## 2 Challenges Associated with Isolating Microbial Biocontrols
The system allowed for more precise genome editing, which has revolutionized the biomedicine and agricultural industries [22; 23]. In recent years, researchers have used the CRISPR/Cas9 system in order to further understand the plant-microb interaction, the role of endophytic microorganisms and their bioactive compounds to serve as potential biocontrol agents. This system provided a promising approach to  
Figure 1: Advantageous microbial activity spectrum for potential beneficial endophytes. Microbial biocontrol has the potential to improve agricultural productivity by exhibiting one or all four of the activities depicted in the image. understanding endophytes mode of action for colonization, growth promotion of plants [24] and understanding how they produce biocontrol agents in responses to phytopathogens to improve plant pathogen tolerance [24; 25]. Hence, firstly to understand the interaction between endophytes and their hosts, the CRISPR/Cas9 system has been used to alter or knock out key genes to understand the role they may play. A study by [PERSON] et al. (2020) [26] used the CRISPR/Cas9 system to disrupt the _PmKKA_ gene associated with the beneficial fungal endophyte _Phomopsis liquidambaris_. The introduction of the _PmKKA_-deficient mutant strain in _Oryza sativa_ (rice) seedlings resulted in increased lytic (chitinase and glucanase) enzyme activity, which the authors associated with a resistance response in rice [26]. The findings of the study indicated that the _PmKKA_ gene (in the fungal endophyte) may be required for the rice seedlings to recognize _Phomopsis Liquidambaris_. It was further noted that the disrupted _PmkkA_ gene encoded the CWI MAPK pathway, which may have been required for improved symbiosis ([PERSON] et al., 2020). The findings in [PERSON] et al. (2020) highlighted the importance of using techniques, such as the CRISPR-Cas9 system, to investigate the genes responsible for host colonization, as these discoveries would play a critical role in whether the adoption of endophytic technology within agricultural practices can and will be implemented. Secondly, the CRISPR/Cas9 system has also been successfully implemented to generate endophytic mutants with increased bioactive compound production [27; 28] and enhanced enzymatic expression [29]. The rationale behind manipulating the expression of the enzymes associated with these endophytic microorganisms is to understand their role in growth promotion, the plant's defense response to abiotic stress, biocontrol responses to invading pathogens and to further improve the endophytes beneficial qualities. A study by [PERSON] et al. (2022) [29] produced two mutant strains OE-Chi and IN-Chi by plasmid transformation and CRISPR/Cas9-mediated integration of the chitinase enzyme into the genome of _Phomopsis liquidambaris_, respectively. Both mutant strains showed the ability to inhibit the expansion of wheat pathogen _Fusarium graminearum_ in plate confrontation assays in vitro [29]. However, the OE-Chi strain showed a better colonization of wheat, and it was hypothesized that the higher rate of colonization led to improved resistance to the Fusarium pathogen [29]. This study highlights one of the drawbacks of the current CRISPR/Cas9 system, as integrating the chitinase enzyme led to a decreased colonization potential of the mutant strain [29]. This is an important finding, as the premise of using endophytic microorganisms for beneficial application often hinges on the fact that the endophyte is able to colonize its host. Endophytic microorganisms have been identified as a good source of bioactive compounds with less side effects when compared to their synthetic counterparts. Research involving bioactive compounds from endophytic microorganisms has increased, which has allowed for their different applications in the field of medicine [30] and agricultural industry [30]. In an effort to expedite the discovery of new bioactive compounds from endophytes, researchers have embraced CRISPR technology.