Hot Plasmids: Winter 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:

• Optimized prime editing in dicots
• Self-labeling tags for single-molecule biophysics
• Fast and responsive voltage indicator
• ONE-GO biosensors kit
• Antibodies for neurodegeneration research

Optimized prime editing in dicots

By Emily P. Bentley

Prime editing (PE), initially developed in human cells, has been uniquely challenging to apply in dicotyledonous plants, or dicots. But Jae-Yean Kim’s lab recently "broke the PE efficiency barrier in tomatoes" by combining several approaches (Vu et al., 2024). Their optimized strategy uses new combinations of PE protein components (Figure 1), an altered epegRNA delivered with an enhanced pU6cm promoter, viral replicon amplification of cargo, and heat treatment. 

Figure 1: Left: recombined prime editing tools PE2max-NC and ePEmax3 optimized for dicots. NC: nucleocapsid RNA chaperone, RT: reverse transcriptase. Right: T1 tomato plants that inherited an edited gene to confer resistance to the herbicide chlorsulfuron (Homo line 2) or wild type (WT), two weeks after herbicide was applied to both plants. Adapted from Vu et al., 2024 bioRxiv, under CC-BY-NC-ND 4.0 International license.

With this approach, the team was able to drive prime editing in both tomatoes and Arabidopsis, suggesting the improvements may be broadly applicable to dicot species. In addition, they generated offspring tomato plants that inherited the genomic edit but not the editing machinery. Together, these advances have the potential to improve rapid and precise plant breeding.

Find dicot prime editing plasmids here!

• Vu, T. V., Nguyen, N. T., Kim, J., Song, Y. J., Nguyen, T. H., & Kim, J.-Y. (2024). Optimized dicot prime editing enables heritable desired edits in tomato and Arabidopsis. Nature Plants, 10(10), 1502–1513. doi: https://doi.org/10.1038/s41477-024-01786-w. (bioRxiv preprint: https://doi.org/10.1101/2024.02.11.579803). 

CLIP, CLAP, SNAP! Self-labeling tags for single-molecule biophysics

By Mike Lacy

In order to study the conformational dynamics of the motor protein kinesin-1, the Twelvetrees Lab took advantage of the self-labeling tags SNAPf and CLIPf. These tags rely on synthetic dye molecules, which are much brighter and more photostable than fluorescent proteins, making them ideal for single-molecule fluorescence techniques. After generating a series of single- and double-tagged constructs (including "CLAP", combining an N-terminal CLIPf tag and a C-terminal SNAPf tag; see Figure 2), the authors developed a cost-effective expression and labeling approach using these constructs in HEK 293 cells (Smith et al., 2024). They then used single-molecule FRET to study the intramolecular dynamics of the proteins in solution and when associated with microtubules.

Figure 2: CLIP-SNAP fusion proteins for single-molecule FRET. A) Schematic of a CLIP-SNAP fusion protein (a.k.a. CLAP) with a flexible linker where a protein of interest can be inserted. B) Protein expression and labeling procedure. Adapted from Smith et al. 2024 under a CC-BY 4.0 license. 

The team deposited an array of constructs for tagging the kinesins KIF5A and KIF5B as well as backbones for generating other SNAPf-, CLIPf-, and CLAP-tagged proteins for mammalian cell expression. These plasmids will be useful not only for future studies of kinesins but for many other proteins of interest too!

Find CLIP, CLAP, and SNAP plasmids here!

• Smith, E.R., Turner, E.D., Abdelhamid, M.A.S., Craggs, T.D., & Twelvetrees A.E. (2024) Kinesin-1 is highly flexible and adopts an open conformation in the absence of cargo. bioRxiv 2024.12.20.629623; doi: https://doi.org/10.1101/2024.12.20.629623. 

A fast and responsive voltage indicator with enhanced sensitivity for unitary synaptic events

By Brian O’Neill

The Lin Lab and collaborators recently created ASAP5, a faster and more responsive genetically-encoded voltage indicator (GEVI) than previous versions. ASAP5 can be imaged using one- or two-photon microscopy to detect action potentials and subthreshold events, including miniature excitatory postsynaptic potentials (mEPSPs) (Hao and Lee et al., 2024).

While studying mEPSPs has been possible with conventional electrophysiological methods, it has remained difficult with GEVIs. Not only was ASAP5 able to detect mEPSPs, it also enabled imaging of their propagation along dendrites for the first time (Figure 3). The team also used AAV9 to deliver ASAP5 to the cortex and imaged voltage fluctuations in awake behaving mice through two-photon microscopy at depths up to 600 µm.

