Journal: Advanced Science

Article Title: CRISPR‐MI and scRNA‐Seq Reveal TREM2's Function in Monocyte Infiltration and Macrophage Apoptosis During Abdominal Aortic Aneurysm Development

doi: 10.1002/advs.202412227

Figure Lengend Snippet: Development of in vivo genome‐wide CRISPR Cas9 screen for monocyte infiltration (CRISPR‐MI). A) Flowchart of the adoptive transfer of BMDM labeled with VivoTrack680 fluorescent dye. B,C) The aortae from recipient mice in (A) were isolated and underwent in vivo imaging (n = 4). Scale bar = 1 cm. D) Flowchart of the adoptive transfer of BMDM from mTmG mice. E,F) The aortae from recipient mice in (D) were digested and analyzed by flow cytometry (n = 4). G, BMDMs were infected with lentivirus encoding EGFP (lenti‐EGFP, 50 MOI) 48 h after isolation with different adjuncts. 22 h after infection, cells were imaged by fluorescent microscopy. Scale bar = 100 µm. H) BMDMs in (G) were analyzed by flow cytometry to quantify EGFP+ cells (n = 3). I) BMDMs, were infected with lenti‐EGFP at different time points (0, 24, 48, 72 h) after isolation with or without F108. EGFP + cells were quantified by flow cytometry (n = 3). J) Rosa26‐Cas9 Tg mice were used as the BMDM donor, and the LentiGuide Cherry lentivirus vector was used to carry sgRNA. K) BMDMs from Rosa26‐Cas9 Tg mice were infected with lentivirus carrying different sgRNAs (50 MOI) 48 h after isolation. 72 h after infection, the genome editing efficiency was determined by CRISPR‐Seq. Data are presented as mean ± SEM. Unpaired t‐test was used for C, F. * , p < 0.05. CD, chow diet; WD, Western diet; AngII, angiotensin II; Tg, transgenic.

Article Snippet: We used the genome‐wide sgRNA library (Mouse Cherry Brie Pooled Library, from Addgene Pooled Library #170511) [ ] to perform the genome‐wide CRISPR knockout screen.

Techniques: In Vivo, Genome Wide, CRISPR, Adoptive Transfer Assay, Labeling, Isolation, In Vivo Imaging, Flow Cytometry, Infection, Microscopy, Plasmid Preparation, Western Blot, Transgenic Assay

Journal: Advanced Science

Article Title: CRISPR‐MI and scRNA‐Seq Reveal TREM2's Function in Monocyte Infiltration and Macrophage Apoptosis During Abdominal Aortic Aneurysm Development

doi: 10.1002/advs.202412227

Figure Lengend Snippet: CRISPR‐MI identified genes regulating monocyte infiltration in the murine AAA model. A) Flowchart of CRISPR‐MI. B) Number of sgRNA and corresponding genes identified by next‐generation sequencing (NGS) in BMDM donor and aorta, heart, and liver from recipient mice. C) Heat plot showing the Pearson correlation coefficient of gRNA counts from different organs and biological replicates. D) Principal component analysis gRNA counts from different organs and biological replicates. E) Scatter plots comparing gRNA targeting genes in aorta versus gDNA in donor BMDMs. Red dots indicated significantly enriched genes in the aorta. F) Scatter plot of overrepresented pathways of genes enriched in the aorta by GSEA.

Article Snippet: We used the genome‐wide sgRNA library (Mouse Cherry Brie Pooled Library, from Addgene Pooled Library #170511) [ ] to perform the genome‐wide CRISPR knockout screen.

