...sites tested when compared to eSpCas9(1.1) and SpCas9-HF1. The HypaCas9 plasmids are available from Addgene...evidenced by all the CRISPR publications, press, and plasmids out there, it’s obvious that CRISPR is a ground-breaking... (2015): aad5227. PubMed PMID: 26628643. Find plasmids from this publication at Addgene. 2. Nishimasu..." Nature (2016).  PubMed PMID: 26735016. Find plasmids from this publication at Addgene. 5. Chen, JS...." Nature (2017). PubMed PMID: 28931002. Find plasmids from this publication at Addgene. Resources on...Find CRISPR Plasmids Browse our CRISPR Guide Pages Find Genome Engineering Resources ...the Cas9 nuclease with engineered variants: eSpCas9, SpCas9-HF1, & HypaCas9. The off-target problem It...

...The original pCas/pTargetF system Our original pCas/pTargetF system consists of one plasmid that expresses...pTargetF into the BL21(DE3)/pCas strain. In response, we modified the pCas/pTargetF system and updated...beneficial for plasmid clearance. It is more convenient to update the spacer(20nt) on pEcgRNA plasmid as only.... pCutamp cures the plasmids by targeting the ampR promoter in all three plasmids. pCutamp is then removed... 13 plasmids with 14 rounds of transformation in 30 days. Figure 2: (A) MUCICAT plasmids for .... In pCas9, we 1) replaced the promoter of gRNA-pMB1 with PrhaB, 2) changed the replicon of pCas into ...the use of CRISPR editing systems in bacteria. Our pCas/pTargetF system has been one of the most popular...

...284 . https://doi.org/10.1038/nature24049 Find plasmids from this paper at Addgene. Amrani N, Gao XD,... https://doi.org/10.1126/science.aaq0180 Find plasmids from this paper at Addgene. Kim D, Kim J, Hur...863–868 . https://doi.org/10.1038/nbt.3609 Find plasmids from this publication at Addgene. Kim E, Koo ...8: . https://doi.org/10.1038/ncomms14500 Find plasmids from this publication at Addgene. Kleinstiver...485 . https://doi.org/10.1038/nature14592 Find plasmids from this publication at Addgene. Kleinstiver...869–874 . https://doi.org/10.1038/nbt.3620 Find plasmids from this publication at Addgene. Hu JH, Miller...191 . https://doi.org/10.1038/nature14299 Find plasmids from this publication at Addgene. Walton RT, ...

.... citrea scarless editing plasmids Sheng Yang’s lab describes a two-plasmid system that combines recombineering...editing takes two days, and the pTargetF and pCas plasmids can be subsequently cured from the bacteria....to the original pCas/pTargetF but has many other advantages including shorter plasmid curing time. pCRISPomyces...system available from Addgene consists of two plasmids: pCas9: carries Cas9 and chloramphenicol resistance... lab M. tuberculosis CRISPRi plasmids Noting that the more common spCas9 system has generally low efficiencies...engineering functions in bacteria, but have no fear - plasmids from Addgene depositors are making it easier than...gRNA and a backbone-targeting gRNA cassette for plasmid curing through "self-destruction" after induction...

... resources on the Addgene blog Plasmid 101 Protein Expression Plasmid 101 Biotinylation Other resources...vivo studies (Che and Khavari, 2017). Find APEX plasmids at Addgene! Since the development of BioID and... specific locations in the cell.   Find BioID2 plasmids here APEX2: A more sensitive ascorbate peroxidase... They named this mutant APEX2.  Find the APEX2 plasmids! TurboID and minturbo: Rapid and non-toxic proximity...allowing for more precise temporal control.  Find plasmids for TurboID and miniturbo Split-BioID: A conditional...Split-BioID combines a protein fragment complementation (PCA) technique with proximity labelling to validate binary...additional interacting proteins in one simple experiment. PCA is an in vivo technique where two proteins are fused...

