...individual A549 cells stably expressing Cas9. A549 cells were transduced with lentiCas9-Blast 1 and then selected...Figure 2: Cas9 expression in monoclonal cell lines generated from A549 cells transduced with lentiCas9-Blast... not express Cas9 and are included as a control (WT). Vinculin is a loading control. 1 lentiCas9-Blast...could lead to reduced transgene expression over time, as the lower expressing clones take over the polyclonal...that are more likely to retain stable transgene expression. Other methods of generating a monoclonal cell...population be monitored regularly for transgene expression. This protocol has been optimized for adherent...sufficiently expanded, screen the lines for transgene expression and/or other phenotypes. For example, perform...

...individual A549 cells stably expressing Cas9. A549 cells were transduced with lentiCas9-Blast and then selected... the most desirable level of expression. Over time, transgene expression in a polyclonal population may...single cells for 13 days. lentiCas9-Blast was a gift from Feng Zhang (Addgene plasmid #52962 ) and is described...Protocol to generate stable cell lines expressing a gene of interest from an integrated lentiviral vector...protocol can be used to generate stable cell lines expressing a gene of interest from an integrated lentiviral...lentiviral vector. Unlike the short-term protein expression observed using transient transfection approaches... lentiviral vectors enables long-term protein expression studies. Moreover, repeating experiments in a...

...produce a plasmid encoding the gRNAs and Cas9. As with the Gersbach lab plasmids, multiple Cas9 variants...  Gersbach Lab multiplexing plasmids: This plasmid set allows you to express 2-4 gRNAs, with four being...with high GFP expression. These cells have the highest levels of Cas9 and gRNA expression, and thus the ...Gibson Assembly Multiplexing Plasmids: These plasmids can be used to express up to 8 gRNAs after Golden ...Golden Gate Assembly Multiplex Plasmids: These plasmids allow expression of 2-5 gRNAs in zebrafish. Custom... ligation step. Plasmids are then digested using BsmBI and ligated into Cas9 or dCas9-containing destination...the multiple gRNAs and Cas9 together into a single plasmid. Although only wt hCas9 entry vectors are supplied...

...approach is to express and purify His-tagged Cas9 from E. coli using a plasmid such as pET-28b-Cas9-His from...promoters must be chosen for both Cas9 and gRNA expression       Plasmid DNA may be incorporated into the... can occur due to prolonged Cas9 expression     The requirement for Cas9 transcription and translation... similar efficiency as compared to plasmid-based expression of Cas9/gRNA and can be used for most of the...pathways. This makes Cas9 RNPs useful for CRISPR applications where limited expression of Cas9 is required and...for experiments where stable Cas9 expression is required, such as when a Cas9 fusion to a fluorescent protein...long-term expression of Cas9, such as visualizing genomic loci using fluorophore tagged dCas9 may require...

...one vector to express the gRNAs and Cas9. Co-transfections can lead to variable expression levels due to...Multiplexed Lentiviral Expression Cassettes lentiviral transduction One vector expresses Cas9, eGFP, and up ...transfection of single plasmids vs pooled plasmids; for multiple edits, single plasmids are generally more...Comparing the Cas9 and Cpf1 CRISPR Nucleases   Cas9 Cpf1 Components crRNAtracrRNACas9 crRNACpf1 ... guide page for a review of Cas9.  For a brief comparison of Cpf1 vs. Cas9, see the table below. Table...Golden Gate Assembly transfection One vector expresses Cas9 and up to 7 gRNAs Each gRNA requires its ...transfection gRNAs expressed from a polycistronic transcript  Requires co-transfection of Cas9 Nissim et alTsai...

...effects” have included using a Cas9 nickase variant, lowering Cas9 expression, and truncating gRNA sequences...improving. Cas9 variants have been modified for genome editing, activating gene expression, visualizing... target effects of CRISPR/Cas9 by altering the nuclease activity of the Cas9 nuclease itself. Design and...non-target strand and Cas9, Kleinstiver et al disrupted interactions between Cas9 and the phophate backbone...governs CRISPR-Cas9 targeting accuracy." Nature (2017). PubMed PMID: 28931002. Find plasmids from this publication...specificity of the Cas9 nuclease with engineered variants: eSpCas9, SpCas9-HF1, & HypaCas9. The off-target...sites tested when compared to eSpCas9(1.1) and SpCas9-HF1. The HypaCas9 plasmids are available from Addgene...

