Hot Plasmids: Summer 2024

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

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

• MagIC-Cryo-EM for enrichment and structure determination
• LEA proteins protect samples during cryo-EM grid freezing
• Engineered and evolved optimizations for prime editing
• CHARM: A compact epigenetic silencer
• Recombinant antibodies for marking astrocytes
  
  

MagIC-Cryo-EM for sample enrichment and structure determination

By Mike Lacy

Conventional cryogenic electron microscopy (cryo-EM) protocols typically require high concentration and purity of the target molecule, which can be problematic for low-abundance targets or natively isolated complexes. To address these limitations, the Funabiki Lab developed a technique called Magnetic Isolation and Concentration (MagIC)-cryo-EM that combines enrichment and imaging of proteins captured on magnetic beads (Arimura et al., 2024).

In MagIC-cryo-EM, target proteins (expressed as a GFP fusion in this proof-of-concept study) are captured on streptavidin-coated magnetic beads through an assembly of biotinylated SpyTag-SpyCatcher linkers and spacer proteins ending with a GFP nanobody (Figure 1). This enrichment streamlines the purification and minimizes sample loss while also supporting imaging, making it easy to identify dozens or hundreds of particles surrounding each of the highly-visible beads on the cryo-EM grid.

Figure 1: MagIC-cryo-EM for sample enrichment and structure determination. A) Schematic of workflow. B) Detail of spacer peptides (3HB: 11-nm 3-helix bundle; SAH: 60-nm single alpha helix) and target capture module (SpyTag-GFP nanobody) assembled on beads. C: Micrograph of H1.8-GFP nucleosomes collected with MagIC-cryo-EM. Nucleosome-like particles identified by Topaz software (green circles), magnetic beads (*), and spacers are labeled; scale bar: 100 nm. D) 3D model of the obtained structure of the H1.8-GFP-bound nucleosome. Images adapted from Arimura et al. 2024 under CC-BY-NC-ND 4.0 International license. 

Arimura et al. used MagIC-cryo-EM to determine several structures (at < 5 Å resolution) of the linker histone H1.8 in Xenopus nucleosomes and in complex with its chaperone NPM2, but the technique should be widely applicable to other targets. The modular system of linkers and spacers could enable many future variations using nanobodies or scFvs for other targets or could be used for applications beyond cryo-EM.

Find plasmids for MagIC-Cryo-EM here!

Arimura, Y., Konishi, H.A., Funabiki, H. (2024) MagIC-Cryo-EM: Structural determination on magnetic beads for scarce macromolecules in heterogeneous samples. bioRxiv 2024.01.21.576499; doi: https://doi.org/10.1101/2024.01.21.576499. 

LEA proteins protect fragile samples during cryo-EM grid plunge freezing

By Rachel Leeson

Cryo-EM samples can be damaged at the air-water interface (AWI) during the freezing process, resulting in sample loss. Ci Ji Lim’s lab found that adding Late Embryogenesis Abundant (LEA) proteins, small proteins that naturally adsorb to the AWI, can protect cryo-EM samples from damage (Abe & Lim, 2024).
 

Figure 2: A) When proteins (blue) interact with the air-water interface (AWI) before vitrification, they can be completely denaturated (left) or complexes may dissociate  (right). B) Protein samples protected from AWI by helical LEAs. C-E) cryo-EM single-particle analysis of PRC2 with RvLEAMshort (1:6 molar ratio) with 10 minutes of glutaraldehyde crosslinking: representative micrograph (C), particle orientation distribution and Fourier-shell correlation curve (D), and the reconstructed 3D map with 3.1 Å global resolution (E). Figure adapted from Abe & Lim, 2024 under CC-BY-NC-ND 4.0 license.

Protection by the LEAs allowed the authors to determine structures at comparable or higher resolution than using more complex protective methods or detergents like CHAPSO, while requiring a lower concentration of sample — nearly ten times less protein. While the addition of LEAs may bias the distribution of particle orientations, this could be overcome with methods such as chemical crosslinking or tilted-stage imaging. A truncated form of a tardigrade LEA, RvLEAM_short, protected samples and was compatible with the buffer additive MgCl₂ and the crosslinking agent glutaraldehyde. Adding RvLEAM_short to the sample before plunge freezing represents a convenient, effective approach to protecting your cryo-EM samples from damage at the AWI. Plus, tardigrades! 