...Addgene.org Find plasmids from the Bethune Lab Find Plasmids from the Roux Lab Find Plasmids for FRET ...called a protein fragmentation complementation assay (PCA). In the Split-BioID method, each BirA* fragment ...BirA* fusion proteins are expressed in cells via plasmids or by the creation of stable cell lines and these...Interested in using Split-BioID with your research? Plasmids are available from Addgene! References Schopp... Central PMCID: 5467174. Find the Split-BioID Plasmids Here! Munter, S.D., Görnemann, J., Derua, R.,...site than in Schopp et al. Find these Split-BioID Plasmids Here!   Burke, B., Kim, D.I., Roux, K.J., & Raida...PubMed Central PMCID: 3308701. Find the BioID Plasmids Here! Macneill, S., & Varnaitė, R. (2016). Meet...

...applications. This plasmid was the top requested plasmid of 2017. Find pMD2.G. pSpCas9(BB)-2A-GFP (PX458...and an envelope expressing plasmid, such as our second most requested plasmid of all time, pMD2.G. Find...the end of this plasmid which indicates that there was a prior version of this plasmid. Indeed the first...envelope expressing plasmid pMD2.G. Find pMDLg/pRPE. scramble shRNA - This plasmid from David Sabatini....1 protocol. Pro-tip: this plasmid grows more slowly than standard plasmids. Find pLKO.1 - TRC cloning...pX330-U6-Chimeric_BB-CBh-hSpCas9. pSpCas9(BB)-2A-Puro (PX459) V2.0 - This plasmid from the Zhang lab is ... that expresses both SpCas9 and your gRNAs and was also the top requested plasmid from 2016. You may notice...

...express both SpCas9 and your gRNA in a variety of mammalian cell types. Purpose  SpCas9 This plasmid expresses...sure, as is obvious from our most requested plasmid, SpCas9 is still going strong as the basis for many...without further ado, the most request plasmid in 2016 was... pSpCas9(BB)-2A-Puro (PX459) V2.0       Similar...the top requested plasmid from 2016 is an expression vector for CRISPR nuclease, SpCas9 from the Zhang Lab...sequence as directed by a gRNA. 2A-Puro In this plasmid, SpCas9 is fused to the puromycin resistance gene through... V2.0. pSpCas9(BB)-2A-Puro (PX459) V2.0: What's not in the name? Not included in this plasmid's name are...resistance gene enables selection of cells with the plasmid post transfection. The 2A "self cleaving" peptide...

...cassette. (b) pCAG-EGxxFP target plasmid and pX330-sgRNA plasmid (Mashiko et al. 2013). The pCAG-EGxxFP target...BbsI site. (c) The pCAG-EGxxFP target plasmid was co-transfected with pX330 plasmids containing sgRNA sequences...your modified pCAG-EGXXFP plasmid along with the individual gRNAs in their PX330 plasmids (or similar plasmids...amplified, you can insert this region into a pCAG-EGXXFP plasmid using standard cloning techniques (The XX...the primers you designed to generate your pCAG-EGXXFP plasmid? They are the perfect primer sets to use ... system floating around, and many pre-designed plasmids containing these variants ready for purchase. ...target plasmid contains overlapping 5′ and 3′ EGFP fragments under the ubiquitous CAG promoter. The approximately...

...highlight a subset of the new plasmids in the repository through our hot plasmids articles. These articles ...recent plasmid deposits and we hope they'll make it easier for you to find and use the plasmids you need... were previously difficult. Find SpCas9 and Target-AID-NG plasmids at Addgene! Nishimasu H et al., Science...need. If you'd ever like to write about a recent plasmid deposit please sign up here.        New toolkit... the Kanemaki lab has created a new toolkit of plasmids for the N- or C- terminal tagging of proteins ...datasets for research and innovation. Find MerMAIDs plasmids at Addgene Opperman J et al., Nature Communications...inducible and reversible nature. Find the LADL plasmids at Addgene Kim et al., Nat Methods. 2019, PubMed...

