillus fumigatus by a CRISPR system. Methods Mol. Biol. 1625, 249e258. Zhang, C., Meng, X., Wei, X., Lu, L.J., 2016. Highly efficient CRISPR mutagenesis by microhomology-mediated end joining in Aspergillus fumigatus. Fungal Genet. Biol. 86, 47e57. Zhang, G.-C., Kong, I.I., Kim, H., Liu, J.-J., Cate, J.H.D., Jin, Y.-S., 2014. Construction of a quadruple auxotrophic mutant of an industrial polyploid Saccharomyces cerevisiae strain by using RNA-Guided Cas9 Nuclease. Appl. Environ. Microbiol. 80 (24), 7694e7701. Zheng, X., Zheng, P., Zhang, K., Cairns, T.C., Meyer, V., Sun, J., Ma, Y., 2018. 5S rRNA promoter for guide RNA expression enabled highly efficient CRISPR/Cas9 genome editing in Aspergillus niger. ACS Synth. Biol. 8 (7), 1568e1574. Zheng, Y.-M., Lin, F.-L., Gao, H., Zou, G., Zhang, J.-W., Wang, G.-Q., Chen, G.-D., Zhou, Z.-H., Yao, X.-S., Hu, D., 2017. Development of a versatile and conventional technique for gene disruption in filamentous fungi based on CRISPR-Cas9 technology. Sci. Rep. 7 (1), 9250. Zhong, J.-J., Xiao, J.-H., 2009. Secondary metabolites from higher fungi: discovery, bioactivity, and bioproduction. Adv. Biochem. Eng. Biotechnol. 113, 79e150.