...compared to DNA transfection of gRNA and Cas9 expression plasmids. These results are in line with previous...releasing Cas9. Nanoblades can also deliver Cas9 variants, such as a transcriptional activator dCas9-VPR, ...Nanoblades by association with the the Cas9 portion of the Gag-Cas9 fusion protein. A single Nanoblade particle...along with the gRNA and Cas9. An “all-in-one” Nanoblades which carries a gRNA, Cas9, and a HDR donor template...centrifugation. Gag-Cas9 fusion protein The MLV Gag protein fused to a Flag-tagged SpCas9 is incorporated ...Gag in place of SpCas9. gRNA A gRNA targeting a DNA sequence of interest is expressed in 293T cells and...infiltrate cells. Options for CRISPR delivery include plasmid transfection, RNP electroporation, and viral transduction...

...labs’ plasmids, including protocols, check out the plasmids at Addgene: pAdSh.PGK.Cas9 (expresses S. pyogenes...target cells. Within target cells, Cas9 machinery is transiently expressed and binds to target DNA. In the...capacity is large enough to carry the Cas9 gene and a gRNA expression cassette in one single viral particle...DNA Adenoviral CRISPR/Cas9 genome editing tools can also be used to deliver Cas9 and DNA templates in ... CRISPR/Cas9 research! To find more information about the adenoviral delivery of CRISPRS/Cas9 using the... also Adeno Cas9 and Adeno EA. Or if you're looking for a broader range of CRISPRs plasmid tools, find...Genome Editing of Pten by CRISPR/Cas9 in Mouse Liver in Spite of Cas9-Specific Immune Responses. Human...

...Staphylococcus aureus Cas9 (Sa-Cas) and deposited plasmid IF311: pMAGIC (R4-R3) NLS-Sa dCas9-NLS-LSD1. LSD1 ...types. They have also fused LSD1 to x-Cas9(3.7). x-Cas9(3.7) is a SpCas9 mutant that exhibits increased PAM... Find plasmids from this publication at Addgene. Additional Resources on the Addgene Blog Cas9 Activators...pCMV-dCas9-D3A) and Tet-dependent (TetO-dCas9-D3A) constructs. For lentiviral expression, Fuw-dCas9-Dnmt3a...deposited mammalian expression constructs including pcDNA-dCas9-p300 Core and pcDNA3.3-Nm-dCas9-p300 Core, as...as well as pLV-dCas9-p300-P2A-PuroR for lentiviral expression. Figure 1: dCas9-p300 adds H3K27ac... created pdCas9-Tet1-CD for targeted cytosine demethylation in mammalian cells. This plasmid is used with...

...Abudayyeh et al., 2016). In the endogenous CRISPR/Cas9 system, Cas9 uses both a tracrRNA and crRNA to facilitate...multiplexing; expressing 5 gRNAs simultaneously produces comparable gene knockdown to single gRNA expression. Although... 27, 2020. CRISPR, and specifically Cas9 from S. pyogenes (SpCas9), is truly an exceptional genome engineering...Perhaps the biggest difference between Cas13a and Cas9 is that Cas13a binds and cleaves RNA rather than...enzymes - Cas13a contains two HEPN domains, whereas Cas9 uses HNH and RuvC domains to cleave target DNA. ...roles for HEPN domains in other proteins. As with Cas9, mutating key residues in the Cas13a molecule results... Guide RNA Target PAM (PFS) Cleavage Mechanism Cas9 HNH, RuvC TracrRNA, crRNA DNA 5' NGG Specific...

... of protein coexpression. By introducing a single plasmid containing both GOIs, expression levels are ...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... recent plasmid deposit please sign up here.          Listen to this episode of Hot Plasmids! Optogenetic...genes-of-interest (GOI) into two independent expression cassettes on a single plasmid (Grefen and Blatt, 2012). Similar...DNA transfer, each GOI is usually expressed from a different plasmid, which may lead to unequal gene dosage...Grefen and Blatt, 2012). The 2in1 Plasmid Toolkit consists of 4 plasmid toolkits generated with 4 different...

... (ARG) plasmids to better monitor gene expression in vivo using ultrasound. These ARG plasmids contain...pXPR_053. They expressed this construct in bone marrow stem cells from Cas9-expressing mice, and used ...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... recent plasmid deposit please sign up here.      Listen to this episode of the Hot Plasmids podcast ...responses in C. elegans. Find the split BoTN/B plasmids at Addgene. Liu Q et al., Neuron. 2019. PubMed... microbe travels through a host organism, the expression of some genes can change depending on the location...

