A review: CRISPR/Cas12-mediated genome editing in fungal cells: advancements, mechanisms, and future directions in plant-fungal pathology

The CRISPR-associated protein system (CRISPR/Cas), characterized by clustered regularly interspaced short palindromic repeats, has revolutionized life science research by providing vast possibilities for altering specific DNA or RNA sequences in various organisms. The present system integrates fragments of exogenous DNA, known as spacers, into CRISPR cassettes. These cassettes are subsequently transcribed into CRISPR arrays, which are further processed to generate guide RNA (gRNA). The CRISPR arrays are genetic loci that are responsible for encoding Cas proteins. The Cas proteins are responsible for supplying the necessary enzymatic machinery to acquire new spacers that are aimed at invading elements. The development of novel genome engineering tools has been made possible by utilizing various Cas proteins, including but not limited to Cas9, Cas12, Cas13, and Cas14, which possess programmable sequence specificity. The emergence of Cas variants has spurred genetic research and advanced the utilization of the CRISPR/Cas tool to manipulate and edit nucleic acid sequences within a wide range of living organisms. This review aims to furnish operational modalities of the Cas12 protein identified thus far. Furthermore, the advantages and disadvantages of Cas12 protein are examined, along with their recent implementations in the plant fungal world.


INTRODUCTION
The advent of CRISPR/Cas genome editing technology has revolutionized the field of molecular biology and opened up new possibilities for precise genetic modifications. Among the various CRISPR/Cas systems, CRISPR/Cas12, also known as Cpf1, has emerged as a powerful tool for genome editing in diverse organisms, including fungal cells. This advanced gene-editing system utilizes a unique RNA-guided endonuclease, Cas12, to introduce targeted DNA modifications, offering unprecedented control over the fungal genome. CRISPR/Cas12, similar to other CRISPR systems, is based on the prokaryotic adaptive immune system found in bacteria and archaea. It protects these microorganisms against invasive genetic elements such as viruses and plasmids. The Cas12 endonuclease acts as the effector protein that carries out the DNA cleavage, while the CRISPR array serves as a memory bank of previously encountered foreign DNA sequences.

