Hot Plasmids Spring 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:

• Multiplexed perturbation and decoding in pooled CRISPR screens
• Improved prime editor PE7
• Antibodies to facilitate SARS-CoV-2 research
• New viral vector preps of Voltron2 voltage sensors
• AAV vectors for targeting microglia
• The REVeRT Dual AAV Vector System
• Neurodegeneration Collection updates

Multiplexed perturbation and decoding in pooled CRISPR screens

By Emily Bentley

Building on their CROPseq method for optical pooled CRISPR screening, Paul Blainey’s lab has developed a multiplexing approach to Cas9-based pooled CRISPR screens called CROPseq-multi (Walton et al. 2024). This platform is compatible with enrichment, single-cell sequencing, and optical pooled screens in human cells. Addgene offers vectors for CROPseq-multi using selection by Puromycin, Zeocin®, or nuclear expression of BFP2.

Figure 1: CROPseq-multi uses two sgRNAs with internal barcodes (iBARs), multiplexed using tRNAs, within the lentiviral 3’ long terminal repeats (LTR). The 3’ LTR is duplicated during lentiviral integration, producing a second copy of the sgRNAs. Image reused from Walton et al. 2024 under a CC-BY-NC-ND license.

In this iteration, the size of the lentiviral insert was minimized to reduce recombination and avoid separation of the multiplexed elements. The team designed sgRNAs that include an internal barcode and separated multiple sgRNAs with tRNAs, which are cleaved out of the RNA transcript by endogenous processing enzymes (Figure 1). To ensure this tRNA processing only occurs on the RNA polymerase III transcript, the sgRNAs were encoded on the lentiviral minus strand in the 3’ LTR. These optimizations produced 90% accurate integration of multiplexed constructs into target genomes and a 10-fold improvement in detection efficiency over the original CROPseq.

Find CROPseq-multi plasmids here

• Walton, R.T., Qin, Y., Blainey, P.C. (2024). CROPseq-multi: a versatile solution for multiplexed perturbation and decoding in pooled CRISPR screens. bioRxiv 2024.03.17.585235; doi: https://doi.org/10.1101/2024.03.17.585235.

Improved prime editor PE7

By Andrew Hempstead

Numerous advances have been made since prime editing was first described in Anzalone et al., 2019, including modifications to the Cas9 or reverse transcriptase enzymes, inhibition of host cell proteins, and modifications to the pegRNA. The Britt Adamson Lab recently used a CRISPRi screen to identify additional host factors impacting prime editing and found the protein La (a small RNA-binding exonuclease protection factor) promotes editing (Yan, J et al., 2024). They developed a new prime editor that incorporates La’s N-terminal domain, PE7 (Figure 2). 

PE7 shows enhanced editing efficiencies in a number of cell types at different genomic loci — in some cases, over 20-fold higher editing than PEmax. The authors hypothesized that the improved efficiency is at least in part due to La binding and stabilizing the 3’ end of the pegRNA, which is not well protected by Cas9. This is similar to the proposed mechanism for engineered pegRNAs (epegRNAs), which add a structural motif to stabilize the 3’ end of the pegRNA. As research has just begun with PE7, it will be exciting to see future studies enabled by this tool as well as additional potential improvements to the editor.

Find PE7 plasmids here!

Figure 2: PE7 improves prime editing. A) Schematic of PE7 prime editor. B, Prime editing efficiencies at different loci in U2OS cells. Image adapted from Yan et al. 2024 under a CC-BY license.

• Yan, J., et al. (2024) Improving prime editing with an endogenous small RNA-binding protein. Nature. 2024 Apr 3. doi: https://doi.org/10.1038/s41586-024-07259-6. PMID: 38570691.
  
  

Antibodies to facilitate SARS-CoV-2 research  

By Ashley Waldron

Ready-to-use recombinant antibodies were recently added to the Addgene repository of tools for SAR-CoV-2 research. Two new antibodies, Anti-SARS-CoV-2 Nucleocapsid Protein [mBG17] and Anti-SARS-CoV-2 Nucleocapsid Protein [mBG86], target distinct epitopes on the SARS-CoV-2 nucelocapsid protein, one of the virus’s most abundant proteins and a critical component for viral genome packaging. Addgene recommends both for use in western blots (Figure 3). 

Figure 3: HeLa cells transduced with SARS-CoV-2 nucleocapsid protein (Plasmid #141391-LV [+N]) or membrane protein (Plasmid #141386-LV [+M], negative control) were immunoblotted against Anti-SARS-CoV-2 Nucleocapsid Protein antibodies #211756 (left panel) or #211757 (middle panel). Samples were also immunoblotted against vinculin as a loading control (right panel). Image from Addgene.

These antibodies are recombinant versions of mouse monoclonal antibodies generated and characterized by the Geiss Lab in their effort to fill a void in available tools for detecting SARS-CoV-2 (Terry et al., 2021). Check out their paper for details on the original antibodies.

Find Anti-SARS-CoV-2 Antibodies here!

• Terry, J.S., et al. (2021). Development of a SARS-CoV-2 nucleocapsid specific monoclonal antibody. Virology. 558, 28–37. https://doi.org/10.1016/j.virol.2021.01.003. PMID: 33714753.
  