...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...-baToN, “sender” cells express GFP on the surface and “receiver” cells express an anti-GFP nanobody on...response 1 (TIR1) expressed under a desired promoter with a specific spatiotemporal expression pattern and ...highlights from recent CRISPR plasmids. To find all of the CRISPR plasmids available from Addgene, head...head over to our CRISPR Plasmids and Resources page.  Plasmids for delivering CRISPRi components by a single.... Once activated, Cas9 cleavage can induce DSBs within minutes. New CRISPR plasmids for Borrelia burgdorferi...

...co-injection of the targeting vector and a Cas9 expression plasmid will insert the desired genetic tag and...as a universal donor plasmid. The CRISPaint toolkit includes several donor plasmids for protein tagging...are then mixed and transfected along with a Cas9 expression vector. They can also be introduced directly...directly into a Drosophila S2 cell line stably expressing Cas9. This PCR toolkit offers C- and N-terminal tagging...this system lies in the ability of the endonuclease Cas9 to create a DNA double stranded break (DSB) at a...The Jorgensen Lab recently developed a modular plasmid assembly toolkit called SapTrap to improve CRISPR...accepts the sgRNA target sequence for U6 promoter expression and the second site accepts the homology arm ...

...in a 1-plasmid system, in which Cas9 is included on the gRNA-containing plasmid, or a 2-plasmid system...CRISPR/Cas9 over previous genome editing techniques is its simplicity and versatility. CRISPR/Cas9 consists...gRNA-containing plasmids, each with a different genomic target. The goal of a CRISPR screen is to use Cas9 and a...to deliver a pooled population of gRNAs to Cas9 expressing cells. Pooled lentiviral CRISPR libraries (...(A) to be used to generate lentivirus (B). Cas9-expressing cells or wild-type cells are treated with lentivirus...contain thousands of gRNA plasmids, discerned only by a unique barcode on each plasmid. As such, sequencing... how CRISPR/Cas9 can be used in other types of experiments, check out our CRISPR Plasmids and Resources...

...ubiquitous expression of sgRNA(s) and mCherry (vector 1) with neuron-specific expression of dCas9-VPR (Figure...system with ubiquitous expression of sgRNA (top) and neuron-specific expression of dCas9-VPR (bottom). Created...et al., 2018). This mouse expresses a modified version of the “Suntag” Cas9 activator that recruits up... for generating the SPH mouse. (B) AAV plasmids used to express CRE under neuron-specific promoters (aCaMKII... (i.e., how many sgRNA expression cassettes will fit). Cre-dependent expression of the SPH activator paired...neuronal populations.  The Yang Lab’s AAV plasmids used to express Cre under neuron-specific promoters, including...Figure 2), a popular Cas9 activator developed by the Church Lab. Neuron-specific expression is mediated by ...

...catalytically inactive form of Cas9 known as dCas9 (Jinek et al., 2012). Like Cas9, dCas9 can bind to a specific... This system utilizes dCas9 fused to a biotinylation tag expressed on a plasmid, allowing for in vivo ...gRNA, which can easily be cloned into a gRNA expression plasmid. Experimental use Since the initial report...CRISPR guide Find CRISPR plasmids by function Find recently deposited plasmids ...in mammalian cells by expressing dCas9 with an N-terminal 3xFLAG tag (3xFLAG-dCas9) and a gRNA targeting...vivo biotinylation through a plasmid-based biotin ligase. Biotinylated dCas9, with bound DNA, can then be...editing, numerous discoveries have been made using the Cas9 nuclease in the absence of genomic alterations. ...

...1). Plasmids: We generated a series of plasmids that are available from Addgene. These plasmids enable...lentiviral expression platform, to assess selective degradation of FKBP12F36V-BRD4 using CRISPR/Cas9-mediated...engineered to express ENL-FKBP12F36V and endogenous ENL was inactivated using CRISPR/Cas9 gene-editing....engineered to express FKBP12F36V-MELK and endogenous MELK was inactivated using CRISPR/Cas9 gene-editing... Cell lines were engineered to express YY1-FKBP12F36V using CRISPR/Cas9-mediated knock-in. Featured molecule...Mouse Models CRISPR 101: Cas9 Nickase Design and Homology Directed Repair Plasmids for endogenous gene tagging...Resources on Addgene.org Browse all CRISPR plasmids CRISPR plasmids for protein tagging Find validated gRNAs...

...vector formats: all-in-one (lentiCRISPRv2, expressing both Cas9 and an individual gRNA from this single ...-only (lentiGuide expressing only an gRNA, for use with cells that already have Cas9). Each library consists...individual lentiGuide gRNA plasmids targeting the human kinome. These plasmids contain the same 3-4 gRNAs...setting. The individual plasmids will also be useful for imaging or gene expression assays necessitating ...true biological insight. Pooled and individual plasmid formats A first for a library at Addgene, you can...first subset of the pooled library. The individual plasmids will be useful when screening for phenotypes, ...necessitating the screening of individual plasmids separately. Additionally, this set can be used for cherry-picking...