...model systems, expression of S. pyogenes Cas9 or Cas9 variants (hereafter referred to as Cas9) and a single...also contain Cas9, which enables expression of both Cas9 and the gRNA off of the same plasmid. Delivering...RNA pol II promoters for Cas9 expression include the ubiquitously expressing cauliflower mosaic virus ...toolkits for creating plant expression plasmids, including plant CRISPR plasmids from the Yiping Qi lab and...drive gRNA expression is dependent upon the cell type in question. In plant cells, gRNA expression is achieved...target genes in plant cells, respectively. Cas9 expression is commonly driven by plant-derived RNA pol...promoters which regulate expression of longer RNAs (such as mRNAs for gene expression). Examples of commonly...

...control RFP expression plasmid.  With this set up, all successfully transfected cells will express RFP while...was cleaved by Cas9 and repaired via HDR using the supplied donor plasmid will express GFP. If there’s...used when transfecting your cells.  A plasmid that contains Cas9. The Madison lab also created an inducible...inducible SRIRACCHA system (iSRIRACCHA) where Cas9 expression is activated using 4-OH-T and doxycycline. ...The ratio of RFP to GFP expressing cells provides a real-time read out of Cas9 activity.  By using the ... labor intensive and quite time consuming.   As Cas9/gRNA activity is crucial for successful CRISPR editing...the scientific community for efficient real-time Cas9 activity assays. Recently scientists have created...

...System Application Mammalian expression vector Promoter driving Cas9 expression can be constitutive or inducible...in vivo viral delivery. Cas9 mRNA and gRNA Plasmids containing gRNA and Cas9 are used in in vitro transcription... CRISPR tools for your application: Wildtype Cas9 or Cas9 nickase are appropriate for generating knockouts...donor plasmid or single-stranded DNA oligonucleotide, so you will need to make sure your expression system...included with each expression system as well as suitable applications. Expression System Components ...generate stable cell lines. Transient or stable expression of Cas9 and/or gRNA in a mammalian cell line that...lentiviral particles. Stable, tunable expression of Cas9 and/or gRNA in a wide variety of mammalian cell ...

...high expression, so adding this G could help with expression, though it is possible for the plasmid to ...double nickase, you could express two spacers and use PX335 to express the Cas9n (nickase). The concept ...nickase system is that you can express two different chimeric gRNAs with the Cas9 nickase which will together...the Cas9 nickase doesn't have the ability to induce double-stranded breaks like the wildtype Cas9 does...articles (3/12/2014). With so many options for CRISPR plasmid tools and numerous experimental design decisions...questions and answers. Here are just 4 of the CRISPR-Cas9 FAQs. If you still have more questions after reading...genome editing using the Zhang lab's CRISPR/Cas plasmids. Designing your CRISPR genome editing experiment...

...variable Cas9-expression was across the different cells (Figure 3).   Figure 2: Cas9 expression in monoclonal...A549 cells stably expressing Cas9 via transduction with Cas9 lentiviral vector, lentiCas9-Blast (52962-LV... Figure 1: Cas9 expression in A549 cells transduced with lentiCas9-blast. Anti-Cas9 Western blotting... Cas9 delivery Overview: Before any editing can begin we need our A549 cells to express Cas9. In these...too many Cas9 integrations per cell, and my stable cell pool had wildly variable Cas9 expression (Figure...Checking Cas9 expression Overview: By Western blotting (Figure 2, pool), I knew the cells expressed Cas9,... 20% of their Cas9-expressing cells. So, even when Cas9 is efficiently editing, don’t expect a 100% knockout...

...and a gRNA. But instead of Cas9, this plasmid expresses Cas9n (a D10A nickase mutant) to nick, rather than...vectors and an envelope expressing plasmid, such as our second most requested plasmid of all time, pMD2.G....PX458) - This plasmid from the Zhang lab expresses Cas9 from Streptococcus pyogenes with an invariant sgRNA... the envelope expressing plasmid pMD2.G. Find pMDLg/pRPE. scramble shRNA - This plasmid from David Sabatini... with lentiCas9-Blast or with cell lines already expressing Cas9. Find lentiGuide-Puro. pSpCas9n(BB)-2A-GFP... Similar to pSpCas9(BB)-2A-Puro (PX458), pSpCas9n(BB)-2A-GFP (PX461) expresses both a Cas9 protein and...applications. This plasmid was the top requested plasmid of 2017. Find pMD2.G. pSpCas9(BB)-2A-GFP (PX458...

