Plasmids for Endogenous Gene Tagging in Human Cells

Posted by Guest Blogger on Apr 6, 2017 9:02:59 AM


This post was contributed by the gene editing team at the Allen Institute for Cell Science. Learn more by visiting the Allen Cell Explorer at allencell.org and the Allen Institute website at alleninstitute.org.

A classic challenge in cell biology is making sure that what we observe through the microscope represents reality as accurately as possible. This is especially true in the case of protein tagging to elucidate cellular structures. Overexpression methods flood the cell with protein, which can both interfere with a cell’s normal function and result in a ubiquitous background signal that makes it hard to visualize the precise location of the protein or structure of interest.

Endogenous gene tagging is an ideal solution because it allows for tagging and visualization of specific, individual proteins under endogenous regulatory control. But even with the advent of CRISPR/Cas9 technology, inserting large tags into a precise location in the genome is still inefficient, particularly in human cell lines. Furthermore, the quality control necessary to ensure the edited cells are behaving normally can be prohibitively expensive for many labs.

We’ve created and tested plasmids that use CRISPR/Cas9 to endogenously tag a wide variety of genes with GFP. These plasmids are available through Addgene, and the stem cell lines we’ve made using them are available as part of the Allen Cell Collection at Coriell. More information and images generated from these cell lines can be found in the Cell Catalog on our data portal. The plasmids should be functional in many different human cell types, and while most of the genes we’ve tagged are widely recognized markers of cellular structures, we provide guidance on how to make your own gene-tagging constructs below. Read on to find out how these plasmids work and to discover more resources for your genomic tagging experiments.

Browse the Allen Institute for Cell Science Plasmid Collection

Fluorescent Tagging Strategy

Molecular_Bio_of_Tagging3-01.png

The first step in creating the plasmids was to identify the gene of interest and decide which terminus, N or C, to tag and which short amino acid linker to use. These decisions were made based on known functional aspects of the protein gleaned from the lterature and engagement with researchers who study the protein or structure of interest. For a C-terminal tag, as shown in the example above, we inserted a linker and GFP tag at the end of the last exon of the gene to ensure it would be transcribed and translated. For an N-terminally tagged protein, we used the same strategy, inserting the tag and the linker preceding the first exon of the gene. In this manner, we have created an initial set of 10 plasmids representing 10 human genes and will add more to the collection later this year.

Designing the Donor Plasmid

Donor Plasmid Design.png

A key feature of our methodology is the use of a donor plasmid that contains long stretches of DNA on either side of the fluorescent protein sequence that is homologous to the sequence into which it is being inserted: one thousand base pairs on each side, in fact. Later, when we transfect the cells, these large regions of homology enable us to effectively utilize the host cell’s inherent homology directed repair (HDR) process following the double strand break CRISPR/Cas9 makes at the target site. The whole segment—a GFP tag and 1kb of homologous DNA flanking both sides (about 2.7kb total)—is inserted into a plasmid backbone for delivery to the cells. As a precautionary measure, it’s also good to make a few single base pair mutations in crRNA target sites found within the homology areas of the plasmid to prevent the plasmid from being cut and destroyed by CRISPR/Cas9 during transfection.

Making the Double Strand Break and Introducing the Tag through Homology Directed Repair

To introduce the fluorescent tag to the cells, we used CRISPR/Cas9 to make a precise cut in the genome near either the beginning or end of the gene of interest —in our case in the genome of an hiPS cell. While the majority of the time the genome will attempt to repair itself without outside influence (non-homologous end joining, or NHEJ), in approximately one percent of cases, the stem cell will seek out a template for the repair process. By flooding the cell with a plasmid containing regions of high homology to the gene with the cut, we trick the cell into using the introduced donor plasmid as a repair template. This results in the insertion of the GFP tag precisely where we need it, in this example, at the C-terminus of the gene of interest. To get the molecular components of CRISPR/Cas9 (Cas9 protein and the crRNA-tracrRNA complex) and the donor plasmid into the cells we use electroporation, a technique that briefly destabilizes the cell’s outer membrane and allows the components to physically enter the cells so they can go to work.

NHEJ_and_HDR-01.png

Plasmids, Plasmids Everywhere

To date, we have created 10 plasmids that can be used to introduce a GFP tag at the endogenous loci of 10 genes (see table below). The genes we chose to tag are widely recognized markers of key cellular structures. In the future, additional plasmids to tag other key proteins will be made available through Addgene. 

Addgene ID Plasmid Allen Institute ID Tag Protein Structure
87420

PXN-EGFP

AICSDP-5 EGFP Paxillin Matrix Adhesions
87421 TUBA1B-mEGFP

AICSDP-12

mEGFP

Alpha-tubulin Microtubules
87422 LMNB1-mEGFP

AICSDP-13

mEGFP

Nuclear laminB1 Nuclear envelope
87423

TOMM20-mEGFP

AICSDP-11 mEGFP TOM20 Mitochondria
87424

DSP-mEGFP

AICSDP-17 mEGFP Desmoplakin Desmosomes
87425

ACTB-mEGFP

AICSDP-16 mEGFP Beta-actin Actin
87426 SEC61B-mEGFP

AICSDP-10

mEGFP

Sec61-beta Endoplasmic Reticulum
87427

FBL-mEGFP

AICSDP-14 mEGFP Fibrillarin Nucleolus
Coming Soon MYH10-mEGFP

AICSDP-24

mEGFP

Non-muscle myosin heavy chain IIB Actomyosin bundles
87429  TJP1-mEGFP AICSDP-23 mEGFP Tight junction protein ZO1 Tight junctions

To find out more about gene edited hiPS cell lines generated using these plasmids, please visit the Cell Catalog on the Allen Cell Explorer, where you can also find the certificate of analysis provided with each cell line. The editing strategy and design described above can be used to create similar donor plasmids for introducing tags into your gene of interest.

Endogenous gene tagging with fluorescent proteins as described here enables live cell imaging to study cellular structures and processes and can lead to the better understanding of cell biology. We hope that these high-quality, highly specific plasmids, as well as the instructions on how to make your own, will make it easier for you to use genomic tagging in your experiments.


Many thanks to our guest bloggers from the Allen Institute for Cell Science.

This post was contributed by the gene editing team at the Allen Institute for Cell Science. Learn more by visiting the Allen Cell Explorer at allencell.org and the Allen Institute website at alleninstitute.org.

References

1. Roberts, Brock, et al. "Systematic gene tagging using CRISPR/Cas9 in human stem cells to illuminate cell organization." bioRxiv (2017): 123042.

Additional Resources on the Addgene Blog

Resources on Addgene.org

Sign Up to Recieve Addgene's CRISPR 101 eBook!

Topics: Plasmid How To, CRISPR, Techniques

Addgene blog logo

Subscribe to Our Blog