...CRISPR/Cas9 is an enormously plastic tool and has taken the scientific world by storm. While Cas9 has been...2018). cDNA libraries, which consist of plasmids that over-express coding sequences from a given cell type...context of the gene, Cas9 activators efficiently solve these problems.   Which Cas9 activator should I ...Best Cas9 Variant for Your Next Experiment Find out More about Genome-wide Screens with CRISPR/Cas9 Learn...with AAV Resources on Addgene.org Find Cas9 Activators Find Cas9 Repressors Catch Up on Your CRISPR Background... cDNA overexpression vector rather than going through all the troubleshooting required for Cas9-based ...there has also been significant work on constructing Cas9 transcriptional activators. These constructs allow...

...light-activated split-Cas9 (FAST) system constitutively expresses the C-terminal fragment of Cas9 while transcription...proteins from N. crassa Split Cas9, Split Cas9 nickase, Split dCas9 Genome editing by NHEJ and HDR, repression...Photoswitchable Cas9 Richter et al., 2016 tsRC9 RsLOV from R. sphaeroides Cas9, dCas9 Gene editing ...Sativa AcrIIA4 anti-CRISPR protein, co-expressed with Cas9 or dCas9 Repression of gene editing and transcription...heterodimerizing domain to each of the Cas9 fragments, the authors created a photoactive Cas9 tool, as shown in the figure...modular nature of Cas9, Nihongaki et al. showed that it was possible to swap out the Cas9 domains in their... is light-dependent (Yu et al., 2020). To express the N-Cas9, Haifeng Ye’s lab used the bacterial phytochrome...

... measure time. These plasmids with different promoters driving expression of Cas9 were used in this study...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...highlights from recent CRISPR plasmids. To find all of the CRISPR plasmids available from Addgene, head...CRISPR/Cas9 constructs. zCas9i is a high efficiency intron-optimized SpCas9-coding gene. SlugCas9 is a ...Hutchinson Cancer Research Center developed a pooled plasmid library encompassing all possible amino acid mutations...anti-SARS-CoV-2 antibodies. In the library, each plasmid encodes the mutated spike protein and a barcode...

...sequence targeted by the Cas9 nuclease. The Streptococcus pyogenes Cas9 (SpCas9) requires an NGG PAM to...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...range of CRISPRCas9 by engineering SpCas9 Article contributed by Shreya Vedantam CRISPR/Cas9 is widely... were previously difficult. Find SpCas9 and Target-AID-NG plasmids at Addgene! Nishimasu H et al., Science...factors tethered to dCas9, require long time scales with constitutively expressed proteins that are not...need. If you'd ever like to write about a recent plasmid deposit please sign up here.        New toolkit...

...iTOP resulting in GFP expression. Image courtesy of Diego D'Astolfo. CRISPR/Cas9: a potential application...would be compatible with CRISPR/Cas9 genome editing. Although CRISPR/Cas9 is all the rage, they had another...which transient protein expression has a long-lasting effect, such as Cre- or Cas9-mediated recombination... mutations and gene expression changes elsewhere in the genome. Second, expression is by nature transient... method and its potential applications to CRISPR/Cas9 genome editing.   Protein-only transduction? Niels...control could enter cultured cells and activate expression of a luciferase reporter. CPP was not required...causes a cell to take up, and continued protein expression would require multiple transduction events. For...

... CHIME works through transduction of Cas9-expressing (Rosa26-Cas9) hematopoietic stem cells with a lentiviral...and lentiviral expression vectors (Table 1). The four systems Name Function Plasmid Transduction...genes pXPR_219 Violet-excited GFP (vex) Rosa26-Cas9 I-CHIME Inducible knockout of a single gene...Violet-excited GFP (vex) Rosa26-FlpO-ERT2; H11-Cas9 L-CHIME Knocks out a single gene in defined... Violet-excited GFP (vex) Lineage-Cre; Rosa26-Cas9 S-CHIME Knocks out pairs of genes sequentially...Violet-excited GFP (vex) Rosa26-FlpO-ERT2; H11-Cas9 Table 1: X-CHIME systems Proof of Concept These...  The X-CHIME deposit also includes pXPR_071, a plasmid that leads to higher titer lentivirus preparations...

