plasmid pgem1  (Promega)

Journal: FEBS Open Bio

Article Title: CRISPR‐based knock‐in mutagenesis of the pioneer transcription factor FOXA1: optimization of strategies for multi‐allelic proteins in cancer cells

doi: 10.1002/2211-5463.13139

Figure Lengend Snippet: General workflow for CRISPR‐based site‐specific mutagenesis of multi‐allelic genes in cancer cell lines. (A) Three plasmids used for mutagenesis. The plasmid expressing spCas9 and the sgRNA (pX459) and the repair template plasmid containing the homology arms to the intended target site as well as the desired nucleotide substitutions for mutagenesis (pGEM1) are transfected into the cells. A third plasmid, containing a larger fragment of the endogenous locus sequence plus the desired nucleotide substitutions for mutagenesis, is used as a positive control for the ASP genotyping strategy. (B) Transfections, antibiotic selection, colony picking, and genomic DNA extraction for genotyping of potential KI clones are discussed in the text. (C) ASP is used as a screening strategy to identify clones that have successfully integrated the repair template sequence with desired nucleotide substitutions (KI). (D) Western blotting and mass spectrometry (LC‐MS/MS) is used to confirm that KI clones successfully produce full‐length KI proteins. (E, F) Allele frequency estimation is performed using TOPO cloning‐based screening of genomic DNA extracted from individual KI clones and/or the use of next‐generation sequencing approaches such as RNA‐sequencing to measure the abundance of RNA transcripts that contain the desired KI mutations. The details of each step are discussed throughout the text.

Article Snippet: Once cloned into the pGEM1 plasmid, we used the Q5 site‐directed mutagenesis Kit (NEB Cat# E0554S) as per the manufacturer's instructions to introduce the specific nucleic acid substitutions needed to create the repair template sequence shown in Fig. .

Techniques: CRISPR, Mutagenesis, Plasmid Preparation, Expressing, Transfection, Sequencing, Positive Control, Selection, DNA Extraction, Clone Assay, Western Blot, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Cloning, Next-Generation Sequencing, RNA Sequencing

Journal: FEBS Open Bio

Article Title: CRISPR‐based knock‐in mutagenesis of the pioneer transcription factor FOXA1: optimization of strategies for multi‐allelic proteins in cancer cells

doi: 10.1002/2211-5463.13139

Figure Lengend Snippet: Repair template plasmid and PCR screening positive control plasmid preparation. The human FOXA1 endogenous locus is shown at the top. Two different plasmids were generated, the repair template plasmid and an ASP positive control plasmid to be used during the screening of potential KI clones. The repair template plasmid was built on the pGEM1 backbone and contains the FOXA1 repair template sequence (shown in Fig. ) along with FOXA1 homology arms of 600 bp upstream and 1200 bp downstream of K295. The ASP ‐positive control plasmid was intentionally designed to contain more of the endogenous sequence of FOXA1 as well as the repair template sequence to be able to test our ASP primers. The forward primer only hybridizes to this plasmid and not the repair template plasmid thus allowing us to detect successful KI editing within the endogenous locus of FOXA1 and not random integration of the repair plasmid.

Article Snippet: Once cloned into the pGEM1 plasmid, we used the Q5 site‐directed mutagenesis Kit (NEB Cat# E0554S) as per the manufacturer's instructions to introduce the specific nucleic acid substitutions needed to create the repair template sequence shown in Fig. .

Techniques: Plasmid Preparation, Positive Control, Generated, Clone Assay, Sequencing