Cas12 protein
The Cas12 protein exhibits a high degree of versatility and can be utilized in various dynamic applications, such as epigenome editing. The Cas12 protein is classified under the type V CRISPR system [1]. The Cas12 protein has been identified as an RNAguided DNA endonuclease that serves as a viable alternative to the Cas9 protein for genome editing [2]. The Cas12 protein, known for its antiviral properties, was obtained from two distinct bacterial sources: Acidaminococcus species (AsCas12a) and Lachnospiraceae bacterium (LbCas12a). According to a study, the Cas9 protein exhibits greater precision in distinguishing mismatches located within the first approximately ten base pairs of the RNA-DNA helix close to the PAM sequence. Additionally, it is noteworthy that Cas12 exhibits dissimilarities from the Cas9 protein. Specifically, this protein can independently process the precursor crRNA, eliminating the need for tracrRNA or RNase III. CRISPR/Cas12 genome editing involves several steps. A fungal genome-targeted single-guide RNA (sgRNA) is expressed first. A trans-activating CRISPR RNA (tracrRNA) segment helps assemble the functional Cas12 endonuclease, while a crRNA segment recognizes the target site. A conformational change occurs when the sgRNA binds to the Cas12 protein, activating it to recognize and bind to the fungal cell's complementary target DNA sequence. This binding unwinds the target DNA into an R-loop structure, exposing the target strand for cleavage [3,4]. At the target site, activated Cas12 endonuclease causes double-strand breaks (DSBs). DSBs activate the cell's DNA repair machinery using NHEJ and HDR. DSBs in fungi are repaired by NHEJ, which creates small insertions or deletions (indels). Indels disrupt target gene reading frames, causing gene knockout or loss-of-function mutations. Choosing and designing the sgRNA can improve CRISPR/Cas12-mediated genome editing in fungal cells. To avoid off-target effects, the target sequence should be unique in the fungal genome, and the sgRNA should have high complementarity and stability to bind to the target DNA site efficiently. In recent years, many studies have used CRISPR/Cas12 technology to edit genes in fungal species, including plant pathogens like Botrytis cinerea, Fusarium, Botrytis, Colletotrichum and Powdery mildew fungi. These advances have allowed researchers to study pathogenicity, secondary metabolism, and drug resistance fungal genes. To utilize the CRISPR/Cas12 system, fungal cells must first acquire the necessary components. The acquisition of the CRISPR/Cas system typically involves the integration of short DNA fragments, known as spacers, derived from foreign genetic elements, such as bacteriophages or plasmids. These spacers are inserted into the fungal genome, allowing the cells to retain a genetic memory of previous encounters with foreign DNA [5] demonstrated the acquisition of CRISPR/Cas systems in diverse fungal species, including Saccharomyces cerevisiae and Candida albicans. They identified the presence of spacers derived from plasmids and viruses within the genomes of these fungi, providing evidence for the acquisition and retention of foreign DNA fragments. In 2015, the application of Cas12a in genome editing was initially documented through the manipulation of the genome of human cells utilizing a conceivably less complex CRISPR system known as Cpf1 (CRISPR-associated endonuclease in Prevotella and Francisella 1). As reported in previous studies, this system was discovered in the bacteria Francisella and Prevotella [2,6,7,33]. Cpf1 proteins from 16 bacterial species, including Acidaminococcus sp. BV3L6 (AsCpf1) and Lachnospiraceae bacterium (LbCpf1) were selected for their efficacy in genome editing in human cells. In contrast to Cas9, it has been observed that Cas12a (also known as Cpf1) does not necessitate the presence of a tracrRNA and RNase III derived from the host cell for the maturation of sgRNA and recognition of the target [8,9]. Cas12a enzyme is responsible for cleaving pre-crRNA molecules located upstream of a hairpin structure that is formed within the CRISPR repeats. This process ultimately results in the production of crRNAs. It has been suggested that Cas12a exhibits RNase activity in addition to its DNA cleavage activity owing to its crRNA processing capability [4]. Variations in hairpin structure recognition exist among Cas12a proteins, and it is crucial to consider this aspect while designing Cas12a for genome editing purposes. Mutations that produce a modified repeat sequence or an unorganized repeat in the pre-cr-RNA have led to suboptimal or nonexistent genome editing by Cas12a [4,10]. These findings indicate that the repeat sequence in the sgRNA may significantly impact the effectiveness of genome editing facilitated by Cas12a proteins. Upon pre-cr-RNA processing by Cas12a, the resulting Cas12a-sgRNA complex identifies a PAM that is rich in T nucleotides (5′ TTN 3′ or 5′ TTTN 3′) located at the 5′ end of the target sequence. In contrast to Cas9, which recognizes a PAM rich in G nucleotides (5′NGG 3′) located at the 3′ end of the target DNA. Cpf1 induces DNA cleavage during genome editing by generating a staggered double-stranded break in DNA, resulting in a 5′ overhang of four or five nucleotides, as illustrated in Figure 1. Studies have reported that Cpf1 exhibits reduced off-target effects in both human and plant cells [11][12][13]. Cpf1, also known as Cas12a, is a compelling option for genome engineering in fungi due to several noteworthy features. Firstly, the absence of tracr-RNA, which spans approximately 80 nucleotides, simplifies the design process. Secondly, the recognition PAM sequence is less restrictive, broadening the range of potential targeting sites within the genome. Lastly, the cleavage of the target DNA at the distal end of the protospacer, located away from the PAM site, preserves the latter for future targeting, enhancing the editing capabilities. Thus far, AsCpf1, LbCpf1, and FnCpf1 have demonstrated effective utilization in genome engineering of S. cerevisiae [14], Y. lipolytica [15], as well as the filamentous fungi Aspergillus [16], A. gossypii, and M. thermophila [17]. In order to guarantee ample nuclease activity, the expression of Cpf1 has been facilitated through the utilization of potent constitutive promoters, such as tef1 of A. nidulans for expression in Aspergillus [16] and TEF-intron for expression in Y. lipolytica [15]. Successful targeting of Cpf1 and modified Cpf1 (dCpf1) to the nucleus has been achieved using a nuclear localization signal located at the C-terminal [14], N-terminal [18,19]. Similar to Cas9, the guide Pol III promoters govern RNA's expression. The efficacy of genome editing through Cpf1 in vivo and in vitro has been investigated in M. thermophila. The efficacy of single-gene targeting is comparable between in vivo and in vitro methods. However, for multi-site targeting, the in vitro approach exhibited lower efficiency. In 2019 a study reported that FnCpf1, AsCpf1, and Cas9 exhibit comparable efficacy in genome editing when directed toward a solitary gene [17]. It has been demonstrated that only FnCpf1 among the Cpf1 enzymes can execute multi-gene targeting. FnCpf1 exhibited the capability to target individual, paired, and triple genes in S. cerevisiae with an efficiency of 95%, 52%, and 43%, respectively in a study conducted by Li et al. [20]. Additional research is necessary to comprehend the variation in editing efficacy among Cpf1 effectors in fungal organisms. The primary distinguishing factor among various Cpf1 proteins pertains to their PAM sites. Specifically, AsCpf1 and LbCpf1 exhibit a PAM site of 5′ TTTN 3' (4 nt), whereas FnCpf1 can identify this PAM site in addition to a shorter PAM of 5′ TTN 3' (3 nt) [21]. A 3-nucleotide protospacer adjacent motif (PAM) sequence increases the number of potential sites available for protospacer selection. Like Cas9, genome editing utilizing Cas12a involves either non-homologous end-joining or homology-directed repair.