Techniques: CRISPR, Next-Generation Sequencing

Journal: The Journal of cell biology

Article Title: Analysis of native Ist1 dynamics reveals multiple pools of ESCRT-III on endosomes

doi: 10.1083/jcb.202407013

Figure Lengend Snippet: (A and B) Cells natively co-expressing Ist1-HaloTag and either GFP-Rab5a (A) or GFP-Rab4b (B) were imaged live using confocal microscopy following labeling with the JF650-HaloTag ligand. Representative images are shown (left) with quantification showing the distribution of distances between structures labeled with each marker (right). Error bars represent the mean ± SEM ( n = 10 cells each; 3 biological replicates each). Bars, 5 μm; inset bars, 2 μm. (C and D) Cells natively expressing Ist1-HaloTag were fixed and stained using antibodies directed against Hrs (C) or CHMP1B (D) following labeling with the JF650-HaloTag ligand. Representative images are shown (left) with quantification highlighting the distribution of distances between structures labeled with each marker (right). Error bars represent the mean ± SEM ( n = 10 cells each; 3 biological replicates each). Bars, 5 μm; inset bars, 2 μm. (E) Cells natively co-expressing Ist1-HaloTag and labeled with the JF650-HaloTag ligand were exposed to Alexa Fluor 555-EGF for 9 min and imaged live using confocal microscopy. Representative images are shown (left) with quantification highlighting the distribution of distances between structures labeled with each marker (right). Error bars represent the mean ± SEM ( n = 10 cells each; 3 biological replicates each). Bar, 5 μm; inset bar, 2 μm.

Article Snippet: Immunofluorescence studies were conducted as described previously using the following antibodies (1 μg/ml final concentration each): CHMP1A (rabbit polyclonal; Proteintech 15761-AP), CHMP1B (rabbit polyclonal; Proteintech 14639-1-AP), CHMP4B (rabbit polyclonal; Proteintech 13683-1-AP), Snx15 (rabbit polyclonal; Proteintech 16049-1-AP), spastin (mouse monoclonal; Santa Cruz Biotechnology SC-81624), GAPDH (mouse monoclonal; Proteintech 60004-1-Ig), and Ist1 (rabbit polyclonal; a gift of Dr. Wesley Sundquist, University of Utah, Salt Lake City, UT, USA).

Techniques: Labeling, Expressing, Confocal Microscopy, Marker, Staining

Journal: The Journal of cell biology

Article Title: Analysis of native Ist1 dynamics reveals multiple pools of ESCRT-III on endosomes

doi: 10.1083/jcb.202407013

Figure Lengend Snippet: (A) Cells natively expressing Ist1-HaloTag were fixed and stained using antibodies directed against Snx15 following labeling with the JF650-HaloTag ligand. Representative images are shown (left) with quantification highlighting the distribution of distances between structures labeled with each marker (right). Error bars represent the mean ± SEM ( n = 10 cells each; 3 biological replicates each). Bar, 5 μm; inset bar, 2 μm. (B) Representative images of natively expressed Ist1-HaloTag in control cells and cells lacking Snx15 following labeling with the JF650-HaloTag ligand (left). Quantification of the number of Ist1-HaloTag–positive structures per unit area under each condition is also shown (right). Error bars represent the mean ± SEM ( n = 10 cells each; 3 biological replicates each). Bar, 5 μm. (C) Quantification of CHMP1B levels in control cells and cells depleted of CHMP1A, CHMP1B, or both CHMP1A and CHMP1B, based on immunoblot analysis ( n = 3). Error bars represent the mean ± SEM. ****P < 0.0001 and ***P < 0.001, as calculated using a one-way ANOVA and Tukey’s post hoc test. (D) Representative images of natively expressed Ist1-HaloTag in control cells treated with a scrambled siRNA (Mock) or siRNAs targeting CHMP1 isoforms following labeling with JF650-HaloTag ligand (left). Quantification of the number of Ist1-HaloTag–positive structures per unit area under the conditions shown is also provided (right). Error bars represent the mean ± SEM ( n = 10 cells each; 3 biological replicates each). ****P < 0.0001, as calculated using a two-sided t test. Bar, 5 μm. (E) Cells natively expressing Ist1-HaloTag were fixed and stained using antibodies directed against CHMP4B following labeling with JF650-HaloTag ligand and treatment with either a scrambled siRNA (Mock) or siRNAs targeting CHMP1A and CHMP1B. Representative images are shown (left) with quantification highlighting CHMP4B fluorescence intensity under each condition (right). Error bars represent the mean ± SEM ( n = 10 cells each; 3 biological replicates each). ****P < 0.0001, as calculated using a two-sided t test. Bar, 5 μm; inset bar, 2 μm.