...highlight a subset of the new plasmids in the repository through our hot plasmids articles. These brief articles...articles provide a synopsis of a plasmid or group of plasmids' functions and applications. We hope that... you to find and use the plasmids you need. You can find all the hot plasmids from 2017 below. With over...over 50,000 plasmids, we can't write posts for every great plasmid that comes into the repository, but...write about your plasmids in a future blog post. No time to read? Listen to our hot plasmids segment on the...editing for precise genome modification. Plasmid ID Plasmid Name Cas9 Variant Speacial Features 85169... to the Addgene blog to get all the latest hot plasmid updates!     Tol2 Gateway-compatiable toolbox...

...Neurosci 2016. PubMed PMID: 26950006 Plasmid ID Plasmid Name Plasmid Type 74309 pcDNA3.0-TRPV4-p2A-ferritin-p2A-mCherry...highlight a subset of the new plasmids in the repository through our hot plasmids articles. These brief articles...features and applications of a partiular plasmid or set of plasmids. We hope that these articles make it ... you to find and use the plasmids you need. You can find all the hot plasmids from 2016 below. With over...over 45,000 plasmids, we can't write posts for every great plasmid that comes into the repository, but...write about your plasmids in a future blog post. No time to read? Listen to our hot plasmids segment on the...stocks in a single plate with every plasmid or as individual plasmids shipped as agar stabs. Iverson, et...

...names of the plasmids you plan to use Enter the base pair size of those plasmids Enter plasmid concentration...: 1 Transfer Plasmid : 1 pHelper: 1 RepCap OR 3 Transfer Plasmid : 5 pHelper: 2 RepCap Select what type...of each plasmid needed per dish or stack. Calculations are based on using 28 μg of total plasmid (Transfer... ug of each plasmid needed for the whole experiment Total volume (μL) of plasmid needed for the whole ...Transfer, helper, and RepCap added together) per 15 cm dish or 2500 μg of plasmids per CF5 stack. Total ug...packaging typically requires transfecting three plasmids at specific molar ratios. To get these ratios ...transfection calculator for AAV packaging Select the plasmid molar ratio you would like to use for your AAV ...

...transcription from the accessory plasmid (AP) encoding phage gene III. The accessory plasmid library contained all...PAM flexibility. In this system, the mutagenesis plasmid (MP6) permits quick, large-scale mutation of the...doi: 10.1038/nature26155 PubMed PMID: 29512652 Plasmids from this paper are available at Addgene.  Additional...Addgene.org Check out our CRISPR Guide Find CRISPR Plasmids for Mammalian Systems Learn about CRISPR History...than SpCas9, in this case, NNGRRT and NNNNGATT, respectively. Researchers also engineered SpCas9 variants...with wt SpCas9 in mammalian cells, finding xCas9 3.7 displayed about the same editing rate as SpCas9 at an...the targeted sequence at the locus of interest. SpCas9’s 3’ NGG PAM occurs frequently in GC-rich genomes...

...changes we’ve made: New CRISPR plasmid pages We’ve given our CRISPR Plasmids and Resources a fresh new design...design, sorting the plasmids we have available by category. Each plasmid page now has a helpful graphic...introduce you to the plasmid class covered on that page. To help you find popular plasmids, we’ve also added... new plasmid pages that cover some of the hottest CRISPR tools. Base Edit: Base editing plasmids allow...revolution, Addgene has distributed over 100,000 CRISPR plasmids. But that’s not our only job - we strive to also...up with new developments (and, of course, find plasmids that will be useful to you.) With these goals...mind, we set out to update our web resources and plasmid pages to make them more useful and easy to navigate...

