Fig 1.
Scheme of CNT-Mediated Gene Knockout in Plants.
Ten-day-old plants were infiltrated with CNTs and CDs containing CRISPR-Cas reagents, followed by sample collection at 15-days post-infiltration (dpi). Edited and control plants had their DNA isolated, PCR-amplified, and analyzed by gel electrophoresis. Sequencing confirmed the presence of mutations, and phenotypic changes served as validation for the knockout (created with BioRender.com).
Fig 2.
Histochemical detection of GUS expression in cowpea leaves infiltrated with carbon nanotubes (CNTs) and plasmid DNA (pDNA) solution.
Cowpea leaves were treated with a mixture of CNTs and pDNA at a 1:1 ratio (500 ng of CNTs to 500 ng of pDNA). After a 72-hour incubation period, GUSPlus enzymatic activity was assessed using a histochemical assay following standard procedures. Panel (A) shows cowpea leaves infiltrated with water as the negative control. GUS expression was observed in cowpea leaves infiltrated with the CNT-pDNA mixture, utilizing both binary (Panels B-C) and non-binary plasmids (Panels D-E). The exposure time was set to 100 ms.
Fig 3.
Histochemical detection of GUS expression in cowpea leaves infiltrated with carbon dots (CDs) and plasmid DNA (pDNA) solution.
Cowpea leaves were treated with a mixture of CDs and pDNA at a 1:1 ratio (500 ng CDs: 500 ng pDNA). After a 72-hour incubation period, GUSPlus enzymatic activity was assessed using a histochemical assay according to standard procedures. Panel (A) shows cowpea leaves infiltrated with water as a negative control. GUS expression was observed in cowpea leaves infiltrated with the CNT-pDNA mixture, using both binary (B-C) and non-binary plasmids (D-E). The exposure time was set to 100 ms.
Fig 4.
Phenotypes of cowpea leaves infiltrated with single-walled carbon nanotube (SWCNT) or carbon dot (CD) plasmid vector mixtures.
Cowpea leaves were infiltrated with a 1:1 mixture of SWCNTs or CDs and VgPDS plasmid DNA solution (a non-binary vector) at a concentration of 500 ng each. After 10 days of incubation, white spots appeared on both the front and back sides of the infiltrated leaves. Genomic DNA was then extracted from these leaves for PCR screening, while non-infiltrated leaves served as negative controls. The exposure time for the infiltration was 100 ms.
Fig 5.
Mutations of the PDS target region identified from cowpea leaves infiltrated with SWCNT-PEI-VgPDS or CD-PEI-VgPDS plasmid vector mixtures.
(A) Schematic illustration of the PDS gene (VgPDS; Vigun01g249800) structure in cowpea, highlighting the translated regions and untranslated regions (UTRs). Translated exons are marked in blue, while UTRs are shown in grey. The positions of four selected guide RNA (gRNA) target sites are indicated by orange arrows for the multiplex editing of VgPDS gene, with each gRNA sequence provided and protospacer adjacent motif (PAM) sequences underlined. Primer sites (F1 and R1) used to detect deletions within the targeted region are represented by arrowheads. The schematic includes the lengths of the entire gene, the targeted region, and the expected PCR amplicon from the wild-type locus. (B) Agarose gel electrophoresis analysis of the VgPDS target region amplified with the F2 and R2 primers from genomic DNA extracted from cowpea leaves (wild type (WT) allele amplicon size: 1,062 bp) infiltrated with SWCNT-PEI-VgPDS or CD-PEI-VgPDS plasmid vector mixtures. The boxed region in the gel image represents the excised fragment subsequently recovered from the gel. The purified DNA was cloned into TOPO vectors, and five plasmid clones per sample were sequenced by Sanger sequencing. The number of plasmids with identified deletions per sample is indicated below the respective lanes in the gel image. The negative clones had unedited sequences similar to the WT allele of VgPDS gene. L: Thermo Scientific GeneRuler 1 kb Plus DNA Ladder (Thermo Fisher Scientific, Cat. No. SM1331). NC: negative control. (C) Alignment of DNA sequences from deletion events identified using different primer sets on DNA extracted from leaves infiltrated with either SWCNTs-PEI-plasmid or CDs-PEI-plasmid vectors targeting the PDS gene, which displayed observable phenotypes. The reference sequence of the unmodified VgPDS locus is displayed at the top, followed by sequences of three representative clones showing deletions. The corresponding leaf for the origin of the amplicon/clone is also indicated in brackets. The sizes of the control site and each deletion are provided. Targeted gRNA sequences are underlined, and PAM sequences are highlighted in red.