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Enrich CRISPR’d Cells with Label IT®

In each SNiP or “Small News in Pieces” we highlight standout research articles we’ve come across that feature Mirus Bio products… in bite-size pieces. Today we SNiP a “classic” Label IT® paper.

Label IT® is a nucleic acid labeling technology developed by Mirus Bio that allows for covalent, non-specific attachment of a label to any kind of nucleic acid. Many labels are available to Label IT®, including molecular tags, like biotin, and fluorophores, like Cy®3. The potential applications for Label IT® are limited only by your imagination!

Nasri and Mir et al. describe an elegant use for Label IT® in enriching CRISPR/Cas-edited cells. One of the challenges with genome editing is selecting the cells that received the intended edit from those cells that did not. This is especially difficult if nonedited cells have a proliferative advantage and if the edit is subtly expressed. In the context of clinical use, it is highly desirable to limit the number of nonedited cells that may compete with the therapeutic, modified cells.

To enable discrimination of edited cells, the authors fluorescently label CRISPR gRNA before complexing it with Cas9 and transfecting cells as shown in the figure below.

 

Diagram showing steps for forming and labeling CRISPR/Cas RNPs with Label IT for sorting.

Enrich CRISPR’d cells with Label IT®. CRISPR gRNA are labeled with Label IT® before complexing with the Cas9 enzyme to form Cas9 RNPs. CRISPR’d cells can then be enriched by sorting for cells that received the labeled Cas9 RNP.

They show that the labeling of gRNA did not affect gene editing efficiency, and that they could enrich edited cells by sorting them by their fluorescence. As a proof of concept, they applied their CRISPR workflow to editing the GADD45B gene in multiple cell types. The percentage of edited cells was 15-40% more in the fluorescent population compared to the total population, depending on the cell type.

Read the paper to learn how to incorporate Label IT® into your CRISPR experimental workflow!

 

Citation

Title: Fluorescent labeling of CRISPR/Cas9 RNP for gene knockout in HSPCs and iPSCs reveals an essential role for GADD45b in stress response
Authors: Masoud Nasri, Perihan Mir et al.
Journal: Blood Adv, Volume 3(1), Jan 2019.
DOI: 10.1182/bloodadvances.2017015511
Product Usage: CRISPR gRNA were labeled using the Label IT® Nucleic Acid Labeling Kit with CX-Rhodamine or fluorescein using a 1:1 ratio of Label IT® reagent to mass of gRNA, resulting in a labeling density of ~1 label per 40 to 120 bases. CRISPR/Cas9 RNP complexes were transfected into HEK 293FT cells with TransIT-X2®. Electroporation was used to deliver CRISPR/Cas9 RNP to Jurkat, human inducible pluripotent stem cells, and CD34+ hematopoietic stem and progenitor cells. TransIT®-LT1 Transfection Reagent was used to deliver a labeled plasmid to HEK 293FT cells.

Read more articles featuring Mirus reagents in our Citations Database!

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