... editing is mediated by ADAR2DD, not PspCas13b. Find the plasmids for the RESCUE RNA editors here! Testing...for viral delivery.  Find the RESCUE RNA editing plasmids here! Future directions Building on previous RNA...Science (2017):pii: eaaq0180. PMID: 29070703. Find plasmids from this paper at Addgene. Abudayyeh, Omar O...550(7675) (2017):280-284. PMID: 28976959. Find plasmids from this paper at Addgene. Abudayyeh, Omar O...27256883. PubMed Central PMCID: PMC5127784. Find plasmids from this paper at Addgene. East-Seletsky, Alexandra...) (2016):270-273. PubMed PMID: 27669025. Find plasmids from this paper at Addgene. Gootenberg, Jonathan...28408723. PubMed Central PMCID: PMC5526198. Find plasmids from this paper at Addgene. East-Seletsky, Alexandra...

...packaging plasmid aka the RepCap plasmid encodes two genes: the replication (rep) gene and the capsid (...the AAV RepCap plasmid! Check out this blog post to learn more about the AAV transfer plasmid! References... AAV.  Where do you start? Plasmids of course! You just need three plasmids to start making the AAVs you...packaging plasmid which contains the AAV structural and packaging genes, the adenoviral helper plasmid which...packaging plasmid and take a look at each of its parts. Key elements of the AAV packaging plasmid The packaging...the rep and cap genes. Figure 1. The RepCap plasmid and its products. Created with BioRender.com... for the virus to replicate,  and the transfer plasmid which contains the viral genome. In today’s blog...

...following components: A plasmid in the host bacterium expressing dCas9 A plasmid in the host bacterium ... of gene III to create infectious particles. A plasmid-based genetic circuit in the bacteria links the...encoding the deaminase TadA-7.10 A mutagenesis plasmid controlling the phage mutation rate Phage infection...Phage-assisted evolution of base-editing activity. A plasmid-based genetic circuit in the bacteria links the...off-target RNA editing rate of ABE7.10. Find the ABE8e plasmids at Addgene! Why engineer and optimize base editors...editors for your research Read our CRISPR guide Find plasmids for your research ...including SpCas9, SaCas9, LbCas12a, enAsCas12a, SpCas9-NG, SaCas9-KKH, CP1028-SpCas9, and CP1041-SpCas9. The...

...Francisella Cpf1 (FnCpf1), they employed an E. coli plasmid depletion assay to discover FnCpf1’s PAM sequence...system." Cell (2015). PubMed PMID: 26422227. Find plasmids from this publication at Addgene. 2. Zetsche,....1 (2016): 31-34. PubMed PMID: 27918548. Find plasmids from this publication at Addgene. 3. Kim, Daesik...Biotechnol. (2016). PubMed PMID: 27272384 Find plasmids from this publication at Addgene. Kleinstiver...Biotechnol. (2016). PubMed PMID: 27347757. Find plasmids from this publication at Addgene. Makarova, Kira...PMID: 26411297. Resources at Addgene Find CRISPR plasmids at Addgene Read Addgene's CRISPR Resource Guide...is similar to that of high fidelity Cas9s eSpCas9 and SpCas9-HF1. Both AsCpf1 and LbCpf1 ribonucleoproteins...

...choices. Check out our SpCas9 containing plasmids, optimized for a variety of species.   Specificity concerns...frequently fused to Cas9 for this purpose. dCas9-KRAB plasmids for repressing target genes in a variety of species...additional RNA-binding transcriptional activator. Plasmids for activating target genes in a variety of species...systems Addgene carries a variety of Cas9 containing plasmids that have been optimized for expression in different... plants, drosophila, worms and mammals (Browse plasmids by model organism, here).  More information on... resources on the Addgene website Find CRISPR Plasmids Catch up on your CRISPR background with our CRISPR... to your experimental target, SpCas9 and its enhanced variants, eSpCas9 and HypaCas9, are great choices...

