Petia Adarska

Protocol for fast antibiotic resistance-based gene editing of mammalian cells with CRISPR-Cas9 Protein tagging with CRISPR-Cas9 enables the investigation of protein function in its native environment but is limited by low homology-directed repair (HDR) efficiency. Here, we present a protocol for fast antibiotic resistance-based gene editing with CRISPR-Cas9 (FAB-CRISPR), which streamlines N/C-terminal tagging using an antibiotic resistance cassette for rapid selection and enrichment of gene-edited cells. We describe in detail guide RNA and HDR donor plasmid cloning, transfection of editin...

Protocol for fast antibiotic resistance-based gene editing of mammalian cells with CRISPR-Cas9 #protocol #starprotocols #cellpress

07.08.2025 21:08 — 👍 3    🔁 2    💬 0    📌 1

Protocol for fast antibiotic resistance-based gene editing of mammalian cells with CRISPR-Cas9 Protein tagging with CRISPR-Cas9 enables the investigation of protein function in its native environment but is limited by low homology-directed repai…

Thrilled to share our step-by-step protocol for endogenous protein tagging with CRISPR/Cas9, featuring an antibiotic resistance cassette to boost knock-in efficiencies 🧫🧬
Now out in STAR protocols: www.sciencedirect.com/science/arti...

11.08.2025 07:01 — 👍 6    🔁 2    💬 0    📌 0

T cell protrusions enable fast, localised initiation of CAR signalling Actin-rich protrusions densely cover the surface of T cells and are well characterised for their role in cell migration. However, recent studies have uncovered their role in antigen surveillance and immune signalling initiation. To investigate how membrane protrusions initiate and contribute to signalling, from the first cell-cell contact to immunological synapse formation, we performed dynamic imaging experiments of endogenously tagged signalling proteins in T cells. To quantitatively capture the early dynamics of cell-cell interactions, we employed HER2‑CAR-expressing T cells targeting HER2⁺ breast cancer cells. By harnessing live-cell imaging and super-resolution stimulated emission depletion (STED) microscopy we were able to capture topological membrane changes and their correlation with mesoscale protein rearrangements over time. Our findings indicate that, prior to activation, key molecular players in T cell activation, including the kinase Lck, the phosphatase CD45 and the adaptor LAT, as well as the exogenously expressed CAR, lack any enrichment in actin-rich protrusions. However, upon initial contact of the T cell with the target cell, a dynamic and fast rearrangement of the surface receptors, phosphatases, and kinases occurs within the protrusions, ensuring a rapid and effective initiation of the immune signalling cascade. The rapid clustering of the HER2-CAR occurs preferentially within protrusions rather than flat membrane regions and is accompanied by enhanced recruitment of the kinase ZAP-70 and LAT. While the localisation of the kinase Lck remained unchanged, protrusion-cell contacts trigger a pronounced exclusion of the phosphatase CD45, an effect observed both with and without the cytosolic signalling domain of the CAR. Overall, the signalling machinery rearranged more rapidly and efficiently at contacts mediated by protrusive structures compared to non-protrusive regions. Together, our data provide a quantitative framework illustrating how signalling proteins are dynamically reorganised to facilitate CAR-mediated activation within these specialised structures. ### Competing Interest Statement The authors have declared no competing interest.

Check out our new preprint on the role of T cell actin-rich protrusion in CAR signalling initiation.
Spoiler alert: Protrusions do it better
www.biorxiv.org/content/10.1...

09.07.2025 12:50 — 👍 29    🔁 5    💬 3    📌 3

Thanks to the Hausmeister for making us a little cool corner!

02.07.2025 09:57 — 👍 8    🔁 1    💬 0    📌 0

Vom 23. bis 29. Juni streicht die #FUBerlin das "E", um zu zeigen, was fehlt, wenn Substanz gestrichen wird. Allein für 2025 sollen die Kürzungen rund 37 Million n uro betragen.
D r B rlin r S nat g fährd t damit L hr, Forschung und
Zukunftssich rh it.
All Infos ➡️ tinyurl.com/aktionswoche...

23.06.2025 05:44 — 👍 172    🔁 67    💬 3    📌 5

A big group of us walked down to the beach today to have a fun kayaking afternoon 🤩 #EMBOcellpol

21.05.2025 22:43 — 👍 12    🔁 1    💬 0    📌 0

Check out our CRISPR/Cas9 methods paper, online on arxiv! 🚀🧬

19.02.2025 09:04 — 👍 6    🔁 3    💬 0    📌 0

Carmen practicing her leadership skills

03.12.2024 13:24 — 👍 8    🔁 1    💬 0    📌 0

The lab is in Prague for two days of discussions, fun and team building!

02.12.2024 11:30 — 👍 25    🔁 2    💬 1    📌 0

lab is off somewhere nice! More updates later 🤩🤩🤩🤩

02.12.2024 08:53 — 👍 9    🔁 1    💬 0    📌 0