How to insert a GFP tag in a genome of a cell lines via TALENS or CRISPR to study the localisation of a specific protein?

Hi, In this paper, the authors fused short epitope tag and fluorescent proteins (GFP, mCherry) into three genes using CRISPR/Cas9. It might help you to design your own strategy to tag your protein.

http://www.cell.com/retrieve/pii/S0092867413010167

Cell. 2013 Sep 12;154(6):1370-9. doi: 10.1016/j.cell.2013.08.022. Epub 2013 Aug

One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering.

Yang H, Wang H, Shivalila CS, Cheng AW, Shi L, Jaenisch R.

Shivam Mishra

Thank you Andrea. I will go through it

Stéphane Roche

You need a donor plasmid with two sequences homologous to the region of your editing. Inside the plasmid, you need GFP sequence, a construct for the expression of GFP and perhaps a resistance gene for the selection.

Karthigeyan Dhanasekaran

I tried inserting a GFP tag in the C-terminus using a donor vector suggested by Stephane but without a resistance gene (RPE cells). It did not work for me. I am not sure what so crucial am i missing in this. Let me know if anybody has met success creating the GFP fusion using CRISPR.

Ryan Dikdan

It might be worth it to look at this paper: 1. Leonetti MD, Sekine S, Kamiyama D, Weissman JS, Huang B. A scalable strategy for high-throughput GFP tagging of endogenous human proteins. Proc Natl Acad Sci . 2016;113(25):E3501-E3508. doi:10.1073/pnas.1606731113 .

Apparently the larger the insertion, the less efficient the incorporation. This paper gets around that problem by using a split GFP construct. The only difficulty is getting the thing you'd need is the cell line they use which constitutively expresses the GFP1-10.

Mario Perez-Medina

Hi, do you get successful in your GFP insertion?

Karthigeyan Dhanasekaran

Yes it worked. We had very poor efficiency but ultimately all you need is one pure clonal population which we could screen and get it in time.

Yang Yu

Inserting a GFP (Green Fluorescent Protein) tag into a specific genomic location in cell lines using TALENs (Transcription Activator-Like Effector Nucleases) or CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) to study the localization of a specific protein involves a series of well-planned steps. Here's how you can approach this task:

Target Gene Identification: Identify the gene encoding the protein of interest in the genome of the cell line. Determine the specific site within the gene where the GFP tag will be inserted, typically at the N-terminus or C-terminus.

Designing TALENs or CRISPR System:TALENs: Design TALEN pairs specific to the target site in the gene. The TALENs will create a double-strand break at this specific location. CRISPR: Design sgRNA (single-guide RNA) that targets the CRISPR-Cas9 system to the desired genomic location.

GFP Tag Construct Preparation: Create a donor DNA template that includes the GFP sequence flanked by homology arms. These arms are sequences identical to the genomic DNA surrounding the cut site, facilitating homology-directed repair (HDR).

Cell Line Preparation: Culture the cell line under optimal conditions to ensure healthy and actively dividing cells. This improves the efficiency of TALENs or CRISPR-mediated editing.

Transfection:Co-transfect the cells with the TALENs or CRISPR-Cas9 components and the GFP donor template. Various transfection methods can be used, such as lipofection, electroporation, or viral vectors, depending on the cell line.

Selection and Isolation of Edited Cells: After transfection, select and isolate cells that have incorporated the GFP tag. This can be done using antibiotic selection (if a selection marker was included in the donor template) or by fluorescence-activated cell sorting (FACS) based on GFP expression.

Molecular Confirmation:Perform PCR and sequencing to confirm the correct integration of the GFP tag at the desired genomic location. Verify that the GFP tag has not disrupted the reading frame of the target gene.

Protein Localization Studies:Once the insertion is confirmed, use fluorescence microscopy to observe the localization of the GFP-tagged protein within the cells. Perform additional experiments to validate the functionality of the tagged protein and to ensure that the GFP tag has not altered its normal behavior or localization.

Controls and Replicates: Include appropriate controls and perform experiments in replicates to ensure the reliability of your observations.

Data Analysis: Analyze the data carefully, comparing the localization of the GFP-tagged protein with known cellular markers or structures, if relevant.

Using TALENs or CRISPR for GFP tagging is a powerful way to study protein localization in living cells. However, it requires careful design and validation to ensure that the GFP tag is correctly inserted without disrupting the normal function of the protein.

Take a look at this protocol list; it could assist in understanding and solving the problem

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