Journal: G3: Genes|Genomes|Genetics

Article Title: Alfalfa ( Medicago sativa L.) pho2 mutant plants hyperaccumulate phosphate

doi: 10.1093/g3journal/jkac096

Figure Lengend Snippet: Schematic representation of the PHO2-B and PHO2-C targets and the reagent components. a) Three gRNA targets were designed for each of the 4 PHO2-B and PHO2-C haplotypes with 2 targets in the first exon and the third target in exon 6 for PHO2-B and exon 3 for PHO2-C. b) The reagent components include; the binary vector backbone [pTRANS_220] containing a 35S: nptII selectable marker for kanamycin selection, a Cas9 [pMOD_A] module, a guide RNA [pMOD_B] module that can utilize either the Csy4 or tRNA splicing mechanism for the release of multiple gRNAs, and the rolD: TREX2 exonuclease, in a [pMOD_C] module. All 3 modules are assembled into the binary vector by AarI-mediated golden gate reaction. c) The completed reagent is sequence confirmed and transformed into the Agrobacterium strain LBA4404 for alfalfa leaf explant transformation (Samac and Austin-Phillips 2006). The following nomenclature was used to indicate the type of reagent used for the gene editing. For example, “PhoM#” and “PhocM#” refer to either the pDIRECT or pTRANS reagents with the “c” indicating Csy4 splicing system. The “t” in “PhotM#” refers to the pTRANS reagent with the tRNA splicing system. Both the pTRANS reagents (PhocM# and PhotM#) harbor the TREX2 exonuclease cassette.

Article Snippet: Target guides and Cas9 components were cloned into the binary vectors pDIRECT_22C (Addgene #91135) or pTRANS_220 (Adgene #91113), and included the 35S:Cas9:tHSP cassette, pMOD_A0101 (Addgene #90998), the CmYLCV:tRNA, or CmYLCV:Csy4 guide RNA module pMOD_B2303 (Addgene #91068), or pMOD_B2103 (Addgene #91060), respectively, and the rol D:TREX2 exonuclease module pMOD_C2911 (Addgene #161764).

Techniques: Plasmid Preparation, Marker, Selection, Sequencing, Transformation Assay

Journal: G3: Genes|Genomes|Genetics

Article Title: Alfalfa ( Medicago sativa L.) pho2 mutant plants hyperaccumulate phosphate

doi: 10.1093/g3journal/jkac096

Figure Lengend Snippet: The validation PhotM65 mutant plants. a) Schematic representation of the PCR assays used to validate mutant haplotypes. The blue triangle represents the reagent target sites and the red and gray boxes indicate deleted or inverted DNA sequences, respectively. The green arrows represent the approximate primer locations used to generate amplicons. The “E,” “X,” and the “NlaIV” indicate the location of the Eco130i, XhoI, and NlaIV restriction sites used to genotype haplotypes. b) Sequence confirmation of the haplotypes was carried out using sequence data from the LAA analysis as well as from cloning and sequencing assays using haplotype specific amplicons. The bold red and black text represents the PAM and target guide RNA sites for each reagent, respectively, and the blue text and lime green highlights indicate inverted or deleted DNA sequence. c) Gel images of mutant and wild-type haplotype-specific amplicons from PhotM65 T0 and T1 plants. The absence of a haplotype specific amplicon indicates the segregation of the mutant haplotype in respective plants. Amplicons for the Cas9 and the nptII selectable marker were used to identify the presence or absence of reagent T-DNA in the mutant plants.

Article Snippet: Target guides and Cas9 components were cloned into the binary vectors pDIRECT_22C (Addgene #91135) or pTRANS_220 (Adgene #91113), and included the 35S:Cas9:tHSP cassette, pMOD_A0101 (Addgene #90998), the CmYLCV:tRNA, or CmYLCV:Csy4 guide RNA module pMOD_B2303 (Addgene #91068), or pMOD_B2103 (Addgene #91060), respectively, and the rol D:TREX2 exonuclease module pMOD_C2911 (Addgene #161764).