  

Voltron2 voltage sensors

By Brian O’Neill

New viral vector preps expressing the Voltron2 voltage indicator, created by the Schreiter Lab and the GENIE Project at Janelia, are now available! Building on their earlier Voltron indicators (a fusion of Ace2N and HaloTag, Figure 4), they determined that an A122D mutation in the rhodopsin domain conferred several new improvements. The new variant has increased signal-to-noise ratio and better ability to record subthreshold voltage fluctuations, while having high fidelity during action potentials of fast-spiking neurons. The same A-to-D mutation in the related voltage indicator Ace2N-mNeon also led to similar improvements in performance. 

Figure 4: Structure of Voltron or Voltron2 conjugated to a JaneliaFluor dye emitting at 525 nm. Image reused from https://www.janelia.org/open-science/voltron-and-positron. 

Both Ace2N-mNeon-A122D and Voltron2 are available as Cre-dependent AAV1 vectors and are suitable for extended imaging of neurons in vivo. Because they are negative-going sensors (with fluorescent signal at baseline voltage), the investigators found that administering these AAVs with a dilute Cre-expressing vector resulted in a sparsely-labeled field of view that was more desirable than having many closely-packed labeled cells. Plus, Voltron2’s fluorescence is tunable by changing the dye that becomes conjugated with its HaloTag domain. 

Find Voltron2 plasmids here!

• Abdelfattah, A.S., et al. (2023) Neuron, 111(10), 1547–1563.e9. doi: https://doi.org/10.1016/j.neuron.2023.03.009. PMID: 37015225. 
  
  

Simple and highly specific targeting of microglia with AAV

By Alyssa Neuhaus

Genetic modification of microglial immune cells is critical for central nervous system research; however, microglia are notoriously resistant to viral transduction. Through in vivo screening of human promoters, the Zhang Lab found a short fragment of human IBA1 promoter (hIBA1a, 466 bp) that drives high microglia-specific gene expression when incorporated in an AAV vector (Figure 5).

Efficiency and specificity of these AAV vectors for microglia was further enhanced by packaging the virus as self-complementary AAV (scAAV). However, scAAV vectors have limited size compared to single-strand AAV (ssAAV). To maintain the packaging capacity and improve the specificity of ssAAV, a targeting sequence of miR124T was inserted into the vector to silence transgene expression in neurons and other non-microglia cells. 

Figure 5: A) Representative confocal images showing GFP expression from ssAAV5s with hIBA1 or hIBA1a promoters in mice. B) Quantifications showing high microglia specificity of GFP expression for the indicated ssAAV5s. C) Quantifications showing high microglia transduction efficiency for the indicated ssAAV5s. Figure adapted from Serrano et al. 2023 under a CC-BY-NC-ND license. 

Integrating hIBA1a promoter into AAV vectors, either alone or in combination with miR124T targeting sequences, provides a simple yet valuable tool for microglia research and broader therapeutic applications. 

Find microglia-targeting AAV plasmids here!

• Serrano, C., et al. (2023). Simple and Highly Specific Targeting of Resident Microglia with Adeno-Associated Virus. bioRxiv 2023.12.12.571321; doi: https://doi.org/10.1101/2023.12.12.571321. 
  
  

Revolutionizing Gene Therapy: The REVeRT Dual AAV Vector System

By Vaibhav Kawde

To circumvent the payload limitations of traditional AAVs, the Becirovic Lab has developed REVeRT, for gene delivery using reconstitution via mRNA trans-splicing. By splitting a large gene into smaller fragments, delivering them by individual AAVs, and then splicing their mRNAs, the functional gene product is reconstituted within the target cells (Figure 6).

Figure 6: Illustration of split fluorophore assay to test reconstitution via mRNA trans-splicing. A cis-splicing vector served as positive control (cis-ctrl). v1, vector 1; v2, vector 2; BD, binding domain; SDS, splice donor site; SAS, splice acceptor site; pA, polyadenylation signal. Image reused from Riedmayr et al. 2023 under a CC-BY license. 

The authors used REVeRT to functionally reconstituting coding sequences for a range of applications, including CRISPR activation by dCas9-VPR, prime editing, and delivery of disease-relevant large proteins. They demonstrated REVeRT in various contexts in vitro and in vivo, including in human retinal organoids and live mice. Importantly, REVeRT avoids the introduction of extraneous genetic material to reconstitute the gene of interest, reducing the immunogenicity typically associated with viral vector-based therapies. REVeRT supports advanced gene expression regulation, enhancing both the mechanisms of delivery and the potential for therapeutic success.

Find REVeRT plasmids here!

• Riedmayr, L.M., et al. (2023). mRNA trans-splicing dual AAV vectors for (epi)genome editing and gene therapy. Nat Commun., 14(1), 6578. https://doi.org/10.1038/s41467-023-42386-0. PMID: 37852949.
  
  

New and notable Neurodegeneration Collection updates

Last but not least, check out the new and noteworthy tools featured in Addgene’s Neurodegeneration Research Collection. This page is regularly updated with useful materials for research into Alzheimer’s, Parkinson’s, ALS, and Huntington’s Disease, including plasmids, antibodies, viral vector preps, CRISPR tools, and more.