...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... our hot plasmid articles from April 2018. If you'd ever like to write about a recent plasmid deposit ...Listen to the Fire-Cas9 Podcast Segment Jerry Crabtree’s lab has developed a new CRISPR/Cas9-based system to...Inducible Recruitment for Epigenome editing by Cas9 (FIRE–Cas9), combines these approaches to enable rapid...epigenetic memory at nearly any locus. The FIRE-Cas9 plasmids can be found here. Braun et al. Nat Communications...PubMed PMID: 28916764. Plasmids for independent control of gene expression in yeast Article contributed...

... this plasmid Cas9 expression is typically cytotoxic, which makes generation of high titer Cas9 lentiviruses...single-vector systems typically express Cas9 and a gRNA from the same plasmid. LentiCas9-blast virus can be used... monoclonal Cas9 background because you’ll have more consistent levels of Cas9 expression between experiments... can be expressed from lentiCas9-Blast. Purpose of LentiCas9-blast LentiCas9-blast is a Cas9 expression...Secondly, you can use a LentiCas9-Blast viral prep to create a single stable Cas9-expressing cell line and then... rate variability when using lentiCas9-blast to generate Cas9-expressing cells. To ensure that this growth...related viral preps. Cas9 is the headliner of most CRISPR applications and can be expressed from your cell ...

... Experiment Cas9 expression systems Addgene carries a variety of Cas9 containing plasmids that have been...using a Cas9 variant with higher specificity (eSpCas9 and HypaCas9) instead.  Choosing a Cas9 for site-specific... which is frequently fused to Cas9 for this purpose. dCas9-KRAB plasmids for repressing target genes in... CRISPR/Cas9 has made it easier than ever to make precise, targeted genome modifications. Cas9 has been...introducing Cas9-mediated frameshift mutations in the gene of interest. The genomic target for Cas9 and the... system. This system uses a “nicking” Cas9, in which one of Cas9’s two critical enzymatic residues has...using Cas9 Don’t want to permanently modify the genome but still want to affect gene expression? dCas9...

...fully functional Cas9 upon dimerization. Splitting wild-type Cas9 into n-terminal (Cas9(N)-2xNLS) and c-terminal...control over CRISPR/Cas9 mediated genome modification and gene expression. The plasmids associated with this...Rapamycin-inducible Cas9 sets (Addgene plasmids 62883 &62884; 62885 & 62886) Rapamycin-inducible dCas9-VP64 activator... direct Cas9 to a specific genomic locus. When fused to the sgRNA and co-expressed with dCas9 – the dCas9...amazing plasmid technologies developed by our community of depositors. With over 40,000 plasmids avaliable...amazing new plasmid tools that have come through our doors in 2015. Do you have a favorite new plasmid tool ... can write a blog post about it! Split Cas9 system The Cas9 protein is composed of an n-terminal DNA...

...selection. Using an inducible Cas9 expression and removing the sgRNA plasmid after identifying successful... Overview of the dual-plasmid CRISPR/Cas9 workflow in M. abscessus. A) Sth1Cas9 mediates double strand...of the new plasmids, antibodies, and viral preps in the repository through our Hot Plasmids articles.  ... in Mycobacterium abscessus using a dual-plasmid CRISPR/Cas9 system By Aryana Khosravani Deborah Hung’...Hung’s lab has developed a novel dual-plasmid CRISPR/Cas9 system for gene disruption in Mycobacterium abscessus... use of CRISPR/Cas9 in M. abscessus. The simplicity and efficiency of this dual-plasmid approach makes...abscessus is first transformed with Cas9 on an integrative plasmid containing tetracycline repressor TetR...

...eukaryotes. Though the plasmids made by Xie et. al are specifically designed for plant expression, PTGs will likely...CRISPR/Cas9 shows great potential for multiplexed genome editing. Interested in using PTG plasmids in your...your research? Check out the PTG plasmids or plant expression systems available from Addgene. If you have...shortens construct size, it requires coexpression of Csy4 with Cas9 and the polycistronic transcript. As...In the short time since its development, CRISPR/Cas9 genome editing has been used to study the effect...recombination. To further increase the utility of CRISPR/Cas9, it will be necessary to improve its multiplexing... pol III promoter is relatively weak, and low expression of gRNAs from these constructs could lower genome...