Find RvLEAMshort here! 

Abe, K. M., & Lim, C. J. (2024). Small LEA proteins as an effective air-water interface protectant for fragile samples during cryo-EM grid plunge freezing. bioRxiv 2024.02.06.579238. doi: https://doi.org/10.1101/2024.02.06.579238. 

Engineered and evolved optimizations for prime editing

By Emily P. Bentley

The David Liu Lab has made two recent deposits focused on optimizing prime editing systems for specific applications!

Delivering prime editors (PEs) via engineered virus-like particles (eVLPs) offers an option to transduce cells directly with ribonucleoprotein cargo. eVLPs contain no genetic material, which ensures the editor will only be transiently present in transduced cells, limiting off-target editing. Cargo size limits are also less stringent for eVLPs than other viral delivery methods, allowing all the PE components to be delivered in a single package. In a recent publication, the Liu lab showed that this method of delivery could be used to partially correct mouse models of genetic blindness (An et al., 2024) — the first demonstrated use of prime editing to rescue a genetic disease phenotype in an animal model.

Find PE-eVLP plasmids here!

The lab’s other recent deposit builds on Prime-editing-Assisted Site-Specific Integrase Gene Editing (PASSIGE), their previously-reported technique for precisely integrating large DNA sequences into a genome (Anzalone et al., 2022). While prime editing and twin prime editing can integrate DNA cargoes of 80–100 bp, PASSIGE can integrate cargoes over a hundred times that size (demonstrated up to 10.5 kb) by relying on a site-specific serine recombinase. First, the prime editor installs the recombinase “landing sites,” then the recombinase handles the installation of the donor sequence. The latest work evolves and engineers Bxb1 recombinase variants to optimize PASSIGE efficiency (Pandey et al., 2024). The best-performing variant yielded an average integration efficiency of 23%, a more than four-fold improvement over the wild-type enzyme.

Find optimized PASSIGE plasmids here!

Figure 3: Schematic of the PASSIGE strategy. First, twin prime editing installs two sequences containing attB/P recombinase landing sites on opposite DNA strands. When the prime editor releases the DNA, the two 3’ flaps containing the edited sequence anneal with each other, while the 5’ flaps containing the original DNA sequence anneal with each other. Excision of the annealed 5’ flaps yields an edited sequence with the attB/P landing site. A recombinase integrates a sequence from a donor plasmid containing a complementary site into the genome at this site, resulting in the integrated sequence between two attR/L sites. Image reused from Pandey et al. 2024 under CC-BY 4.0 license.

An, M., Raguram, A., Du, S. W., Banskota, S., Davis, J. R., Newby, G. A., Chen, P. Z., Palczewski, K., & Liu, D. R. (2024). Engineered virus-like particles for transient delivery of prime editor ribonucleoprotein complexes in vivo. Nature Biotechnology, 10.1038/s41587-023-02078-y. doi: https://doi.org/10.1038/s41587-023-02078-y. 

Anzalone, A. V., Gao, X. D., Podracky, C. J., Nelson, A. T., Koblan, L. W., Raguram, A., Levy, J. M., Mercer, J. A. M., & Liu, D. R. (2022). Programmable deletion, replacement, integration and inversion of large DNA sequences with twin prime editing. Nature Biotechnology, 40(5), 731–740. doi: https://doi.org/10.1038/s41587-021-01133-w

Pandey, S., Gao, X. D., Krasnow, N. A., McElroy, A., Tao, Y. A., Duby, J. E., Steinbeck, B. J., McCreary, J., Pierce, S. E., … & Liu, D. R. (2024). Efficient site-specific integration of large genes in mammalian cells via continuously evolved recombinases and prime editing. Nature Biomedical Engineering, 10.1038/s41551-024-01227-1. doi:  https://doi.org/10.1038/s41551-024-01227-1

CHARM: A compact epigenetic silencer

By Emily P. Bentley

The Weissman and Gilbert Labs have previously deposited their CRISPRoff system — a programmable epigenetic editor consisting of dCas9 fused to a DNA methyltransferase — that can drive lasting transcriptional silencing (Nuñez et al., 2021). Now, in a collaboration with the Vallabh/Minikel Lab, the Weissman Lab has developed a new epigenetic editor, Coupled Histone tail for Autoinhibition Release of Methyltransferase (CHARM), with several advantages over the earlier system. 