Article Snippet: Immunofluorescence studies were conducted as described previously using the following antibodies (1 μg/ml final concentration each): CHMP1A (rabbit polyclonal; Proteintech 15761-AP), CHMP1B (rabbit polyclonal; Proteintech 14639-1-AP), CHMP4B (rabbit polyclonal; Proteintech 13683-1-AP), Snx15 (rabbit polyclonal; Proteintech 16049-1-AP), spastin (mouse monoclonal; Santa Cruz Biotechnology SC-81624), GAPDH (mouse monoclonal; Proteintech 60004-1-Ig), and Ist1 (rabbit polyclonal; a gift of Dr. Wesley Sundquist, University of Utah, Salt Lake City, UT, USA).

Techniques: Expressing, Staining, Labeling, Marker, Control, Western Blot, Fluorescence

Journal: Journal of Virology

Article Title: Three Amino Acid Mutations (F51L, T59A, and S390L) in the Capsid Protein of the Hepatitis E Virus Collectively Contribute to Virus Attenuation

doi: 10.1128/jvi.02278-10

Figure Lengend Snippet: FIG. 1. Immunofluorescence staining of a subclone of Huh7 cells transfected with similar amounts of capped full-length RNA transcripts. (A) pSHEV-3; (B) mutant rF51L; (C) mutant rT59A; (D) mutant rS390L; (E) mock transfection. Cells were stained for HEV ORF2 protein using chimpanzee 1313 anti-HEV immune serum.

Article Snippet: Briefly, a truncated recombinant ORF2 capsid protein containing the HEV immunodominant region from aa 452 to 617 (GenWay Biotech Inc., San Diego, CA) was used as the antigen.

Techniques: Staining, Transfection, Mutagenesis

Journal: Nature

Article Title: Transposase-assisted target-site integration for efficient plant genome engineering

doi: 10.1038/s41586-024-07613-8

Figure Lengend Snippet: The ORF1 and ORF2 proteins are expressed from the Pong transposon and bind the mPing element to form a transposition complex , . ORF1 is a Myb-like DNA binding protein that binds to at least 15 base pairs of the mPing terminal inverted repeat (TIR) sequence . ORF2 is the canonical transposase (TPase) with the DDE catalytic motif necessary for mPing excision and insertion , . The flanking nucleotides (TTA or TAA) that are immediately adjacent to the TIRs at the donor site are necessary for efficient mPing excision . The ORF1 and ORF2 proteins directly interact and are both required for mPing excision from the donor site , . After excision, the donor site is repaired by NHEJ using the microhomology of the staggered cut overhangs left by excision . This allows for very precise repair of the excision site, often reestablishing the coding frame of the mPing donor site , , . The transposition complex remains associated with the extra-chromosomal mPing DNA as it is also responsible for catalysing insertion.

Article Snippet: ORF1 (ORF1SC1 ONE ), ORF2 ( Pong TPase L418A, L420A) and Cas9 were supplied separately or as fusions cloned into pAG423 GAL (Addgene, 14149) and pAG425 GAL (Addgene, 14153) plasmids using standard gateway cloning. p426-SNR52p-gRNA.CAN1.Y-SUP4t (Addgene, 43803) was used to supply the CAN1 gRNA .

