grna expression cassettes  (GenScript corporation)

Journal: Frontiers in Plant Science

Article Title: dCas9-SPO11-1 locally stimulates meiotic recombination in rice

doi: 10.3389/fpls.2025.1580225

Figure Lengend Snippet: Induction of double-strand breaks by dCas9-SPO11-1 and gRNAs transgenes. (A) Schematic representation of hypothetical DNA repair following dCas9-SPO11-1-induced Double Strand Break (DSB) at prophase of meiosis. Left: dCas9 (gray), fused to the SPO11-1 subunit (light blue), binds the target site and recruits the other components of the transesterase Spo11 complex, including SPO11-2 (orange) and MTOPVIB (grey). Right: Recombinational repair of Spo11-dependent DSB in meiosis: Spo11-dependent DSB formation on one of the homologous chromosomes (dark) and ssDNA resection allow strand invasion of the homologous chromosome. Progression and resolution of the recombination intermediates yield recombined non-crossover (NCO) and crossover (CO) molecules adapted from Yelina et al. (2022) . (B) Structure of the T-DNA constructs co-introduced in rice. Up: T-DNA for dCas9-OsSPO11-1 expression. Down: T-DNA for transcription of the multiplexed tRNA:gRNA scaffold, which once managed by the natural tRNA processing system is supposed to release 11 gRNAs targeting either Chr.7 or Chr.9 regions (gRNAs and tRNAs are shown as purple and black boxes, respectively).

Article Snippet: The gRNA expression cassettes (11 and 6 gRNAs) based on tRNA:gRNA processing system ( ) were synthesized by Genscript and inserted into a pCambia2300 using Fok I and Bsa I restriction sites.

Techniques: Construct, Expressing

Journal: Frontiers in Plant Science

Article Title: dCas9-SPO11-1 locally stimulates meiotic recombination in rice

doi: 10.3389/fpls.2025.1580225

Figure Lengend Snippet: Molecular and functional characterization of the transgenic plants 7a and 7b. (A) Schematic representation of the recombination context of the targeted Chr.7 region established by whole genome sequencing of a 379 F2 progeny population of KalingaIII/Kitaake (Unpublished). The position of the dPCR (7#1, 7#2, 7#3 and 7#4) and Kasp (Z1 to Z4) markers with their coordinates on the Kitaake genome are detailed on the zoom. Kasp markers ZA to ZD were only used to genotype plant 7a offsprings ( Figure 5 ). The green bar represents the area containing the recombinant identified in the progeny. The sequence of the polymorphic markers and 11 gRNAs (pink bars) are given in Supplementary Tables S4 , S9 . The 11 gRNAs target the 5’ promoter region of the expansin-like A3 gene ( OsKitaake07g138900.1 ). (B) RT-qPCR quantification of the dCas9 and SPO11-1 transcripts in leaf tissues of the transgenic KalingaIII/Kitaake plants 7a and 7b relative to OsKitaake07g010600.1 Expressed protein gene as reference (n=2). Values follow a Log10 scale. (C) dCas9-SPO11-1 accumulation in leaf tissues of plants 7a and 7b revealed by western blot analysis using an anti-V5 antibody. Expected gel migration positions of dCas9 (161 kDa) and dCas9-SPO11-1 (206 kDa) are pointed by blue and red arrowheads, respectively. (D) RT-qPCR accumulation of the gRNAs in leaf tissues of transgenic plants 7a and 7b. Values follow a Log10 scale and use the OsKitaake07g010600.1 gene as a reference (n=2). (E) ChIP-qPCR of the targeted chromosome 7 region. Chromatin DNA of WT KalingaIII and Kitaake parents and independent transgenic KalingaIII/Kitaake hybrid plants 7a and 7b were immuno-precipitated using the anti-v5 antibody and quantified by qPCR. The sonicated fragments are in the range of 200-900 bp. Target sequences of the 11 gRNA scaffold are shown in purple. The qPCR amplified regions (area 1 and 2) are indicated by red arrowheads. The enrichment values are normalized by the input (10% of total chromatin). The 3’ region of the ubiquitously expressed gene OsKitaake06g078500.1 residing on Chr.6 was used as a non-target control (n=3).

Article Snippet: The gRNA expression cassettes (11 and 6 gRNAs) based on tRNA:gRNA processing system ( ) were synthesized by Genscript and inserted into a pCambia2300 using Fok I and Bsa I restriction sites.

Techniques: Functional Assay, Transgenic Assay, Sequencing, Recombinant, Quantitative RT-PCR, Western Blot, Migration, ChIP-qPCR, Sonication, Amplification, Control

Journal: Frontiers in Plant Science

Article Title: dCas9-SPO11-1 locally stimulates meiotic recombination in rice

doi: 10.3389/fpls.2025.1580225

Figure Lengend Snippet: Molecular and functional characterization of the transgenic offspring plants 7a1 and 7a2. (A) RT-qPCR quantification of the dCas9 and SPO11-1 transcripts in leaf tissues of the transgenic plants 7a1 and 7a2 relative to OsKitaake07g010600.1 Expressed protein gene as reference (n=3). Values follow a Log10 scale. (B) RT-qPCR accumulation of the gRNAs in leaf tissues of transgenic plants 7a1 and 7a2. Values follow a Log10 scale and use the Kitaake Os07g010600.1 gene as a reference. (C) dCas9-SPO11-1 accumulation in leaf tissues of plants 7a1 and 7a2 revealed by western blot analysis using an anti-V5 antibody. Expected gel migration positions of dCas9 (161 kDa) and dCas9-SPO11-1 (206 kDa) are pointed by blue and red arrowheads, respectively. (D) ChIP-qPCR of the targeted chromosome 7 region. Chromatin DNA of WT hybrid and offspring of the hybrid plants 7a1 and 7a2 were immuno-precipitated using the anti-v5 antibody and quantified by qPCR. The sonicated fragments are in the range of 200-900 bp. Target sequences of the 11 gRNA scaffold are shown in purple. The qPCR amplified regions (area 1 and 2) are indicated by red arrowheads. The enrichment values are normalized by the input (10% of total chromatin). The 3’ region of the ubiquitously expressed gene OsKitaake06g078500.1 residing on Chr.6 was used as a non-target control (n=3). (E) Recombination frequencies over the target region of chromosome 7 deduced from genotyping nuclei WT and dCas9-SPO11-1 plant 7a1 or 7a2 pollen. Values are corrected by removing the background (Methods). Frequencies are compared over the whole region and then over each interval. The detailed data set is reported in Table 1 . The corrected number of recombinant nuclei is indicated under recombination rate values. Fisher exact test was performed between WT and plant 7a1 or 7a2 (p values are shown, p>0.05*, ns, not significant).

Article Snippet: The gRNA expression cassettes (11 and 6 gRNAs) based on tRNA:gRNA processing system ( ) were synthesized by Genscript and inserted into a pCambia2300 using Fok I and Bsa I restriction sites.

Techniques: Functional Assay, Transgenic Assay, Quantitative RT-PCR, Western Blot, Migration, ChIP-qPCR, Sonication, Amplification, Control, Recombinant