Hot Plasmids: Spring 2025

Every few months we highlight some of the new plasmids, antibodies, and viral preps in the repository through our Hot Plasmids articles.

Here's what you'll find in this post:

• Glutamate indicators with increased sensitivity and tailored deactivation rates
• Addgene's own empty lentiviral backbones
• New recombinant antibodies for Glypicans
• DNA editing with STITCHR
• Dynamic protein labeling with TurboCas
• Enhancer-AAV viral preps targeting interneurons

Glutamate indicators with increased sensitivity and tailored deactivation rates

By Mike Lacy

Glutamate is one of the most important and abundant neurotransmitters, but it is challenging to monitor because only a small number of glutamate molecules are released in each synaptic event and they are rapidly taken up by postsynaptic receptors and transporters. The intensity-based Glutamate-Sensing Fluorescent Reporter (iGluSnFR) designed and improved by the Loren Looger and Kaspar Podgorski Labs now has a new version: iGluSnFR4, developed by the GENIE Project team at HHMI's Janelia Research Campus (Aggarwal et al., 2025).

In this latest work, the team designed and screened a large library of iGluSnFR3 mutants, identifying the two  best-performing variants as iGluSnFR4f (fast deactivation) and iGluSnFR4s (slow deactivation). Both have significantly improved brightness, kinetics, and sensitivity. iGluSnFR4f is well-suited for imaging rapid synaptic events such as in the somatosensory cortex, while iGluSnFR4s produces larger signal-to-noise ratios in two-photon imaging, making it ideal for video recordings of large populations of synapses and deep-brain fiber photometry. 

Figure 1: Characterization of iGluSnFR4 in mice. A) Scheme for two-photon (2P) imaging and loose-seal, cell-attached recordings in primary visual cortex (V1) layer 2/3 (L2/3). B) Fluorescence from axonal boutons of neurons expressing iGluSnFR3, iGluSnFR4f, and iGluSnFR4s. C) Spike-triggered averages for single action potential-evoked axonal glutamate signals. Shading denotes SEM. D) Summary of ΔF/F₀, Time-to-peak, Rising τ, and Decay τ of the three sensors. Image adapted from Aggarwal et al. 2025 under a CC-BY-NC-ND 4.0 International license.

While the GENIE team tested and deposited versions with both PDGFR and NGR fusions for membrane display, they recommend the NGR vectors for most experiments. These new tools promise to be a popular choice for a variety of synaptic imaging applications.

Find iGluSnFR4 plasmids here!

• Aggarwal, A., Negrean, A., Chen, Y., Iyer, R., … Podgorski, K. (2025). Glutamate indicators with increased sensitivity and tailored deactivation rates. bioRxiv, 2025.03.20.643984. https://doi.org/10.1101/2025.03.20.643984. 

Build your way: Explore Addgene's new empty backbones

By Meghan Rego

You asked, we listened! One of the most powerful tools in a scientist's toolkit is the empty plasmid backbone: the DNA vector that contains everything needed to replicate inside a host cell but with space to insert your genetic cargo of choice. After hearing about researchers' favorite features and limitations of existing backbones, we're excited to share our very own set of empty, HA-, and EGFP-expressing lentiviral backbones designed right here at Addgene. Here's what makes these new backbones special:

• Strong expression: A CMV promoter drives robust gene expression in mammalian cells.
• Flexible tagging: Choose untagged or add a versatile N- or C-terminal HA tag.
• Selectable and stable: A puromycin resistance gene makes stable cell line creation a breeze.
• Modular by design: Key elements (promoter, tag, polyA site) are flanked by unique restriction sites so you can swap sequences effortlessly. 

We built these plasmids for flexibility and speed, so you can design, test, and innovate faster than ever. Plus, we've already taken them for a test drive! We validated viral packaging and strong expression after inserting EGFP and provide step-by-step protocols for viral generation and stable cell line creation (Figure 2). 

Looking for more? Explore our empty and EGFP retroviral plasmid set, our bacterial plasmid set for expressing tagged proteins, and stay tuned for mammalian expression and AAV plasmid sets launching later this year! 

Find Addgene's own Lentiviral backbone plasmids here!