Techniques: Binding Assay, Sequencing

Journal: Nature

Article Title: Transposase-assisted target-site integration for efficient plant genome engineering

doi: 10.1038/s41586-024-07613-8

Figure Lengend Snippet: a . Diagram of fusion proteins tested. Twelve different transgenes were created and transformed into Arabidopsis. Cas9 and derivative proteins were fused either to the Pong transposase ORF1 or ORF2 protein coding regions. Both N- and C-terminal translational fusions were created using the G4S flexible linker. Three different versions of Cas9 were used: double-strand cleavage Cas9, the single stranded nickase, and the catalytically dead dCas9. When a functional transposase protein is generated by expression of ORF1 and ORF2, it excises mPing out of the 35S-GFP donor location in the Arabidopsis genome, producing fluorescence. b . Excision of the mPing TE from GFP restores the plant’s ability to generate fluorescence. Images of representative Arabidopsis seedlings showing GFP fluorescence for all 12 fusion proteins. The cotyledons are outlined with a white dashed line. Size bars represent 500 μm. A subset of this experiment is shown as Fig. . c . Excision of mPing assayed by PCR of pooled seedlings of the twelve different translational fusions from part a , and controls. The top band represents mPing within GFP (donor position), and the smaller band is generated upon mPing excision. The arrows indicate the pair of primers used for PCR. d . Sanger sequencing of the PCR product upon mPing excision. Grey bars behind the sequencing peaks represent quality scores for each base call.

Article Snippet: ORF1 (ORF1SC1 ONE ), ORF2 ( Pong TPase L418A, L420A) and Cas9 were supplied separately or as fusions cloned into pAG423 GAL (Addgene, 14149) and pAG425 GAL (Addgene, 14153) plasmids using standard gateway cloning. p426-SNR52p-gRNA.CAN1.Y-SUP4t (Addgene, 43803) was used to supply the CAN1 gRNA .

Techniques: Transformation Assay, Functional Assay, Generated, Expressing, Fluorescence, Sequencing

Journal: Nature

Article Title: Transposase-assisted target-site integration for efficient plant genome engineering

doi: 10.1038/s41586-024-07613-8

Figure Lengend Snippet: a , Excision of the mPing TE from a GFP reporter restores fluorescence. Arabidopsis seedlings were imaged; the cotyledons are outlined with a white dashed line. ‘ORF2–Cas9’ represents a translational fusion of these proteins. Scale bars, 500 μm. b , Excision of mPing assayed by PCR in pooled seedlings. The top band represents mPing within GFP (donor position), and the smaller band is generated after mPing excision. c , PCR primer design for detecting targeted insertions of mPing at the PDS3 locus. U and D are PDS3 primers that surround the CRISPR target site. R and L are mPing primers. TIR, terminal inverted repeat. d , PCR amplification analysis of targeted insertions of mPing at the PDS3 locus in pooled seedlings. At ADH1 was the PCR control. e , Sanger sequencing of the insertion junctions generated after mPing insertion into PDS3 . The light grey bars behind the DNA-sequencing peaks represent quality scores for each base call. Bases highlighted in red are mismatches compared with the reference sequence. The flanking TTA sequence that comes with mPing from the donor site is annotated. f , Model of targeted insertion of mPing at the PDS3 locus. A functional ORF2–Cas9 fusion protein excises mPing out of the 35S–GFP donor site, cuts the PDS3 gene guided by the gRNA and mPing is inserted into the PDS3 target site. The diagram in f was created using BioRender.

Article Snippet: ORF1 (ORF1SC1 ONE ), ORF2 ( Pong TPase L418A, L420A) and Cas9 were supplied separately or as fusions cloned into pAG423 GAL (Addgene, 14149) and pAG425 GAL (Addgene, 14153) plasmids using standard gateway cloning. p426-SNR52p-gRNA.CAN1.Y-SUP4t (Addgene, 43803) was used to supply the CAN1 gRNA .