... is well suited for genome editing.  Find Cpf1 Plasmids at Addgene   Image attribution: Zetsche...Biotechnol. 2016 Jun 6. PubMed PMID: 27272384 Find plasmids from this publication at Addgene. 2. Kleinstiver...Biotechnology (2016). PubMed PMID: 27347757. Find plasmids from this publication at Addgene. 3. Zetsche ....cell.2015.09.038. PubMed PMID: 26422227 Find plasmids from this publication at Addgene. 4. Makarova...is similar to that of high fidelity Cas9s eSpCas9 and SpCas9-HF1. Both AsCpf1 and LbCpf1 ribonucleoproteins... Cpf1 and its guide RNAs are smaller than their SpCas9 counterparts, so they will be easier to deliver...have a low genetic capacity of ~4.5 kb. At 4.1 kb, SpCas9 barely fits into AAV when you add in a gRNA and...

...Want to get your hands on SpRY? Find a mammalian plasmid here and edit on! References and Resources References..., 6175 (2023). DOI:10.1038/s41467-023-41829-y (Plasmids available here) Walton, R. T., et al., Unconstrained...368,290-296(2020). DOI:10.1126/science.aba8853 (Plasmids available here)   Nishimasu, H., et al.,Engineered... on Addgene.org Addgene’s CRISPR Guide CRISPR Plasmids and Resources Resources on the Addgene blog ...injury, the most utilized PAM sequence, that of SpCas9, is NGG. Assuming all nucleotides are represented...that recognize NG PAMs. The Nureki lab generated SpCas9-NG, a rationally engineered Cas9 with NG PAM compatibility...capable of editing at frequencies comparable to SpCas9. SpRY can also edit NYN PAMs, but with less efficiency...

...EsCas13d. Find the Yan et al. plasmids here Find the Konermann et al. Plasmids here How do Cas13d enzymes...S0092-8674(18)30207-1. PubMed PMID: 29551272 Plasmids from this paper are available at Addgene. Yan...S1097-2765(18)30173-4. PubMed PMID: 29551514 Plasmids from this paper are available at Addgene. Learn... Resources on Addgene.org: Find RNA Knockdown Plasmids Check out our CRISPR Guide Read our CRISPR History...Eubacterium siraeum (EsCas13d) and Ruminococcus sp. (RspCas13d), while Konermann et al. characterized orthologs...WYL-domain-containing accessory protein WYL1 increases RspCas13d and EsCas13d cleavage activity The majority of... et al. from Arbor Biotechnologies found that RspCas13d accessory protein RspWYL1 increases both targeted...

...you want to give GeoCas9 a try, as always, the plasmids are available from Addgene. Happy CRISPRing! ...Background with Addgene’s CRISPR Guide Page Find CRISPR plasmids at Addgene ...than the engineered approach used to make eSpCas9 and SpCas9-HF1. By mining the treasure trove of naturally...GeoCas9 vs. SpCas9, see the table below and read on to learn more about GeoCas9.   SpCas9 GeoCas9 ...temperatures Figure 1: Thermostability of SpCas9 vs GeoCas9. SpCas9 or GeoCas9 RNPs were incubated in reaction... Figure 2: Human plasma longevity of SpCas9 vs GeoCas9. SpCas9 or GeoCas9 RNPs were incubated in human...loving) bacteria, such as Streptococcus pyogene’s SpCas9. These Cas9 proteins don’t work well at high temperatures...

...27256883. PubMed Central PMCID: PMC5127784. Find plasmids from this paper at Addgene. 2. East-Seletsky,...) (2016):270-273. PubMed PMID: 27669025. Find plasmids from this paper at Addgene.  3. Gootenberg, Jonathan...28408723. PubMed Central PMCID: PMC5526198. Find plasmids from this paper at Addgene. 4. East-Seletsky,...) (2017):373-83. PubMed PMID: 28475872.  Find plasmids from this paper at Addgene.  5. Abudayyeh, Omar...550(7675) (2017):280-284. PMID: 28976959. Find plasmids from this paper at Addgene. 6. Shmakov, Sergey...32511521. PubMed Central PMCID: PMC7273289. Find plasmids from this paper at Addgene. Additional Resources...Cpf1 Resources on Addgene.org Browse All CRISPR Plasmids Find Validated gRNAs for Your Next Experiment ...