Figure 2: Addgene cloned EGFP into pAG Lenti CMV N-HA Puro (Addgene #236079) to create pAG Lenti CMV HA-EGFP Puro (Addgene #236081), then generated lentiviral particles in HEK293T cells with pMD2.G (Addgene #12259) and psPAX2 (Addgene #12260). HeLa cells were infected with 96 h lentiviral supernatant and selected with puromycin. Puromycin-resistant cells were fixed and labeled with primary antibody (0.01 µg/mL Anti-GFP [N86/38.1R] (Addgene #180084), 0.01 µg/mL Anti-HA [12CA5] - Chimeric (Addgene #198003)) and Goat anti-Mouse IgG2a Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (Thermo Fisher A-21131) or Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 594 (Thermo Fisher A-11012), and counterstained with DAPI.

New recombinant antibodies targeting Glypicans

By Ashley Waldron

Glypicans are important modulators of cell signaling pathways during embryonic development and cancer. But there is still much to learn about their myriad functions and therapeutic potential. Our partners at the Institute for Protein Innovation (IPI) recently released a set of highly-specific recombinant antibodies against three of the six human glypican proteins (GPC1, GPC3, and GPC4). 

All IPI antibodies undergo extensive characterization at IPI as well as community-led evaluation, and the results are available to view from the antibody pages or in the Addgene Antibody Data Hub. Perhaps these antibodies are just the tools you need to push the field forward!

Browse the IPI Glypican Collection here!

Figure 3: General glypican domain structure. Glypicans are proteoglycans composed of a protein core, 2–4 glycosaminoglycans (GAG), and are (typically) tethered to the cell surface by a GPI-anchor. Image courtesy of IPI.

Repurposing retrotransposons for DNA editing

By Emily P. Bentley

For RNA-guided genome editing, think beyond Cas9 with STITCHR: a combined CRISPR / retrotransposon system with the pithy name "site-specific target-primed insertion through targeted CRISPR homing of retroelements." The Abudayyeh-Gootenberg Lab developed this tool based on several new site-specific retrotransposon elements they identified and characterized (Fell et al., 2025).

In retrotransposon insertion, a combined endonuclease-reverse transcriptase uses homology arms flanking an RNA payload to insert the payload sequence into the genome. The team established that they could reprogram the genomic target by swapping the homology arms for new targets. And, importantly, they found that moving the RNA untranslated regions (UTRs) outside of the homology elements allowed for scarless insertion — no UTR sequence added to the genome (Figure 4).

Figure 4: STITCHR consists of a nicking Cas9 fused to a shortened R2Tocc transposon protein, and can "retarget" the transposon to new loci in the genome. The modified transposon RNA has untranslated regions (U, blue) on the outside of the homology arms (red) to allow for scarless insertion of the payload (teal). The Cas9 gRNA targeting guide and the transposon RNA homology both help direct the fusion construct to the new genomic locus. Image courtesy of Christopher Fell, PhD.

After minimizing the system's size, the scientists fused the retrotransposon protein R2Tocc to a Cas9 nickase to improve target recruitment and nicking, producing STITCHR. STITCHR can make a wide variety of genomic edits — single-base changes, small deletions, and large insertions up to 12 kb — with efficiencies between 5% and 11%. It has higher efficiency and lower indel formation than methods relying on homology-directed repair (HDR), and insertion is scarless, unlike previous integrase-based approaches.

Find STITCHR plasmids here!

• Fell, C. W., Villiger, L., Lim, J., Hiraizumi, M., … Abudayyeh, O. O., & Gootenberg, J. S. (2025). Reprogramming site-specific retrotransposon activity to new DNA sites. Nature. doi: https://doi.org/10.1038/s41586-025-08877-4. 

Two powerhouse tools combine for dynamic protein labeling

By Alyssa Shepard

To fully understand how genes are regulated, it's important to know what proteins interact with each other and where those interactions occur in the genome. This requires both targeting a specific genome location and isolating the interacting proteins. Lucky for us, we now have a two-for-one solution: TurboCas!

TurboCas provides the genomic targeting power of dCas9 with the dynamic labeling power of miniTurboID, a proximity ligation tool (Cenik et al., 2024). While dCas9 has been used in combination with proximity labeling enzymes before, such as CasID, TurboCas improves on these in both efficiency and specificity. With TurboCas, the Ali Shilatifard Lab was able to identify both new and known regulators at the FOS gene promoter and the MYC locus (Figure 5).