Techniques: Fluorescence, Generated, CRISPR, Amplification, Control, Sequencing, DNA Sequencing, Functional Assay

Journal: Nature

Article Title: Transposase-assisted target-site integration for efficient plant genome engineering

doi: 10.1038/s41586-024-07613-8

Figure Lengend Snippet: a . High Resolution Melt (HRM) analysis to test gRNA efficiency. Mutations created by Cas9 were detected for genomic loci PDS3 , ADH1 , or the region upstream of ACT8 . PCR product melting dynamics differed between the WT plants (pink) and Cas9 positive control lines with the indicated gRNA (purple). The melting temperature difference is caused by the generation of short indels and SNPs upon Cas9 cleavage and repair by NHEJ, verifying that all three gRNAs are functional in Arabidopsis plants. b . Representative pds3 homozygous mutant white seedling from plants with the catalytically-active Cas9 fusion protein. c . The ratio of white pds3 T2 seedlings for all Cas9 fusion proteins tested. d . Efficiency of ORF2-Cas9 fusion proteins in yeast. mPing excision frequency (blue, left Y-axis) and Cas9 mutation frequency (orange, right Y-axis) measured for unfused and fused ORF2 and Cas9 with three different protein-protein linker sequences. The ORF2 protein’s C-terminus is fused to Cas9 via a 1xG4S, 3xG4S or 16AA linker. mPing excision was measured as the number of ADE2 revertant colonies due to mPing excision per million cells. The average and standard deviation for multiple biological replicates (n = 6) are shown (blue). Cas9 mutation frequency was measured by testing the gRNA-targeted canavanine resistance of the ADE2 revertant colonies (n = 48). This experiment was performed two times independently to ensure reproducibility.

Article Snippet: ORF1 (ORF1SC1 ONE ), ORF2 ( Pong TPase L418A, L420A) and Cas9 were supplied separately or as fusions cloned into pAG423 GAL (Addgene, 14149) and pAG425 GAL (Addgene, 14153) plasmids using standard gateway cloning. p426-SNR52p-gRNA.CAN1.Y-SUP4t (Addgene, 43803) was used to supply the CAN1 gRNA .

Techniques: Positive Control, Functional Assay, Mutagenesis, Standard Deviation

Journal: Nature

Article Title: Transposase-assisted target-site integration for efficient plant genome engineering

doi: 10.1038/s41586-024-07613-8

Figure Lengend Snippet: a . PCR assay as described in Fig. for the 12 fusion proteins generated in Extended Data Fig. and controls. PCR negative controls include a line lacking the Cas9, ORF1 and ORF2 proteins (-ORF1,-ORF2), a line with ORF1 and ORF2 but no Cas9 (+ORF1, + ORF2), and a no-template DNA PCR reaction (water). Among the 12 fusion proteins, only ORF2-Cas9 displays the correct size band for targeted insertions. Insertions were verified by Sanger sequencing of the PCR products. b . Western blots using the Cas9 and Actin11 antibodies, showing that the ORF2-Cas9 and ORF2-dCas9 proteins are expressed in transgenic plants as full-length fusion proteins. Upper panel shows that both ORF2-Cas9 and ORF2-dCas9 have the expected size of ~216 kDa (Cas9 is 150 kDa and ORF2 is 66 kDa). Lower panel compares the size of the unfused Cas9 with the ORF2-Cas9 fusion protein. Raw images of the Westerns are shown in Supplementary Fig. . c . Sanger sequencing of the junctions of targeted integration events into the PDS3 gene. PCR products from panel a were cloned into the pCR4_TOPO TA vector and 9 individual colonies were sequenced per PCR reaction. The triangle represents where Cas9 cuts in the gRNA target sequence. The flanking TTA/TAA sequence is present at some insertion junctions and absent in others. The PDS3 sequence is shown in blue, the gRNA target site is highlighted in grey, mPing is shown as red text, and the flanking TTA/TAA sequences are shown in black text.

Article Snippet: ORF1 (ORF1SC1 ONE ), ORF2 ( Pong TPase L418A, L420A) and Cas9 were supplied separately or as fusions cloned into pAG423 GAL (Addgene, 14149) and pAG425 GAL (Addgene, 14153) plasmids using standard gateway cloning. p426-SNR52p-gRNA.CAN1.Y-SUP4t (Addgene, 43803) was used to supply the CAN1 gRNA .

