Gene-editing by CRISPR/Cas systems has revolutionized plant biology by serving as a functional genomics tool. It has tremendously advanced plant breeding and crop improvement by accelerating the development of improved cultivars, creating genetic variability, and aiding in domestication of wild and orphan crops. Gene-editing is a rapidly evolving field. Several advancements include development of different Cas effectors with increased target range, efficacy, and enhanced capacity for precise DNA modifications with base editing and prime editing. The existing toolbox of various CRISPR reagents facilitate gene knockouts, targeted gene insertions, precise base substitutions, and multiplexing. However, the major challenge in plant genome-editing remains the efficient delivery of these reagents into plant cells. Plants have larger and more complex genome structures compared to other living systems due to the common occurrence of polyploidy and other genome re-arrangements. Further, rigid cell walls surrounding plant cells deter the entry of any foreign biomolecules. Unfortunately, genetic transformation to deliver gene-editing reagents has been established only in a limited number of plant species. Recently, there has been significant progress in CRISPR reagents delivery in plants. This review focuses on exploring these delivery mechanisms categorized into Agrobacterium-mediated delivery and breakthroughs, particle bombardment-based delivery of biomolecules and recent improvements, and protoplasts, a versatile system for gene-editing and regeneration in plants. The ultimate goal in plant gene-editing is to establish highly efficient and genotype-independent reagent delivery mechanisms for editing multiple targets simultaneously and achieve DNA-free gene-edited plants at scale.

Mitragyna speciosa ("kratom") is used as a natural remedy for pain and management of opioid dependence. The pharmacological properties of kratom have been linked to a complex mixture of monoterpene indole alkaloids, most notably mitragynine. Here, we report the central biosynthetic steps responsible for the scaffold formation of mitragynine and related corynanthe-type alkaloids. We illuminate the mechanistic basis by which the key stereogenic center of this scaffold is formed. These discoveries were leveraged for the enzymatic production of mitragynine, the C-20 epimer speciogynine, and fluorinated analogues.

The monoterpene indole alkaloid (MIA) mitragynine has garnered attention as a potential treatment for pain, opioid use disorder, and opioid withdrawal because of its combined pharmacology at opioid and adrenergic receptors in humans. This alkaloid is unique to Mitragyna speciosa (kratom), which accumulates over 50 MIAs and oxindole alkaloids in its leaves. Quantification of 10 targeted alkaloids from several tissue types and cultivars of  M. speciosa revealed that mitragynine accumulation was highest in leaves, followed by stipules and stems, but was absent, along with other alkaloids, in roots. While mitragynine is the predominant alkaloid in mature leaves, juvenile leaves accumulate higher amounts of corynantheidine and speciociliatine. Interestingly, corynantheidine has an inverse relationship with mitragynine accumulation throughout leaf development. Characterization of various cultivars of M. speciosa indicated altered alkaloidal profiles ranging from undetectable to high levels of mitragynine. DNA barcoding and phylogenetic analysis using ribosomal ITS sequences revealed polymorphisms leading M. speciosa cultivars having lower mitragynine content to group with other mitragyna species, suggesting interspecific hybridization events. Root transcriptome analysis of low- and high-mitragynine-producing cultivars indicated significant differences in gene expression and revealed allelic variation, further supporting that hybridization events may have impacted the alkaloid profile of M. speciosa.

Mitragyna speciosa (Kratom) is used as a natural remedy for pain and management of opioid dependence. The pharmacological properties of Kratom have been linked to a complex mixture of monoterpene indole alkaloids, most notably mitragynine. Here, we report the central biosynthetic steps responsible for the scaffold formation of mitragynine and related corynanthe-type alkaloids. We illuminate the mechanistic basis by which the key stereogenic centre of this scaffold is formed. These discoveries were leveraged for the enzymatic production of mitragynine, the C-20 epimer speciogynine, and a series of fluorinated analogues.

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