...transfection of single plasmids vs pooled plasmids; for multiple edits, single plasmids are generally more...transfected on a single plasmid. This is versus 2.4% of cells transfected with a pool of plasmids that contained...sites. This eliminates bulk from crRNA expression plasmids resulting in smaller, more streamlined vectors...contained one crRNA per plasmid plus a Cpf1 expression vector.  See the graph in figure 2 for a comparison of...your research? Check out the Cpf1 multiplexing plasmids available from Addgene. If you have any questions... Our CRISPR Multiplexing Resources Find CRISPR Plasmids for Your Research Catch up on Your CRISPR Background...crRNAtracrRNACas9 crRNACpf1 Endonuclease size spCas9: ~4 kbsaCas9: ~3 kb ~3.9kb crRNA/gRNA length...

...gene-targeting capabilities of full-length SpCas9. This set of plasmids includes AAV-Cas9C-VPR for targeted ...bacterial immune system cleaves bacteriophage and plasmid DNA. Nature 468:67–71 . https://doi.org/10.1038...Addgene.org Check out our CRISPR Guide Find CRISPR Plasmids ...compared to the full-length SpCas9. In 2016, Chew et al. developed a split intein spCas9-AAV toolbox that retains...tissues. However, packaging Streptococcus pyogenes (SpCas9) and a gRNA together (~4.2 kb) into an AAV vector...approximately 1350 amino acids in size, which includes SpCas9, and shortern orthologs approximately 1000 amino...SaCas9 produced indels at a similar efficiency to SpCas9, leading the group to focus their efforts on SaCas9...

...exact Cas9 plasmid used by Jinek et al. 2012, or browsing for the latest AsCas12a plasmids, Addgene and...find a link to search Addgene's collection for plasmids containing that enzyme, and embedded in the References...lists on every page you can find direct links to plasmids from the article if they're available through ...Once upon a time, not so long ago, spCas9 was the only Cas enzyme widely available and applied by researchers...getting the desired features and cutting (note: spCas9 isn’t always the winner!).   Fig. 3 – Comparison...The PAM Requirement and Expanding CRISPR Beyond SpCas9 More resources from the Innovative Genomics Institute...

...receive transfer plasmid DNA that you can use along with lentiviral packaging plasmids to create your own...rough approximations of the total number of CRISPR plasmids for that particular organism available at Addgene...helped distribute many of these technologies in plasmid form and are proud to have facilitated their fast...fast adoption. However, in many cases the plasmids themselves are only the starting point for the production... use; after receiving a pooled gRNA library in plasmid format, a researcher must amplify the library, ... makes it unuseable. With the large size of the SpCas9 nuclease (~4.2 kb) this is a particularly prominent...Institute to provide lentiviruses for delivery of SpCas9 and Cas9 nickase for genome editing. You can also...

...Visit our CRISPR plasmids and resources page Read our CRISPR guide Find CRISPR plasmids for mammalian systems...Zhang Lab had described eSpCas9, while the Keith Joung Lab had developed SpCas9-HF1. Both of the enzymes... similar off-target activity, relative to eSpCas9 and SpCas9-HF across a number of different sites. Using...the nuclease. For the most frequently used Cas9, spCas9 from Streptococcus pyogenes, this sequence is 3...The PAM Requirement and Expanding CRISPR Beyond SpCas9 described the PAM specificity of Cas9s isolated...Nureki Lab described a rationally engineered Cas9, SpCas9-NG, which required only the di-nucleotide PAM NG...