# Releasing the Full Potential of Cannabis through Biotechnology
## 3 The Genetic Improvement of Cannabis  
The current breakthroughs in GE tools, especially the CRISPR/Cas system, are characterized with the high efficiency, simple target design, multiplex roles in gene knock-in/-out, and low cost, which has significantly boosted research across plant science and crop improvement by optimizing functional genes and creating genetic variations.  
THCAS and CBDAS genes were mapped to the loci with tight linkage and multiple copies of CBDAS- and THCAS- related sequences were cloned within the same region. Some of them contributed to the final chemotype, while the others were pseudogenes or partially functional alleles [50]. The CRISPR/Cas9 system enables the knockout of several homologous genes in one editing step. [PERSON] et al. (2017) edited eight targeted rice genes simultaneously and isolated mutants carrying these homozygous mutated alleles [51]. The high efficiencies in specific editing would be beneficial for Cannabis metabolic engineering.  
Gene editing reagents are delivered to plant cells by agroinfiltration, which can express a gene of interest rapidly. To achieve transient gene expression, agroinfiltration protocols for Cannabis have been optimized recently. Using optimized _Agrobacterium_-mediated transformation system, T-DNA of the corresponding plasmid was delivered into the hairy roots and tumors of Cannabis successfully [52]. VIGS-vectors were reported to be delivered to mature leaf with a 70% reduction of gene expression in magnesium chelatase subunit I [53]. Besides, the agroinfiltration system was developed and utilized to overexpression and silence genes with high efficacy in the aerial parts of Cannabis [31].  
The RNA interference (RNAi) tool is an alternative way to knockdown targeted gene expression, which enables the generation of novel cannabinoid profiles. The bottlenecks for the success are efficient delivery of dsRNA into the cell and instability of naked dsRNA in plants. Attempts regarding the RNAi spray or soaking protocols were reported from some companies, but details were not further disclosed. Most recently, [PERSON] et al. (2021) modulated cannabinoid biosynthesis genes using RNAi via agroinfiltration [54]. The vacuum infiltrated and transfected leaf segments with different RNAi constructs targeting _CBDAS_ and _THCAS_ genes, which showed significant downregulation quantified by real-time qPCR. Although RNAi technology provides an exceptional tool to study the gene function, the stable transformation of exogenous genes is still the barrier to enhance Cannabis phenotypes through genetic engineering.  
Despite several critical genes involved in cannabinoid biosynthesis being identified [47], the functions of these genes were not fully validated yet, mainly due to few reports of stable transformation in this species. Recently, several approaches were used in the transient transformation to introduce foreign DNA fragments, such as the vacuum infiltration or nanoparticle-based method [55]. In addition, the regeneration of transgenic plantlets from transformed cells is a time-consuming process, as well as varied between different Cannabis varieties [40]. Although there are some problems to solve, editing of THCAS is still an attractive target for researchers. Transgenic plants with zero THC production and increased production of other cannabinoids could be guaranteed simultaneously if the function of THCAS can be completely silenced.  
Recently, [PERSON] et al. (2021) reported the first generation of edited Cannabis plants by optimizing the _Agrobacteriumium_-mediated transformation method [44]. The shoot regeneration efficiency was increased through the integration of co-expression of DR chimera and CRISPR/Cas9 tools in the embryo hypocotyls of immature grains. The phytoene desaturase gene was edited to generate four seedlings with albino phenotype, which validated the stable integration of T-DNA in the Cannabis genome. Further optimization of this approach will help to release the potential of Cannabis.