ArticleGenes2025
Utilizing Target Sequences with Multiple Flanking Protospacer Adjacent Motif (PAM) Sites Reduces Off-Target Effects of the Cas9 Enzyme in Pineapple.
Article in Genes, 2025. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 1 paper.
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The abstract states no effect estimate the extractor could read, or names no intervention and outcome on the map, so this paper lights no cell and moves no belief. It is still indexed, cited and linked below.
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Who cites it
1 citing paper in PubMed.
- CRISPR-Cas technologies for precision genome editing in plants: advances, applications, and future perspectives.Frontiers in plant science · 2026Review
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Authors and funding
9 authors.
Funding
Abstract
BACKGROUND/
objectivesCRISPR-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.
methodsIn 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.
resultsSelecting 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 (
conclusionsWe 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.
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