Challenges and considerations
Similar to Cas9, the Cas12 protein was previously regarded as the only constituent of the CRISPR family utilized for genome editing purposes. In the majority of cases, Cas12 has been regarded as superior to Cas9 due to its ability to produce staggered double-strand breaks and facilitate the homologydirected repair mechanism, as opposed to both non-homologous end joining and homology-directed repair, as observed with Cas9 [22]. Efficient delivery of the CRISPR components, including Cas12a and crRNA, into fungal cells, is critical to successful genome editing. Several methods have been explored, including transformation-mediated delivery and viral vectors. However, optimizing the delivery methods to ensure efficient uptake and expression of the CRISPR components remains an ongoing challenge. A study by Rhijn et al. [23], reported successfully delivering CRISPR components into the fungal pathogen Aspergillus fumigatus. They employed electroporation-based transformation to introduce Cas9 and sgRNA (single-guide RNA) into the fungal cells, resulting in efficient gene editing. This study demonstrated the feasibility of delivering CRISPR components into fungal cells and their functional expression. One of the concerns in CRISPR/Cas applications is the potential for off-target effects, where the Cas12a endonuclease may cleave unintended DNA sequences that resemble the target site. Minimizing off-target effects is crucial to ensure the specificity and accuracy of gene editing in fungal cells. Lewis et al. [24], examined the off-target effects of CRISPR/Cas12a in Saccharomyces cerevisiae. Through genome-wide analysis, they identified potential off-target sites and evaluated their cleavage efficiency. The results showed a low frequency of off-target cleavage, demonstrating the specificity of CRISPR/Cas12a in fungal cells. The CRISPR/Cas12 system relies on the DNA repair machinery of the host cell, regardless of the presence or absence of a template [25]. The efficacy of this system in achieving precise DNA insertion at specific genomic loci has been demonstrated, albeit with variable outcomes across different cell types. The efficacy of HDR-mediated DNA repair is hindered in cell types that exhibit low levels of active cell division, such as neurons [26]. Nevertheless, a considerable ongoing research effort is to refine the Cas12 system to achieve precise DNA integration into the intended genome. Despite this limitation, the system exhibits a broad spectrum of potential applications, and current efforts are dedicated to developing improved CRISPR/ Cas12 variants to facilitate more reliable genome manipulation.

Future directions
CRISPR/Cas12-mediated gene editing provides a powerful tool to unravel the mechanisms underlying fungal pathogenesis. By targeting and modifying specific genes associated with virulence identified using mapping studies [27][28][29], researchers can investigate their roles in infection processes and hostpathogen interactions, shedding light on the molecular basis of fungal diseases [30,31]. Developing strategies to avoid or reduce off-target effects would be valuable. This understanding can aid in developing effective disease management strategies and identifying potential therapeutic intervention targets [32]. Furthermore, CRISPR/Cas12 technology provides a valuable platform for elucidating the complex molecular mechanisms underlying fungal infection and disease progression. By systematically studying the function of individual genes and their interactions, researchers can gain valuable insights into the intricate processes involved in fungal pathogenicity. This knowledge can inform the development of novel strategies to control and manage fungal diseases effectively. The successful implementation of CRISPR/Cas12 in various fungal pathogens, such as Botrytis cinerea, Fusarium graminearum, and Magnaporthe oryzae, demonstrates its versatility and potential for broad application. The ability to modify multiple genes simultaneously using CRISPR/Cas12 further expands its utility in understanding the genetic basis of fungal pathogenicity and host defense. However, there are still challenges and limitations to address. Improving the efficiency and reliability of CRISPR/ Cas12 delivery systems, enhancing target specificity, and minimizing off-target effects are ongoing research areas. Additionally, ethical and regulatory considerations surrounding using CRISPR/Cas12 in agriculture must be carefully addressed to ensure this technology's responsible and safe deployment. Looking ahead, the future of CRISPR/Cas12 in plant-fungal pathology holds tremendous promise. Advancements in technology and continued research will likely lead to further optimization and refinement of CRISPR/Cas12 tools for fungal genome editing. Exploring novel CRISPR/Cas systems, such as Cas12b and Cas14, may expand the range of targetable genes and improve the precision of editing. Moreover, integrating CRISPR/Cas12 technology with other approaches, such as RNA interference (RNAi) or antifungal compounds, could yield synergistic effects and provide more effective and sustainable strategies for fungal disease management. By combining multiple tools and techniques, researchers can develop comprehensive and integrated solutions to the ever-evolving challenges of fungal pathogens.

CONCLUSION
In conclusion, using CRISPR/Cas12 in plant-fungal pathology can revolutionize our understanding and management of fungal diseases. The scientific evidence presented in this section supports the utilization of CRISPR/Cas12 for precise genome editing in fungal cells. The ability to target specific genes involved in fungal pathogenicity and host defense mechanisms opens up new possibilities for developing resistant crop varieties, biocontrol strategies, and antifungal compounds. Using CRISPR/Cas12 in plant-fungal pathology offers several advantages over traditional methods of disease management. It enables precise and targeted genome editing, allowing for modifying or disrupting specific genes involved in fungal pathogenicity. CRISPR/Cas12 can attenuate fungal pathogenicity and enhance plant resistance by selectively targeting essential virulence factors or disrupting vital regulatory genes.

DeCLARATIONS
Ethics approval and consent to participate

AvAILABILITy Of DATA AND mATeRIAL
No datasets were generated during the study