...modification. Plasmid ID Plasmid Name Cas9 Variant Speacial Features 85169  pJL-SaBE3  SaCas9 (NNGRRT)   ...Bacterial Expression Construct SpCas9 Inhibition (E. coli) Mammalian Expression Construct SpCas9 Inhibition...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...

...fragments. The pX330 plasmid contains humanized Cas9 expression cassette and sgRNA expression cassette. The ...reconstituted the EGFP expression cassette. (b) pCAG-EGxxFP target plasmid and pX330-sgRNA plasmid (Mashiko et al...pCAG-EGXXFP plasmid along with the individual gRNAs in their PX330 plasmids (or similar plasmids for gRNA...mice by pronuclear injection of circular plasmid expressing Cas9 and single guided RNA." Scientific reports...variants of the CRISPR/Cas9 system floating around, and many pre-designed plasmids containing these variants...EGFP expression cassette reconstitution. When the target sequence was digested by sgRNA guided Cas9 endonuclease... (c) The pCAG-EGxxFP target plasmid was co-transfected with pX330 plasmids containing sgRNA sequences ...

...CRISPR plasmids available at Addgene. We will still periodically focus on specific CRISPR plasmid tools...capsid-based bionanoparticle Transient expression of CRISPR/Cas9 in cells has advantages like reduced off-target...bionanoparticle system for packaging Cas9/gRNA ribonucleoproteins for transient expression. They replaced the tetraloop...CRISPR guide Find CRISPR plasmids by function Find recently deposited plasmids ...toolkit for epitope tagging Lentiviral capsid-based Cas9/gRNA ribonucleoprotein delivery Cytosine deaminase...of bacteria that can cause disease in humans. Expression of this system is tunable via a rhamnose-inducible... a catalytically impaired Streptococcus pyogenes Cas9. They called this editor Target-ACE (adenine and...

...Kit contains Cas9, Cas9 nickase, and Cas9 activator expressing plasmids that can co-express 1-8 gRNAs. ...lines expressing it. The Church lab fixed this issue by creating a new dCas9-VPR expression plasmid that...then transfected with a Cas9-expressing plasmid to generate a population of mutant cells. The mutant cells...the Liu lab’s own pAc-sgRNA-Cas9 insect expression backbone, which expresses the gRNA from a Drosophila...deposited their Cas9 and gRNA backbone plasmids with Addgene. Additional control plasmids for the TKO library...parasites that constitutively express Cas9 with a library of sgRNA expression vectors (containing ten guides...Neurosci 2016. PubMed PMID: 26950006 Plasmid ID Plasmid Name Plasmid Type 74309 pcDNA3.0-TRPV4-p2A-ferritin-p2A-mCherry...

...editing within a single expression cassette is advantageous to Cas9’s larger expression requirements (Figure...individually expressed proteins run in separate lanes. B: individually expressed and coexpressed proteins ...subset of the new plasmids and viral preps 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...Image from Santilli, et al. 2021 Plasmids are used to express 10, 15, 20, 30, 40, 50, 60, 80 and 100...through two polycistronic expression vectors (3570 lanes/50 mLs) coexpressing a set of either 10, 30, 50...need. If you'd ever like to write about a recent plasmid deposit please sign up here.  Here's what you'...

...available from Addgene consists of two plasmids: pCas9: carries Cas9 and chloramphenicol resistance pCRISPR...technology (Ronda et al., 2016). pMA7CR_2.0 expresses lambda Red and Cas9, which are separately inducible by L-arabinose.... citrea scarless editing plasmids Sheng Yang’s lab describes a two-plasmid system that combines recombineering...pCRISPomyces-1 includes Cas9, a tracrRNA, and a CRISPR array, while pCRISPomyces-2 contains Cas9 and a gRNA cassette...functions more similarly to CRISPR/Cas9 systems adapted for eukaryotes in that Cas9-mediated cleavage induces ...Tet responsive promoter to drive expression of a catalytically dead Cas9 enzyme allows reversible repression...knockdown expression of multiple target transcripts simultaneously. Marraffini lab's CRISPRi/a plasmids In addition...

