15 Years of Addgene: The Top 15 Plasmids

By Jennifer Tsang

15 years of plasmid sharing has certainly taken us on many adventures...From moving office locations three times, to opening our UK office in 2014, to starting our viral vector service in 2016...we’re excited to help scientists share their reagents with the scientific community.

15 years addgene 

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As we look ahead to the things to come, we wanted to take a look back at some of the contributions from our depositors. Below are the 15 top requested plasmids over the last 15 years - the majority of these being lentiviral or CRISPR tools. How many times have these plasmids been shared in total? Just over 49,000 times! Their long-lasting popularity illustrates the utility these tools have in many different areas of biology as they grow.

The top 15 plasmids requested from Addgene

  1. psPAX2  - psPAX2 is a 2nd generation lentiviral packaging plasmid from the Didier Trono lab that expresses the HIV gag, pol, rev, and tet genes. It can be used with 2nd or 3rd generation lentiviral vectors and an envelope expressing plasmid, such as our second most requested plasmid of all time, pMD2.G. Find psPAX2.

  2. pMD2.GAlso from the Didier Trono lab is pMD2.G, a VSV-G envelope expressing plasmid. Envelope proteins determine which types of cells a virus can infect. The VSV-G envelope protein enables this virus to infect many different cell types so it is useful in a wide range of applications. This plasmid was the top requested plasmid of 2017Find pMD2.G.

  3. pSpCas9(BB)-2A-GFP (PX458) - This plasmid from the Zhang lab expresses Cas9 from Streptococcus pyogenes with an invariant sgRNA scaffold and cloning sites for inserting the guide sequence (pSpCas9(BB)). The 2A-GFP allows for screening of transfected cells. Find pSpCas9(BB)-2A-GFP (PX458).

  4. lentiCRISPR v2 - This 3rd generation lentiviral transfer vector also from the Zhang lab is used to deliver Cas9 and your gRNA of interest into mammalian cells. This vector was the top requested plasmid of 2015. What makes lentiCRISPR v2 great? It produces a ~10 fold higher titer virus, making it ideal for CRISPR pooled library screening experiments. Find lentiCRISPR v2.

    (If you’ve peeked through the rest of the list at this point, you may have noticed that many other CRISPR plasmids are from the Zhang lab. In fact, the Zhang lab has shared over 40,000 CRISPR-Cas9 components over the years on their own or through Addgene!)

  5. pX330-U6-Chimeric_BB-CBh-hSpCas9 - The S. pyogenes Cas9 (SpCas9) has been widely popular in genome engineering. For use in human cell lines, the Zhang lab codon-optimized SpCas9. Using the BbsI restriction site, a pair of annealed oligos can be cloned to create a chimeric guide RNA. Find pX330-U6-Chimeric_BB-CBh-hSpCas9.

  6. pSpCas9(BB)-2A-Puro (PX459) V2.0This plasmid from the Zhang lab is a mammalian vector that expresses both SpCas9 and your gRNAs and was also the top requested plasmid from 2016. You may notice the “V2.0” at the end of this plasmid which indicates that there was a prior version of this plasmid. Indeed the first version is the 15th most popular plasmid of all time, but was found to have a point mutation in the puromycin cassette that makes it less efficient in some cell lines. pSpCas9(BB)-2A-Puro (PX459) V2.0 was made available through Addgene in 2015, making this the most recently available plasmid in the top 15. Find pSpCas9(BB)-2A-Puro (PX459) V2.0.
  7. pCMV-VSV-GpCMV-VSV-G was deposited by the Bob Weinberg lab and contains an envelope protein for producing lentiviral and MuLV retroviral particles. It can be used with packaging plasmids such as pCMV-dR8.2 dvpr (lentiviral) or pUMVC (MuLV retroviral). Fun fact: published in 2003, this plasmid predates Addgene’s founding. Find pCMV-VSV-G.

  8. pRSV-Rev - This 3rd generation lentiviral packaging plasmid from the Trono lab contains the rev gene. It’s meant to be used with pMDLg/pRRE and the envelope expressing plasmid pMD2.G, both deposited from the same lab. Find pRSV-Rev.

  9. pMDLg/pRRE - This is a 3rd generation lentiviral packaging plasmid from the Trono lab that contains the gag and pol genes. It also requires pRSV-Rev and the envelope expressing plasmid pMD2.GFind pMDLg/pRPE.

  10. scramble shRNA - This plasmid from David Sabatini’s lab is a 3rd generation lentiviral vector that contains a scrambled shRNA. This scrambled shRNA is used as a negative control for mammalian RNAi experiments.  Find the scrambled shRNA vector.

  11. pLKO.1 - TRC cloning vector - This vector from the David Root lab is a 3rd generation lentiviral transfer plasmid for cloning and expressing new shRNA sequences. For more information, see the Addgene’s pLKO.1 protocol. Pro-tip: this plasmid grows more slowly than standard plasmids. Find pLKO.1 - TRC cloning vector.

  12. Tet-pLKO-puro - Tet-pLKO-puro contains all the necessary elements for packaging, reverse transcription, and integration needed for the production of lentiviral particles. It’s capable of inducible expression of shRNA in target cells by the addition of tetracycline/doxycycline to the grown media resulting in target gene knockdown. Note: this plasmid was called pLKO-Tet-On in the original publication but its name was subsequently changed to clarify that it does not contain the trademarked Tet-On(R) sold by Clontech. For additional information, see the manual provided by the Weiderschain labFind Tet-pLKO-puro.

  13. lentiGuide-Puro - This plasmid expresses the S. pyogenes CRISPR chimeric RNA element with customizable sgRNA from the U6 promoter and puromycin resistance from the EF-1a promoter. It’s a 3rd generation lentiviral backbone that must be used with lentiCas9-Blast or with cell lines already expressing Cas9. Find lentiGuide-Puro.

  14. pSpCas9n(BB)-2A-GFP (PX461)Similar to pSpCas9(BB)-2A-Puro (PX458), pSpCas9n(BB)-2A-GFP (PX461) expresses both a Cas9 protein and a gRNA. But instead of Cas9, this plasmid expresses Cas9n (a D10A nickase mutant) to nick, rather than cleave DNA. Find pSpCas9n(BB)-2A-GFP (PX461).

  15. pSpCas9(BB)-2A-Puro (PX459)This mammalian expression vector encodes both SpCas9 and your gRNA. It’s the former version of our sixth most popular plasmid pSpCas9(BB)-2A-Puro (PX459) V2.0. It contains a point mutation in the puromycin cassette that makes it less efficient in some cell lines so it may be best to go with pSpCas9(BB)-2A-Puro (PX459) V2.0. Find pSpCas9(BB)-2A-Puro (PX459).

Thank you!

We would like to thank all of our depositors and requestors for making their reagents available and for supporting scientific sharing. We’re very excited for more reagent sharing projects in the years to come.

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