Hot Plasmids: Summer 2025

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

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

• Bright and photostable yellow fluorescent protein mGold2
• Viral delivery of TnpB for plant genome editing
• LARRYv2 pooled libraries for cell barcoding
• Addgene’s own AAV6 and AAV11 packaging plasmids
• Mapping anterograde synaptic connections with ATLAS

Bright and photostable yellow fluorescent protein mGold2

By Mike Lacy

Get your sunglasses — these proteins are bright! Most current yellow fluorescent proteins tend to photobleach rapidly, which hinders their use for long-term imaging or advanced techniques like super-resolution microscopy. To make this segment of the color spectrum more powerful, the lab of Francois St-Pierre recently developed a pair of new yellow fluorescent proteins: mGold2s and mGold2t (Lee & Lai, et al., 2025).

The team characterized and deposited a variety of constructs for visualizing cellular components (nucleus, endosomes, lysosome, golgi, ER, inhibitory synapses, and cytoskeleton components) and in a FRET-based biosensor for cAMP dynamics. The mGold2 variants significantly enhanced photostability — 25-fold over mVenus and 4-fold over the previous best performer mGold — while maintaining high brightness and fast maturation time (Figure 1). See the paper for more details on small differences between mGold2s and mGold2t depending on the application.

Figure 1: Super-resolution imaging of actin filaments with mGold2. Top: TIRF-SIM imaging of COS-7 cells expressing LifeAct fused to mVenus, mGold2s, or mGold2t. Bottom: Quantification of photostability. Image reused from Lee & Lai et al. (2025) under CC BY-NC-ND license.

If you’re currently using mVenus or mCitrine, or if existing yellow fluorescent proteins just haven’t met your needs, consider trying mGold2s or mGold2t for your next experiments and see the difference!

Find mGold2 plasmids here!

• Lee, J., Lai, S., et al. (2025). Bright and photostable yellow fluorescent proteins for extended imaging. Nature Communications, 16(1), 3241. https://doi.org/10.1038/s41467-025-58223-5.

TnpB, so hot right now

By Alyssa Shepard

While CRISPR has become a household name, the search for smaller genome editors is heating up and TnpB has found itself in the spotlight. TnpBs are gaining popularity, especially among plant biologists, due to their compact size and ability to generate heritable edits (learn more in our recent blog post on TnpB). Similar to CRISPR-Cas, TnpB is directed to genomic targets using an RNA guide (known as an omega RNA, or ωRNA) (Figure 2), but TnpB is small enough to be carried by viral vectors (~400 amino acids, less than a third of the size of Cas9).

Figure 2: Comparison of TnpB (A) and Cas9 (B) genome editors. Effector nucleases are shown bound to the target DNA, recruited by either the ωRNA (TnpB) or a gRNA (Cas9). Drawings not to scale. Created with BioRender.com.

Steven Jacobsen and Jennifer Doudna’s labs engineered the tobacco rattle virus (TRV), a bipartite RNA virus, to deliver a TnpB (named ISYmu1) to the popular plant model Arabidopsis (Weiss et al., 2025). By co-delivering TRV1 with TRV2 that encodes ISYmu1 and an ωRNA under the same promoter, they introduced heritable genome edits in a single step with minimal off-target effects. With this proof-of-concept, they believe that the strategy will be applicable with other TnpBs and in other plant species, thanks to the broad host range of TRV.

Find TRV-TnpB plasmids here!

• Weiss, T., et al. (2025). Viral delivery of an RNA-guided genome editor for transgene-free germline editing in Arabidopsis. Nature Plants, 11(5), 967–976. https://doi.org/10.1038/s41477-025-01989-9.

Make a splash with new LARRY barcoding libraries

By Mike Lacy

Whether you’re planning on lounging poolside or swimming laps, Addgene has pooled libraries for any summer project. While many barcoding libraries are available for tracking cell lineage, different applications require careful selection or design to ensure reliable readout and avoid biases.

The lab of Alejo Rodriguez-Fraticelli recently developed an approach called Simultaneous Tracking of Recombinase Activation and Clonal Kinetics (STRACK) to study stem cell differentiation and cancer fates (Singh et al., 2025). Key to this method is their updated Lineage and RNA Recovery (LARRYv2) barcoding libraries. The team used these libraries to analyze pre-existing heterogeneity of hematopoietic stem cells and track clonal dynamics during differentiation after they introduced mutations that drive leukemia (Figure 3).