...cell types. Purpose  SpCas9 This plasmid expresses Streptococcus pyogenes (Sp) Cas9 from a Cbh promoter...the top requested plasmid from 2016 is an expression vector for CRISPR nuclease, SpCas9 from the Zhang Lab...a versatile mammalian expression vector that you can use to express both SpCas9 and your gRNA in a variety...a versatile mammalian expression vector that you can use to express both SpCas9 and your gRNA of interest...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...sequence as directed by a gRNA. 2A-Puro In this plasmid, SpCas9 is fused to the puromycin resistance gene through...

...or apoptosis with Cas9/gRNA expression. A pooled analysis found no evidence of Cas9 cleavage at predicted...promoter-driven GFP expression. In cell lines that can produce HIV-1 toxic proteins, CRISPR/Cas9 SAM caused apoptosis...target the HIV-1 5’ and 3’ LTRs and expressed them along with Cas9 in the T-cell line 2D10. PCR amplification...portion of the LTRs joined together. Clonal Cas9/gRNA-expressing cell populations were also immune to HIV...with HIV-1. For two HIV-1 strains, Cas9/gRNA lentiviral expression significantly reduced the HIV-1 copy...preventing re-infection would require sustained expression of Cas9/gRNAs, which could result in higher rates...were located near the Cas9 cut site, leading Wang et al. to conclude that some Cas9-derived indels may not...

...permanently induce expression? Check out our Retro expression vector and Lenti expression vector. Turn it...blog Plasmid 101: Inducible Promoters Plasmid 101: Repressible Promoters CRISPR 101: Which Cas9 Do I...turn off expression permanently is to knock out the gene for your protein of interest. CRISPR-Cas9 and a ...ablate protein expression. If you need the ability to control when you turn off expression for good, the...inducible expression (more on this below) will both achieve temporary repression of protein expression. Addgene... a gene (cDNA) can be done by either plasmid or virus. A plasmid carrying your cDNA can be targeted for...appropriate promoter to express your cDNA, depending on the level of expression you would like. Check out...

...downregulate expression of specific Caulobacter genes using a catalytically dead Cas9 (dCas9) derived from...CRISPRa depended on continued expression of dCas9 and gRNAs whose expression varies depending on transgene...CRISPR plasmids available at Addgene. We will still periodically focus on specific CRISPR plasmid tools... lower average Cas9-independent off-target DNA editing, ~5–50-fold lower levels of Cas9-dependent off-...CRISPR guide Find CRISPR plasmids by function Find recently deposited plasmids ...fluorescent dCas9-KRAB and dCas9-VPR for quantifying and tracking cells that express both the dCas9 construct...repression of gene expression in Saccharomyces cerevisiae. The system consists of dCas9, the mammalian transcriptional...

...determined by comparing cells expressing only eGFP expression to cells expressing eGFP and mCherry. ACE was...single base editing by APOBEC-Cas9 Though the ACE reporter quantifies APOBEC-Cas9 efficiency in real-time, ...mCherry and a T2A site and expressed in cells. mCherry is constitutively expressed to identify successfully...efficiency of plasmid delivery to a cell. In contrast, TREE provides a read out for plasmid delivery and...integrated it into human and mouse cells that expressed either Cas9 or an optimized cytidine base editor. The...and enrichment of DNA editing by APOBEC–Cas9 or cleavage by Cas9 in living cells. Nucleic Acids Research... a APOBEC-Cas9n-UGI complex. So how exactly does this work? Figure 1: APOBEC- and Cas9-mediated ...

...ease and efficiency for all-in-one CRISPR/Cas9 vectors expressing up to seven guide RNAs (gRNAs). Sakuma ...They went on to show that their CRISPR/Cas9-nuclease and CRISPR/Cas9-nickase vectors work successfully to...based on earlier evidence showing that a CRISPR/Cas9 plasmid could be injected directly into mice for genome...versions of Cas9, nuclease and nickase, but we are now adopting dCas9, dCas9-VP64, and dCas9-KRAB in our...all-in-one vector construction system for CRISPR/Cas9-mediated multiplex genome engineering is now available...one of the most advantageous properties of CRISPR/Cas9 compared to ZFNs and TALENs,” said Tetsushi Sakuma...Figure 1: Schematic overview of the all-in-one CRISPR/Cas9 vector construction system for multiplex genome ...