Techniques: Mutagenesis, Sequencing, Clone Assay, Amplification, Marker

Journal: Frontiers in Plant Science

Article Title: Evaluation of Methods to Assess in vivo Activity of Engineered Genome-Editing Nucleases in Protoplasts

doi: 10.3389/fpls.2019.00110

Figure Lengend Snippet: Constructs for targeted mutagenesis of ALS1 and EPSPS1 loci in potato protoplasts using CRISPR/Cas9 and TALEN reagents. (A) Map of potato target gene Acetolactate synthase1 ( ALS1 ) and (B) 3-Phosphoshikimate 1-carboxyvinyltransferase ( EPSPS1 ) used in the study. The sequence of the target site is shown with sgRNA spacer in red, PAM in blue, TALEN binding sites are underlined, and restriction enzyme sites are in bold italicized. (C) Structure of the constructs used for expressing sgRNAs and TALENs co-expressing green fluorescent protein (GFP) for both target genes. PCaMV35S, cauliflower mosaic virus 35S promoter, AtCas9, Arabidopsis codon optimized Cas9 nuclease; tHSP, Arabidopsis heat shock protein 18.2 terminator; AtU6, Arabidopsis U6 promoter; sgRNA, single guide RNA ; PFMV34S, figwort mosaic virus 34S promoter; GFP, green fluorescent protein; tE9, pea ribulose bisphosphate carboxylase small subunit terminator; P2A, ribosomal skipping sequence.

Article Snippet: Module A vector, pMOD_A0101 (Addgene #90998) was used for AtCas9 expression cassette. eCas9(1.1) and Cas9-HF1 (pMOD_A6101 and pMOD_A6201, respectively) were made by amplification of the vector sequence from pMOD_A0101 for the backbone and most part of AtCas9 and sequence specific for Cas9 variants, was synthesized and fragments joined via Gibson assembly ( ).

Techniques: Construct, Mutagenesis, CRISPR, Sequencing, Binding Assay, Expressing, TALENs

Journal: Frontiers in Plant Science

Article Title: Evaluation of Methods to Assess in vivo Activity of Engineered Genome-Editing Nucleases in Protoplasts

doi: 10.3389/fpls.2019.00110

Figure Lengend Snippet: Determination of protoplast viability and transformation efficiency of genome-editing nucleases targeting ALS1 . (a) Protoplasts isolated from in vitro grown potato leaves. (b) Protoplasts stained with Evans blue to test viability. Arrows indicate defective protoplast cells into which the dye permeated. Scale bar = 20 μ. (c) Transformation efficiencies are compared between the CRISPR/Cas9 and TALEN plasmid constructs targeting ALS1 . Each bar represents the % mean value of three independent transformations, each with five technical replicates ± Standard deviation, Student’s t -tests ( P ≤ 0.05). (d–f) Confocal laser scanning microscope images showing, merged images of GFP fluorescence (green) and bright field (gray). CRISPR/Cas9 targeting ALS1 (d) , TALENs targeting ALS1 (e) , and no plasmid control (f) are shown. Scale bar = 100 μ.

Article Snippet: Module A vector, pMOD_A0101 (Addgene #90998) was used for AtCas9 expression cassette. eCas9(1.1) and Cas9-HF1 (pMOD_A6101 and pMOD_A6201, respectively) were made by amplification of the vector sequence from pMOD_A0101 for the backbone and most part of AtCas9 and sequence specific for Cas9 variants, was synthesized and fragments joined via Gibson assembly ( ).

Techniques: Transformation Assay, Isolation, In Vitro, Staining, CRISPR, Plasmid Preparation, Construct, Standard Deviation, Laser-Scanning Microscopy, Fluorescence, TALENs