Figure 3: Using LARRYv2 libraries for state-fate analysis in ex vivo HSC expansion cultures. A) Experimental design. B) UMAP of cell states in ex vivo expansion cultures from days 7, 14, and 27. Reproduced from Singh et al. (2025) under CC BY license.

Libraries are available with T-Sapphire, EGFP, or mScarlet reporters, so you can choose a color compatible with the other tools in your experiment. These libraries will be useful for studying the impacts of other cancer mutations and non-genetic diversity in human cancer stem cells.

Find the LARRYv2 Libraries here!

• Singh, I., Fernandez-Perez, D., Sanchez, P. S., & Rodriguez-Fraticelli, A. E. (2025). Pre-existing stem cell heterogeneity dictates clonal responses to the acquisition of leukemic driver mutations. Cell Stem Cell, 32(4), 564–580.e6. https://doi.org/10.1016/j.stem.2025.01.012. 
  
  

New AAV6 and AAV11 packaging plasmids from Addgene

By Meghan Rego

Attention AAV users: in an effort to expand your gene delivery toolkit, Addgene now offers packaging plasmids for AAV6 and AAV11!

Why these serotypes? AAV6 offers broad tissue tropism for gene therapy and ex vivo cell engineering applications and is especially well-suited for lung cells via airway administration. AAV11 can transduce neurons for retrograde labeling, making it a powerful tool for circuit tracing and other neuroscience applications (Figure 4). Check out our blog post on AAV serotypes for more about the tissue tropism of these and other AAVs.

Figure 4: AAV6 and AAV11 tropism and applications. A) AAV6 is reported to have broad tissue tropism, including skeletal and cardiac muscle, airway epithelial cells, liver, CNS (limited penetration), retina, hematopoietic cells, and immune cells. B) AAV11 is reported to target skeletal muscle, kidney, spleen, lung, heart, stomach, and brain. C) Recent studies have shown that AAV11 enables potent retrograde labeling of projection neurons. Created with BioRender.com.

Addgene has tested these capsid plasmids in vitro and uses them in our own AAV production processes. Check out our step-by-step protocols for AAV production, purification, and titration. Can't wait? Browse our In-Stock AAV inventory or use our Packaged on Request service for thousands of eligible AAV plasmids to order viral vectors ready to use in your lab.

Find Addgene’s AAV6 and AAV11 packaging plasmids here!

An ATLAS for anterograde synaptic connections

By Emily P. Bentley

While several viral tools can perform neuronal tracing, anterograde tracing poses challenges, from non-selective anterograde transport to a lack of genetic control. ATLAS (anterograde transsynaptic label based on antibody-like sensors) changes that.

Developed by the Arnold Lab, ATLAS is based on an antibody-like protein AMPA.FingR (AF) that binds to AMPA receptor subunit GluA1 found at excitatory synapses (Rivera & Huang, et al., 2025). AF was fused to a presynaptic vesicle protein (VAMP2) and an epitope tag (ALFA-tag, or At) for easy visualization. A cleavage site for endogenous BACE1 protease allows VAMP2-At to remain on the presynaptic cell membrane while AF, fused to a payload, is released into the synapse. Once AF binds to the postsynaptic AMPA receptor and is endocytosed, the Cre recombinase payload activates a floxed reporter gene (genetically encoded or delivered separately).

Figure 5: Mechanism of ATLAS_Cre. VAMP2 = synaptobrevin 2; At = ALFA-tag; BACE = β-secretase; AF = AMPA.FingR; FingR = fibronectin intrabody generated with mRNA display. Modified from Rivera & Huang, et al. (2025) under CC BY-NC-ND license, created with BioRender.com.

When delivered by AAV to various brain regions in mice, ATLAS mediated anterograde labeling across single synapses, a key attribute for neuronal tracing. Labeling was nontoxic and activity-dependent, with more labeling across active synapses. In addition, ATLAS itself could be expressed Cre-dependently to allow tracing exclusively from genetically determined cells. This version of ATLAS works only at excitatory synapses; however, its modular design allows components to be swapped out, potentially broadening its applications.

Find ATLAS plasmids here!

• Rivera, J. F., Huang, H., et al. (2025). ATLAS: a rationally designed anterograde transsynaptic tracer. Nature Methods, 22(5), 1101–1111. https://doi.org/10.1038/s41592-025-02670-x.