Figure 5: TurboCas protein labeling compared to immunoprecipitation. The top panel depicts the identification of heat-shock proteins at the FOS gene promoter using TurboCas, streptavidin immunoprecipitation (IP), and tandem mass tag (TMT) mass spectrometry (MS) in HCT116 cells (colorectal cancer). The bottom panel depicts the identification of general heat-shock proteins using IP with CycT1 or PolII-CTD antibodies and unlabeled MS in DLD-1 cells (colorectal cancer). Image courtesy of Brianna Monroe, MS.

Proximity ligation tools like TurboID and its mini counterpart label nearby proteins with biotin. The biotinylated proteins can then be isolated and identified with tools like mass spectrometry, providing a snapshot of interacting proteins at a certain time and genomic location. TurboID improved on other proximity ligation tools with better kinetics and less background labeling. By fusing miniTurboID with dCas9 to create TurboCas, the Shilatifard Lab created a very quick and efficient way to learn more about gene regulation.

Find the TurboCas plasmid here!

• Cenik, B. K., Aoi, Y., Iwanaszko, M., Howard, B. C., Morgan, M. A., Andersen, G. D., Bartom, E. T., & Shilatifard, A. (2024). TurboCas: A method for locus-specific labeling of genomic regions and isolating their associated protein interactome. Molecular Cell, 84(24), 4929-4944.e8. https://doi.org/10.1016/j.molcel.2024.11.007.
• Branon, T. C., Bosch, J. A., Sanchez, A. D., Udeshi, N. D., Svinkina, T., Carr, S. A., Feldman, J. L., Perrimon, N., & Ting, A. Y. (2018). Efficient proximity labeling in living cells and organisms with TurboID. Nature Biotechnology, 36(9), 880–887. https://doi.org/10.1038/nbt.4201.

Hot viral preps: Enhancer-AAVs targeting interneurons

By Mike Lacy

Targeting gene expression to specific cell types in the brain can be a challenging — but critical — factor for successful experiments or therapies. Using AAV vectors with enhancers that drive cell-type-specific expression has been a promising solution. Recently, the labs of Gord Fishell and colleagues characterized a series of new enhancer-AAV vectors targeting specific classes of cortical and striatal interneurons (Furlanis, Dai, Leyva Garcia et al., 2025).

More than a dozen of these enhancer-AAV constructs are now available as in-stock viral preps using the systemic PHP.eB capsid, to express either the optogenetic activator channelrhodopsin-2 or fluorescent reporter dTomato under the control of one of seven enhancers specific for different interneuron populations. The enhancers span all four major classes of GABAergic interneurons — those marked by parvalbumin (PV), somatostatin (SST), vasoactive intestinal peptide (VIP), or lysosomal-associated membrane protein 5 (LAMP5) — as well as cholinergic (choline acetyltransferase, ChAT) neurons. The authors characterized these tools and validated their specificity in mice, non-human primates, and human tissue.

Figure 6: Enhancer-based targeting of PV interneurons in mice. A) AAV expression of dTomato reporter (magenta) under the control of the BiPVe3 enhancer and parvalbumin (PV) immunostaining (green), in the primary somatosensory cortex (S1) of the mouse brain following retro-orbital AAV injection at 4 weeks. Cortical layers (L1-6) and scale bar are indicated. B) Specificity, as percentage of BiPVe3-dTomato and PV-positive cells out of all BiPVe3-dTom cells. C) Confocal stack of a biocytin-filled cell expressing BiPVe3-dTomato, displaying characteristic PV basket cell morphology. Adapted from Furlanis, Dai, Leyva Garcia et al. 2024 bioRxiv, under a CC-BY-NC-ND 4.0 International license.

These plasmids are part of the BRAIN Initiative Armamentarium Collection, a major collaborative effort to develop molecular tools for studying different brain cell types. Look for more of these interneuron-targeting constructs and other serotypes available later this year!

Find the viral preps here, and all plasmids from the article!

• Furlanis, E., Dai, M., Leyva Garcia, B., Tran, T., … Dimidschstein, J., Fishell, G., Wang, Y. (2024). An enhancer-AAV toolbox to target and manipulate distinct interneuron subtypes. Neuron, 113(10), 1525–1547.e15. doi: https://doi.org/10.1016/j.neuron.2025.05.002. bioRxiv preprint: https://doi.org/10.1101/2024.07.17.603924.