Rather than overexpressing an exogenous methyltransferase for epigenetic silencing, which can be toxic, CHARM uses a cofactor fused to a histone tail to recruit and stimulate the endogenous methyltransferase. The strategy is generalizable to a variety of DNA-targeting proteins, so the team created versions using dCas9, zinc finger proteins, and transcription activator-like effectors (TALEs), with fifteen total variants deposited with Addgene. These variants, including a “miniCRISPR” version, are much smaller proteins than SpyCas9, allowing them to be packaged in AAV vectors, potentially with multiplexed targets.

Figure 4: CHARM uses a fusion of the DNMT3L cofactor (D3L) with a histone tail (H3K4me0) to a DNA-binding protein (dCas9 shown here). D3L recruits the endogenous, autoinhibited methyltransferase DNMT3A to the genomic location specified by the DNA-binding protein. The ADD domain of the methyltransferase binds to H3K4me0 on D3L, releasing the autoinhibition and stimulating the methylation of nearby CpG sites on the target DNA, and thus inactivating the promoter. Image reused with permission from Neumann et al. 2024.

The team showed that AAV-delivered CHARM could silence the expression of prion protein in mouse brains for at least six months. Unlike genome editing, this epigenetic strategy doesn’t induce DNA damage or permanently alter a gene product; it simply turns the target protein expression off. Plus, DNA methylation is a long-lasting repressive marker, allowing the team to develop self-silencing CHARM variants that stop their own expression after silencing their target — potentially improving safety without compromising their efficacy.

Find CHARM plasmids here!

Neumann, E. N., Bertozzi, T. M., Wu, E., Serack, F., Harvey, J. W., Brauer, P. P., Pirtle, C. P., Coffey, A., Howard, M., Kamath, N., … & Weissman, J. S. (2024). Brainwide silencing of prion protein by AAV-mediated delivery of an engineered compact epigenetic editor. Science, 384(6703), ado7082. doi: https://doi.org/10.1126/science.ado7082. 

Nuñez, J. K., Chen, J., Pommier, G. C., Cogan, J. Z., Replogle, J. M., Adriaens, C., Ramadoss, G. N., Shi, Q., Hung, K. L., Samelson, A. J., … & Weissman, J. S. (2021). Genome-wide programmable transcriptional memory by CRISPR-based epigenome editing. Cell, 184(9), 2503–2519.e17. doi: https://doi.org/10.1016/j.cell.2021.03.025. 

Recombinant antibodies for marking cell types in the nervous system

By Ashley Waldron

The mammalian nervous system is composed of myriad cell types and subtypes, and a familiar challenge for anyone who has done histology on brain tissue is identifying the cells you are studying. Antibodies can help reveal cellular identities by targeting marker proteins that are uniquely expressed in a given cell type. For example, glial fibrillary acidic protein (GFAP) is a glia-specific intermediate filament expressed in astrocytes throughout the brain, making it a popular target for labeling these cells. Check out Addgene’s Neuroscience Antibody Collection to find a recombinant anti-GFAP [N206A/8R] antibody shared by James Trimmer’s lab (Figure 5) as well as antibodies for identifying other cell types in the nervous system! 

Find recombinant anti-GFAP [N206A/8R] antibody here!

Figure 5: Free-floating rat brain sections stained with Anti-GFAP. The staining patterns from recombinant antibodies produced at Addgene (N206A/8R) were compared to the hybridoma-derived parent antibody (N206A/8 TC Supernatant). Image attribution: James Trimmer, UC Davis.