Hot Plasmids Spring 2024

By Multiple Authors

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

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.


Three schematic panels show the design, integration, and expression of a lentiviral vector for conducting pooled screens with CROPseq-multi.  The first panel shows the design of a CROPseq-multi vector. It includes a 5’ LTR, an EF1a promoter, a selection gene, and an expanded 3’ LTR. The 3’ LTR contains the multiplexed screening insert in reverse orientation, including a U6 promoter, tRNA 1, sgRNA 1, tRNA 2, and sgRNA 2. Each sgRNA includes a matching spacer and iBAR. The second panel shows the sequence after lentiviral integration into the genome. The 3’ LTR has been duplicated to the 5’ end of the sequence. The full sequence now includes the reversed multiplexed insert under control of the U6 promoter (left), the selection gene under control of the EF1a promoter (center), and the original 3’ copy of the reversed multiplexed insert (right).  The third panel shows how this sequence is transcribed and processed. (1) The duplicated U6 promoter, now on the leftmost end of the sequence, drives minus-strand pol III transcription of the multiplexed insert. This produces an RNA sequence with tRNA 1, sgRNA 1, tRNA 2, and sgRNA 2. Endogenous RNases P and Z cleave the tRNAs out of this sequence, yielding two different mature sgRNAs. (2) In the center, the EF1a promoter drives plus-strand pol II transcription. This produces a barcoded mRNA sequence that includes the selection gene and the reverse complement of the multiplexed insert. (3) The rightmost U6 promoter is not shown driving transcription.


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:


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!


Panel A shows a diagram of PE7 components: an NLS, Cas9-R221K-N394K-H840A, linker, MMLV-RT, linker, La (full-length or aa 1-194), and NLS. Panel B shows bar graphs comparing the % of sequencing reads with edit, for PEmax or PE7 with pegRNA or epegRNA for eight different genomic targets. Values range widely from near-zero to 25%. The increases of PE7 over PEmax are highlighted, ranging from 9.8x to 50.0x. Data values are available in Supplementary Table 7 of Yan et al. 2024.


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.

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). 


Three western blots. Left, labeled 211756, has no signal in the +M lane, while the +N lane has a major band just above 50 kDa with a few very faint minor bands. Middle, labeled 211757, has no signal in the +M lane, while the +N lane has one clean band just above 50 kDa. Right, labeled Vinculin, has two identical sets of bands in the +M and +N lanes with the major band between 115 and 185 kDa.


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!

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. 


A protein structure showing the Ace2N rhodopsin voltage sensing domain as a ribbon diagram with seven alpha helix bundle. The end of one of the helices is linked to a gray oval with a green wedge labeled HaloTag with JF-525 dye.


Figure 4: Structure of Voltron or Voltron2 conjugated to a JaneliaFluor dye emitting at 525 nm. Image reused from

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!

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. 


Panel A shows microscopy images of GFP expressed in brain cells with the full hIBA1 promoter versus the hIBA1a promoter fragment in mice with stroke and with sham. Green GFP signal largely overlaps with red IBA1 signal in both hIBA1 and hIBA1a in all conditions, but the intensity of the GFP signal is much brighter in stroke than sham condition. Panel B shows bar graphs of GFP specificity, with values of around 70 to 85% for both hIBA1 and hIBA1a in all conditions. Panel C shows bar graphs of the transduction efficiency, with values around 35% for both hIBA1 and hIBA1a in sham and around 70% for both hIBA1 and hIBA1a in stroke.


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!

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).


The upper row of the diagram shows two vectors, v1 and v2, carrying gene segments labeled 5’ Cerulean and 3’ Cerulean, respectively, along with regulatory elements including promoters, binding domains, a splice donor site on v1, a splice acceptor site on v2, and polyadenylation signals. When the resulting two mRNAs are bound via their binding domains, they undergo mRNA trans-splicing where the 5’ and 3’ Cerulean segments from v1 and v2 become joined into one mature mRNA. In the lower part of the diagram, the cis-splicing control vector has 5’ Cerulean and 3’ Cerulean separated by a splice donor site, an intron, and a splice acceptor site. This mRNA undergoes cis-splicing to remove the intron. In each case, the successful splicing results in a full-length mRNA that is translated into a full-length protein.

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!

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.


Topics: Hot Plasmids

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