Background/Objectives: CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats)-associated protein 9 is now widely used in agriculture and medicine. Off-target effects can lead to unexpected results that may be harmful, and these effects are a common concern in both research and therapeutic applications. Methods: In this study, using pineapple as the gene-editing material, eighteen target sequences with varying numbers of PAM (Protospacer-Adjacent Motif) sites were used to construct gRNA vectors. Fifty mutant lines were generated for each target sequence, and the off-target rates were counted. Results: Selecting sequences with multiple flanking PAM sites as editing targets resulted in a lower off-target rate compared to those with a single PAM site. Target sequences with two 5′-NGG (“N” represents any nucleobase, followed by two guanine “G”) PAM sites at the 3′ end exhibited greater specificity and a higher probability of binding with the Cas9 protein than those only with one 5′-NGG PAM site at the 3′ end. Conversely, although the target sequence with a 5′-NAG PAM site (where “N” is any nucleobase, followed by adenine “A” and guanine “G”) adjacent and upstream of an NGG PAM site had a lower off-target rate compared to sequences with only an NGG PAM site, their off-target rates were still higher than those of sequences with two adjacent 5′-NAG PAM sites. Among the target sequences of pineapple mutant lines (AcACS1, AcOT5, AcCSPE6, AcPKG11A), more deletions than insertions were found. Conclusions: We found that target sequences with multiple flanking PAM sites are more likely to bind with the Cas9 protein and induce mutations. Selecting sequences with multiple flanking PAM sites as editing targets can reduce the off-target effects of the Cas9 enzyme in pineapple. These findings provide a foundation for improving off-target prediction and engineering CRISPR-Cas9 complexes for gene editing.

A large number of pineapple (Ananas comosus) fruits are discarded in China every year due to softening. However, the underlying molecular mechanism is still unknown. AcoPG3 (GenBank accession number: XM020243935), a pineapple gene of polygalacturonase, was found to be the major gene responsible for the softening of pineapple fruit. Fruit of AcoPG3-overexpressing tomato (Solanum lycopersicum var. Jingfan 101) begins to soften 9 days earlier than that transformed with a net vector. Fruit of AcoPG3-overexpressing pineapple (APG3-2) begins to soften 6 days earlier than that transformed with a net vector. Fruit of MPG3-1, a pineapple line in which AcoPG3 is mutated, begins to soften 31 days later than that transformed with a net vector. The sequence of polygalacturonase activities in fruit from the highest to the lowest was APG3-2, wild type, MPG3-1. The same sequences were also found in the liquid content of apoplast and the electrolyte leakage of pineapple pulp. The order of methyl-esterified pectin content in the pulp cell wall, from the highest to the lowest, was MPG3-1, wild type, and APG3-2. The same order was also observed for the contents of non-methyl-esterified homogalacturonan and rhamnogalacturonan-I in the pulp cell wall. The AcoPG3 mutation resulted in a decrease in polygalacturonase activity in pineapple fruit, decreasing the degradation of methyl-esterified pectin, non-methyl-esterified homogalacturonan, galactan and rhamnogalacturonan-I in the pulp cell wall. Fruit softening can be deferred, and the shelf life can be extended by mutating the AcoPG3 gene.

Mango is a vital fruit crop in tropical and subtropical regions, yet pests and diseases cause 30–70% production losses. Developing disease-resistant cultivars through transgenic methods could mitigate these issues. Agrobacterium-mediated callus transformation is a common genetic engineering approach, but successful transgenic mango plants from callus remain unreported due to severe browning and necrosis post-infection. We hypothesized that Agrobacterium-induced immune responses trigger callus death, hindering transformation. To improve efficiency, we engineered an Agrobacterium strain carrying the type III secretion system (T3SS) and effector gene AvrPto. Compared to controls, infected calluses exhibited elevated reactive oxygen species (ROS), along with up-regulated ROS-related, gallic acid biosynthesis, and defense genes. Calluses infected with T3SS-AvrPto-harboring Agrobacterium showed delayed browning and necrosis versus those infected with the empty vector (NV). The transformation rate with Agrobacterium (T3SS-AvrPto-EGFP) reached 1.6%, while Agrobacterium (NV-EGFP) failed entirely. These findings demonstrate that T3SS and AvrPto enhance mango transformation efficiency, offering a promising strategy for breeding multi-resistant varieties.

Mango industry played important roles in the economy of tropical and subtropical regions in China. Embryo abortion, flesh rotten, pericarp stain and so on always result in heavy loss. Transgenic breeding is a potential way for solving these problems. However, dependable protocol for mango transgene has not been reported until now. In this research, we found that after mango embryogenic callus were infected by Agrobacterium, most callus necrotized and died. No seedling was differentiated from the survived callus. Contents of 106 compounds in mango callus infected by Agrobacterium were found significantly different from those in control. The top 7 up-regulated compounds were gallic acid, phosphoenolpyruvic acid, dihydrotricetin, ubiquinone, dihydrokaempferol, D-erythrose-4-phosphate and cyanidin. The expressions of ROS-related genes, gallic acid synthesis genes, and defense related genes in callus infected by Agrobacterium were significantly higher than those in control. The growth of Agrobacterium on culturing plate pipetting gallic acid was inhibited. Mango callus infected by Agrobacterium transformed with type III secretion system gene cluster (T3SS) and effector gene AvrPto isolated from Pseudomonas syringae pv. tomato strain DC3000 brown and necrotize slower than those infected by Agrobacterium transformed with net vector (NV). The contents of ROS in callus in-fected by Agrobacterium (T3SS-AvrPto) were less than those in callus infected by Agrobacterium (NV). The expressions of genes participated in immune responses in mango callus infected by Agrobacterium (T3SS-AvrPto) were also less than those in mango callus infected by Agrobacterium (NV). The transformation rate of mango callus infected with Agrobacterium (T3SS-AvrPto-gfp) was 1.6 %. No callus infected with Agrobacterium (NV-gfp) was transformed successfully. The efficiency of mango transformation can be improved by constructing Agrobacterium strains harboring type III secretion system gene cluster and its effector gene.