Techniques: Generated, Sequencing, Western Blot, Transgenic Assay, Clone Assay, Plasmid Preparation

Journal: Nature

Article Title: Transposase-assisted target-site integration for efficient plant genome engineering

doi: 10.1038/s41586-024-07613-8

Figure Lengend Snippet: a . Diagram of the multiplexed vector cassette that generates two distinct Cas12a gRNAs, one that targets ADH1 and one that targets upstream of ACT8 . b . PCR assay to detect excision of mPing generated by functional ORF1 and ORF2 proteins. Fusing these proteins to Cas12a does not stop excision activity. c . Diagram of the four PCR reactions to detect targeted insertions into ADH1 . Arrows indicate primers used to detect targeted insertions: U + L, D + R, U + R, D + L. d . Diagram of the four PCR reactions to detect targeted insertions into the region upstream of ACT8 . e . PCR assay to detect targeted insertion of mPing into ADH1 . Targeted insertions are detected for both protein fusions to Cas12a as well as in the unfused configuration. f . PCR assay to detect targeted insertion of mPing into the region upstream of ACT8 . Targeted insertions are detected for both protein fusions to Cas12a as well as in the unfused configuration. g . Sanger sequencing of a mPing targeted insertion into ADH1 mediated by Cas12a cleavage. h . Sanger sequencing of a mPing targeted insertion into the region upstream of ACT8 mediated by Cas12a cleavage.

Article Snippet: ORF1 (ORF1SC1 ONE ), ORF2 ( Pong TPase L418A, L420A) and Cas9 were supplied separately or as fusions cloned into pAG423 GAL (Addgene, 14149) and pAG425 GAL (Addgene, 14153) plasmids using standard gateway cloning. p426-SNR52p-gRNA.CAN1.Y-SUP4t (Addgene, 43803) was used to supply the CAN1 gRNA .

Techniques: Plasmid Preparation, Generated, Functional Assay, Activity Assay, Sequencing

Journal: Nature

Article Title: Transposase-assisted target-site integration for efficient plant genome engineering

doi: 10.1038/s41586-024-07613-8

Figure Lengend Snippet: a , The dashed line marks the Cas9 cleavage site on the PDS3 target sequence before TE integration. Insertion sites are assayed at the 5′ (relative to PDS3 ) (left) or 3′ junction (right) of mPing insertions. The ‘0’ site marks insertion at the exact Cas9-cleavage site. PAM, protospacer adjacent motif. b , Sequencing analysis of targeted insertion junction points mapped to mPing indicates how much of the mPing element was delivered to the targeted insertion site. The x axis shows the nucleotide position along the mPing element. The break in the x axis represents the interior of mPing that was not assayed. c , Model mPing excision by an ORF2 transposase-generated staggered break, blunt cleavage of the target site by Cas9, then integration, repair and resolution of mPing at the target site by NHEJ. The diagram was created using BioRender. d , Nucleotide (nt) variation at the junction of mPing insertions into PDS3 . The precision of each nucleotide at the insertion site was determined on the 5′ junction (left) or 3′ junction (right). The size of the circle represents the percentage of reads in which that nucleotide is as expected ( y = 0), has an insertion ( y ≥ 1) or deletion ( y ≤ −1). The number of SNPs at the insertion site is shown at the top of the y axis. Pearson’s χ 2 tests were used to test the statistical significance of the difference in polymorphism between the two protein configurations. e , mPing insertion sites in pooled seedlings. The Arabidopsis nuclear genome is displayed on the x axis. The PDS3 target site is shown with an arrow and red datapoint. The scale of each y axis was determined by the maximum datapoint. A dashed line at 10,000 reads per million (RPM) is shown for each sample. Chr., chromosome; Rep., distinct biological replicates; WT, wild type. f , Quantitative analysis of the number and read support of free-transposition sites in pooled replicates for each genotype.