Journal: Frontiers in Plant Science

Article Title: Evaluation of Methods to Assess in vivo Activity of Engineered Genome-Editing Nucleases in Protoplasts

doi: 10.3389/fpls.2019.00110

Figure Lengend Snippet: Targeted mutagenesis of ALS1 and EPSPS1 detected by PCR/RE and T7EI assays. Three replications of CRISPR/Cas9 and TALENs are used in the assays. (A) Schematic of PCR/restriction enzyme digestion assay (PCR/RE) and resulting gel images of (B) ALS1 and (C) EPSPS1 in which the amplicons were digested with BslI and XcmI , respectively. Mutant bands resistant to digestion in (B,C) are indicated by red arrow and were cloned for Sanger sequencing. (D) Schematic of T7 Endonuclease I assay (T7EI) and resulting gel images of (E) ALS1 and (F) EPSPS1 . Arrows indicate expected cleavage products from targeted mutagenesis and indel percentage for each sample is indicated below. Sanger sequences of the targeted mutagenesis site of (G) ALS1 and (H) EPSPS1 . ∗ Denote the bands present in mutagenized samples only. Protospacer adjacent motif (PAM) is indicated in red. 100 bp NEB ladder; WT/D, wild type digested; WT/U, wild type un-digested.

Article Snippet: Module A vector, pMOD_A0101 (Addgene #90998) was used for AtCas9 expression cassette. eCas9(1.1) and Cas9-HF1 (pMOD_A6101 and pMOD_A6201, respectively) were made by amplification of the vector sequence from pMOD_A0101 for the backbone and most part of AtCas9 and sequence specific for Cas9 variants, was synthesized and fragments joined via Gibson assembly ( ).

Techniques: Mutagenesis, CRISPR, TALENs, Clone Assay, Sequencing, T7EI Assay

Journal: Frontiers in Plant Science

Article Title: Evaluation of Methods to Assess in vivo Activity of Engineered Genome-Editing Nucleases in Protoplasts

doi: 10.3389/fpls.2019.00110

Figure Lengend Snippet: Quantification of targeted mutagenesis at ALS1 locus by amplicon sequencing. (A) Percentage of mutagenized reads at ALS1 locus are shown using CRISPR/Cas9 and TALENs. The samples represent three biological replicates and three technical replicates of the PCR for each reagent, Student’s t- test ( P ≤ 0.005). (B) Frequency of the types of mutations in each sample are shown. Pie charts below represents the total average for each kind of mutation.

Article Snippet: Module A vector, pMOD_A0101 (Addgene #90998) was used for AtCas9 expression cassette. eCas9(1.1) and Cas9-HF1 (pMOD_A6101 and pMOD_A6201, respectively) were made by amplification of the vector sequence from pMOD_A0101 for the backbone and most part of AtCas9 and sequence specific for Cas9 variants, was synthesized and fragments joined via Gibson assembly ( ).

Techniques: Mutagenesis, Amplification, Sequencing, CRISPR, TALENs

Journal: Frontiers in Plant Science

Article Title: Evaluation of Methods to Assess in vivo Activity of Engineered Genome-Editing Nucleases in Protoplasts

doi: 10.3389/fpls.2019.00110

Figure Lengend Snippet: Detection of dsODNs integration by PCR using AtCas9, eCas9 (1.1) and Cas9-HF1 targeting ALS1 and ALS2 . Gel images showing dsODN integration at ALS locus at the DSB induced by variants of Cas9 including AtCas9, eCas9(1.1) and Cas9-HF1. (A) sg751 targets both ALS1 and ALS2 and (B) sg746 is specific for ALS1 . The order of PCR reactions for each SSN is according to . dsODN only is wild type/negative control without nuclease but with dsODNs in the protoplast transformation reactions to account for background DSB. PCR amplicon sizes assuming one dsODN integration are given which are same for all Cas9 variants. eCas9(1.1), enhanced specificity Cas9; Cas9-HF1, high fidelity Cas9. M, 100 bp NEB ladder.

Article Snippet: Module A vector, pMOD_A0101 (Addgene #90998) was used for AtCas9 expression cassette. eCas9(1.1) and Cas9-HF1 (pMOD_A6101 and pMOD_A6201, respectively) were made by amplification of the vector sequence from pMOD_A0101 for the backbone and most part of AtCas9 and sequence specific for Cas9 variants, was synthesized and fragments joined via Gibson assembly ( ).

Techniques: Negative Control, Transformation Assay, Amplification