Journal: bioRxiv

Article Title: Live-cell imaging of circadian clock protein dynamics in CRISPR-generated knock-in cells

doi: 10.1101/2020.02.28.967752

Figure Lengend Snippet: (A) Donor plasmid design and genome editing strategy: The tag (e.g. fluorescent protein) to be integrated and a floxed positive selection cassette (CFP + blasticidin-resistance) are flanked by arms which are homologous to the genomic target region. When Cas9/sgRNA-mediated DNA double strand breaks are repaired by HDR, tag and positive selection marker are integrated into the target region. The negative selection cassette (hCD4, a cell surface protein exclusively expressed on immune cells) is only integrated into the genome by unwanted random integration of the whole donor plasmid. (B) Selection strategy. Cells are transfected with Cas9, sgRNA, i53bp (see text) and donor plasmid. Stable transfectants are selected by blasticidin selection and fluorescence activated cell sorting (FACS) of CFP positive cells, while unwanted hCD4 positive cells are depleted. Subsequently, cells are transiently transfected with CRE recombinase to remove the positive selection cassette from the genomic locus, and only CFP negative cells are clonally expanded and screened for successful knock-in. (C) Chimeric mRNA was detected in selected batch cultures by RT-PCR using a RT- and a reverse primer specific to the insertion and a gene specific forward primer. (D) Loss of CFP expression after removal of the positive selection cassette monitored by microscopy and flow cytometry. (E) Fluorescence microscopy images of successful knock-in clones. (F) Indicated knock-in cells were either left untreated or transduced with shRNA targeting either CRY1 or PER2 . Images were acquired 10 h after synchronization. Scale bars: 20 µm. mCl3 = mClover3, mSca = mScarlet-I, FP = fluorescent protein, HF: His-tag/FLAG-tag

Article Snippet: The hTK was exchanged for hCD4, amplified from the pMSCV-IRES-hCD4plasmid (Addgene #35712 ( )). mClover3 was subcloned from Addgene #74252 , mScarlet was subcloned from Addgene #98839 ( ).

Techniques: Plasmid Preparation, Selection, Marker, Transfection, Fluorescence, FACS, Knock-In, Reverse Transcription Polymerase Chain Reaction, Expressing, Microscopy, Flow Cytometry, Clone Assay, Transduction, shRNA, FLAG-tag

Journal: bioRxiv

Article Title: Live-cell imaging of circadian clock protein dynamics in CRISPR-generated knock-in cells

doi: 10.1101/2020.02.28.967752

Figure Lengend Snippet: (A-B) Analysis of phase difference between CRY1 and PER2 nuclear accumulation in individual double knock in cells. Phases were calculated either including (A) or excluding (B) the first 24 hours of the three days’ time series. p-values: paired Student’s t-test. (C) Live-cell bioluminescence recordings of knock-in cells expressing either CRY1-luciferase or PER2-luciferase fusion proteins. Depicted are mean ±SD of 3 individual traces from 2 clones of each knock-in. Representative data from 2 experiments. (D) Relative nuclear peak intensity of fluorescence in cells expressing either PER2- or CRY1-mScarlet fusion protein. (E) Ratio of normalized expression of CRY1-mClover3 versus PER2-mScarlet-I in individual cells expressing both fusion proteins. Lines in scatter plots depict median. SKI: single knock-in. DKI: double knock-in.

Article Snippet: The hTK was exchanged for hCD4, amplified from the pMSCV-IRES-hCD4plasmid (Addgene #35712 ( )). mClover3 was subcloned from Addgene #74252 , mScarlet was subcloned from Addgene #98839 ( ).

Techniques: Knock-In, Expressing, Luciferase, Clone Assay, Fluorescence