Article Snippet: ORF1 (ORF1SC1 ONE ), ORF2 ( Pong TPase L418A, L420A) and Cas9 were supplied separately or as fusions cloned into pAG423 GAL (Addgene, 14149) and pAG425 GAL (Addgene, 14153) plasmids using standard gateway cloning. p426-SNR52p-gRNA.CAN1.Y-SUP4t (Addgene, 43803) was used to supply the CAN1 gRNA .

Techniques: Sequencing, Generated

Journal: Nature

Article Title: Transposase-assisted target-site integration for efficient plant genome engineering

doi: 10.1038/s41586-024-07613-8

Figure Lengend Snippet: a . Amplicon deep sequencing of enzymatic-converted DNA methylation patterns. The average methylation level across the amplicon is shown for each cytosine context (CG, CHG, CHH (H = A,T,C)), with 95% confidence intervals calculated using the Wilson score interval method. On the left is the ADH1 insertion site before mPing insertion, broken where mPing will insert and either side of the insertion site is analysed separately. On the right is the methylation after mPing insertion. A dash line denotes the background non-conversion rate of the enzymatic reaction determined for each sample by sequencing an unmethylated region of the genome. This conversion percentage is also listed below each genotype. Biological replicates are denoted as “Rep 1” vs. “Rep 2”. n= the number of total cytosines assayed for each amplicon. b . Map of a single vector containing the mPing donor element, the gRNA and protein machinery required to obtain mPing targeted insertions (+ORF1, + ORF2-Cas9). c . PCR-based targeted insertion assay (as in Fig. ) in pooled seedlings using the one-component transgene system. Targeted insertions are detected in each reaction. d . Sanger sequencing of the junctions of a targeted insertion event in the Arabidopsis PDS3 gene generated from the single vector one-component transgene system shown in panel b .

Article Snippet: ORF1 (ORF1SC1 ONE ), ORF2 ( Pong TPase L418A, L420A) and Cas9 were supplied separately or as fusions cloned into pAG423 GAL (Addgene, 14149) and pAG425 GAL (Addgene, 14153) plasmids using standard gateway cloning. p426-SNR52p-gRNA.CAN1.Y-SUP4t (Addgene, 43803) was used to supply the CAN1 gRNA .

Techniques: Amplification, Sequencing, DNA Methylation Assay, Methylation, Plasmid Preparation, Generated

Journal: Nature

Article Title: Transposase-assisted target-site integration for efficient plant genome engineering

doi: 10.1038/s41586-024-07613-8

Figure Lengend Snippet: a . Transgene design and PCR primer placement for “PCR1” to “PCR6” used to genotype for the presence of the mPing _bar/gRNA/ORF1/ORF2/Cas9 parent transgene in R0 transformed soybean plants. b . PCR assay to genotype for the presence of the mPing _bar/gRNA/ORF1/ORF2/Cas9 parent transgene in R0 transformed soybean plants. “PCR1” detects both the original mPing _bar donor and its excision product. “PCR2” to “PCR6” detect different locations on the mPing _bar/gRNA/ORF1/ORF2/Cas9 transgene. GmLe1 is a control gene. The combined data demonstrates that R0 plant #1 has the full transgene in the genome, plant #2 has a partial transgene insertion that lacks the mPing _bar donor site, and plant #3 does not have the mPing _bar/gRNA/ORF1/ORF2/Cas9 transgene.

Article Snippet: ORF1 (ORF1SC1 ONE ), ORF2 ( Pong TPase L418A, L420A) and Cas9 were supplied separately or as fusions cloned into pAG423 GAL (Addgene, 14149) and pAG425 GAL (Addgene, 14153) plasmids using standard gateway cloning. p426-SNR52p-gRNA.CAN1.Y-SUP4t (Addgene, 43803) was used to supply the CAN1 gRNA .

Techniques: Transformation Assay, Control