Journal: Plant Methods

Article Title: A multiplex guide RNA expression system and its efficacy for plant genome engineering

doi: 10.1186/s13007-020-00580-x

Figure Lengend Snippet: Two-step cloning system for multiplex guide RNA expression in plants. a Cloning procedures of multiplex guide RNAs (gRNAs). b The single gRNA-cloning vector, pGRNA, is designed for a PCR-free multiplex gRNA cloning method. The target-binding sequence of gRNA is prepared by annealing two complementary oligos. A pair of annealed oligos is directly cloned into the BsaI-digested pGRNA (blue triangles) between the tRNA sequence and gRNA scaffold. Two AarI sites (red triangles) are used in step 2. c The Golden Gate assembly for preparing a plant binary vector expressing five gRNAs under a single U6 promoter. Each tRNA-gRNA unit is excised from pGRNA by cutting the pGRNA with AarI. All tRNA-gRNA units and one of the plant binary vectors—pECO100, pECO200, or pECO300—is connected in the Golden Gate assembly mixture described in " "

Article Snippet: The digested pGRNA vector was ligated together with the annealed oligos using T4 DNA ligase (NEB).

Techniques: Clone Assay, Multiplex Assay, RNA Expression, Plasmid Preparation, Binding Assay, Sequencing, Expressing

Journal: International Journal of Molecular Sciences

Article Title: The Combinational Use of CRISPR/Cas9 and Targeted Toxin Technology Enables Efficient Isolation of Bi-Allelic Knockout Non-Human Mammalian Clones

doi: 10.3390/ijms19041075

Figure Lengend Snippet: ( A ) Schematic representation of expression vectors, pgRNA vector, pCAG-NFL- hCas9 , pCAG/ EndoGalC -29, and pPGK-pac. CAG, cytomegalovirus enhancer + chicken β-actin promoter; hCas9, humanized Cas9 gene; p(A), poly(A) tail; EndoGalC , Clostridium perfringens -derived endo-β-galactosidase gene; Kan r , kanamycin resistance gene; Amp r , ampicillin resistance gene; U6, human U6 promoter; PGKp, mouse phosphoglycerol kinase promoter; pac , puromycin N -acetyltransferase gene. ( B ) Flowchart of the experiments used to test the feasibility of a new system for the enrichment of genome-edited cells. Cells are co-transfected with three vectors, namely pgRNA, pCAG-NFL-hCas9, and pCAG/ EndoGalC -29, as the experimental group. Three days after transfection, cells are treated with IB4SAP for a short period, prior to cultivation in a normal medium. For control group-1, cells are transfected with pgRNA and pCAG-NFL- hCas9 . Three days after transfection, they are split to one tenth of the total amount, prior to cultivation in a normal medium. For control group-2, cells are transfected with pgRNA, pCAG-NFL- hCas9 , and pPGK- pac . Three days after transfection, they are split to one sixth of the total amount, prior to cultivation in a medium containing puromycin for 2 days. The emerging colonies are propagated for molecular biological and cytochemical analyses.

Article Snippet: The plasmid structure used in this study is schematically illustrated in A. pgRNA vector (Addgene, Cambridge, MA, USA) has a human U6 promoter to drive transcription of the downstream gRNA.

Techniques: Expressing, Plasmid Preparation, Derivative Assay, Transfection