Figure 3: ASAP5 for voltage imaging. A) ASAP5 selected for its improved kinetics and responsivity in screening ASAP3 variants. B) Electrical and optical recordings of spontaneous activity in human stem cell-derived neurons expressing ASAP5-Kv. C) Cultured rat hippocampal neuron expressing pan-membrane ASAP5. Solid blue circle marks the initiation site and open circles mark pixels used to measure signal along the dendrite. D) Fluorescence signal as a function of the distance between measured pixel and the soma. The initiation site identified in panel (C) is indicated; red line is exponential fit. Adapted with permission from Hao and Lee et al., 2024.

The authors have deposited AAV plasmids for Cre-dependent and -independent versions that are trafficked to the soma via the Kv2.1 targeting motif as well as a pan-membrane construct without the Kv motif. Altogether, if you want a negative-going GEVI that can be used in different illumination regimes with fast activation kinetics and high responsivity near the resting membrane potential, ASAP5 is a great new option.

Find ASAP5 plasmids here!

• Hao, Y. A., Lee, S., Roth, R. H., Natale, S., Gomez, L., Taxidis, J., O'Neill, P. S., Villette, V., Bradley, J., Wang, Z., Jiang, D., Zhang, G., … Lin, M. Z. (2024). A fast and responsive voltage indicator with enhanced sensitivity for unitary synaptic events. Neuron, 112(22), 3680–3696.e8. doi: https://doi.org/10.1016/j.neuron.2024.08.019. 

ONE-GO biosensors kit

By Mike Lacy

G protein-coupled receptors (GPCRs) are critical components of many physiological pathways and represent the largest family of human drug targets. To enable large-scale profiling of many GPCRs, Mikel Garcia-Marcos’ lab recently developed a collection of BRET-based ONE vector G-protein Optical (ONE-GO) biosensors (Janicot and Maziarz et al., 2024). Each plasmid expresses all the components of the biosensor in optimal proportions from a vector backbone that allows lentiviral packaging as a single payload (Figure 4).

Figure 4: ONE-GO biosensors measure the formation of Gα-GTP by BRET. The biosensor consists of a YFP-tagged G protein and a NanoLuc® (Nluc) Luciferase-fused detector that specifically binds to active (GTP-bound) Gα. Gα-YFP assembles into functional heterotrimers with endogenous Gβγ, enabling detection of activation triggered by endogenous GPCR. Example applications include large-scale pharmacological profiling or interrogation of context-dependent signaling in primary cells.

The authors used this platform to assay dozens of GPCRs across many cell types in varying conditions, revealing context-dependent responses and molecular patterns of selectivity. With 10 plasmids available as a kit, plus 21 optional accessory plasmids, the collection targets members of all four G protein families: Gs, Gi/o, Gq/11, and G12/13.

Find the ONE-GO Biosensors plasmids here!

• Janicot, R., Maziarz, M., Park, J. C., Zhao, J., Luebbers, A., Green, E., Philibert, C. E., Zhang, H., Layne, M. D., Wu, J. C., & Garcia-Marcos, M. (2024). Direct interrogation of context-dependent GPCR activity with a universal biosensor platform. Cell, 187(6), 1527–1546.e25. doi: https://doi.org/10.1016/j.cell.2024.01.028.

New recombinant antibodies for neurodegeneration research  

By Ashley Waldron

We recently added two antibodies against important targets for neurodegeneration research: Anti-MAPT [AT8] and Anti-APP [6E10-A5]. Both are recombinant mouse monoclonal antibodies targeting the human genes and are recommended for use in immunohistochemistry (Figure 5). Anti-MAPT [AT8] specifically recognizes the disease-associated version of Tau (phospho-Tau). We hope these will become valuable tools for your research! If they do (or if they don't!), please consider sharing your experience with other researchers by contributing to the Addgene Antibody Data Hub. 

Figure 5: Immunohistochemistry with Addgene's Anti-APP [6E10-A5] recombinant antibody in human postmortem brain samples. Results show positive staining in tissues from patients who had confirmed neurodegenerative diseases (Alzheimer’s disease or Hoarding disorder), but not in a patient with non-neurodegenerative diseases (Diabetes & cancer). Image adapted from Wu and Wang 2024 under a CC-BY 4.0 license.

Find more neuroscience antibodies here!

• Wu, Z., Wang, W. (2024). Anti-APP [6E10-A5] in Immunohistochemistry (Human). Addgene Report, doi: https://doi.org/10.57733/addgene.6ncijt.