... found led them back towards Cas9 fused base editors (nCas9-BE), as Cas9 is mechanistically more favored...promoter, which is used to drive the expression of the array. After expression, the individual gRNAs are released...also exhibited reduced Cas9-dependent off-target editing and do not cause higher Cas9-independent off-target...leading the edge of the precision gene editing. CRISPR-Cas9, the key component to enable efficient base editing...organisms and has previously been used for multiplex Cas9-nuclease editing. Typically, a single array encoding...demonstrated that the tRNA-gRNA array can drive the expression of itself, without using any additional promoter..., that rely on an upstream lengthy promoter to express the gRNA array, DAP arrays use tRNA itself, as ...

... preassembled Cas9/gRNA complexes or with guide RNA- and Cas9-expression plasmids. Cas9 guide RNA delivery...genome, which seem resistant to Cas9 cleavage (1). Both the CRISPR-Cas9 and NgAgo genome engineering methods...cells with 5’-P-ssDNA guides and an NgAgo expression plasmid to edit a gene in vivo. Try NgAgo for Yourself...Targeting Specificity with eSpCas9 and SpCas9-HF1 See How Cpf1 Stacks Up to Cas9 and NgAgo Resources on ...sequencing on select regions of all distributed plasmids as part of quality control, but does not perform...engineer sharing the stage with much-lauded CRISPR-Cas9 is DNA-guided endonuclease NgAgo! We'll discuss ...head-to-head.  Exciting features of NgAgo First, unlike Cas9, NgAgo does not require a PAM sequence for target...

...packaging plasmids as well as transfer plasmids. This concept ensures safe handling and expression of these...cloning and expressing your gene of interest with GFP expression.  Cloning and expressing RNAi pMKO.1 ...humanized (Plasmid #44246) Expression of a catalytically inactive (dead), human codon-optimized Cas9 under the...separating viral genes and transgene expression across several plasmids. The most commonly used viral vector...gamma-retroviral packaging plasmids are not interchangeable. General envelope plasmids, such as VSV-G, may be...gamma-Retroviral Transfer Vectors   Expressing your gene of interest pMKO.1 GFP (Plasmid #10676) Retroviral vector...Learn more about CRISPR plasmids for transcriptional inhibition. Retroviral Plasmid Kit Retroviral Barcoding...

...prime editor consists of a Cas9 nickase fused to a reverse transcriptase. The Cas9 nickase is guided to the... that most Cas9-induced DSBs are repaired by non-homologous end joining. As a result, Cas9 treatment causes... ~ ~ ✓ ✓ ✓ R221K N394K Cas9 mutations Improve Cas9 nuclease activity ~ ~ ~ ~ ...this evolving field! Find prime editing plasmids! Find plasmids from David Liu's lab! This post was originally...the CRISPR guide Find CRISPR plasmids by function Browse our entire plasmid collection ...homology to the genomic DNA locus. After nicking by Cas9, the homologous pegRNA sequence hybridizes to the...-MLV) reverse transcriptase to the C-terminus of Cas9 H840A nickase (PE1). But innovations by the Liu ...

...genome editing, activating gene expression, repressing gene expression, and much more. But there’s one.../35++ Adenovirus Vector Expressing Anti-CRISPR Peptides Decreases CRISPR/Cas9 Toxicity in Human Hematopoietic...Addgene.org Find CRISPR Plasmids for Your Research Interested in light-inducible Cas9 activity? Check out ...et al., 2016). While Streptococcus pyogenes Cas9 (SpyCas9) is the most used and well studied CRISPR system... study, the labs found that anti-CRISPR prevents Cas9 activity in HEK293 by reducing editing from ~30%...interact with CRISPR-Cas systems such as Cascade-Cas3, Cas9, Cas12, and Cas13 (Marino et al., 2020). Some Acr...ways: Inhibit DNA binding Example:  AcrIIA4 blocks Cas9’s interaction with the PAM site (Dong et al., 2017...

... delivery of SpCas9 and Cas9 nickase for genome editing. You can also enhance expression of your gene ...must be used to infect a cell line already expressing Cas9. As John Doench (Broad Institute and member...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...Like all things in science, getting a high titer Cas9 lentivirus prep takes a bit of time and effort that...

...one AAV, Cas9 must be made smaller. Previous attempts to “shrink” Cas9 include the use of St1Cas9 (~3.3 ...amino acids in length, Cas9 from Campylobacter jejuni (CjCas9) is the smallest Cas9 ortholog characterized...vehicle for Cas9. However, to allow for more room for regulatory sequences while still fitting Cas9 and its...equally potent Cas9 protein, the Zhang lab took a different approach. They analyzed over 600 Cas9 orthologs...Trans-spliced Cas9 allows cleavage of HBB and CCR5 genes in human cells using compact expression cassettes... aureus Cas9 (SaCas9, 1053 amino acids) displayed cleavage activity in mammalian cells. SaCas9 produced...capabilities of full-length SpCas9. This set of plasmids includes AAV-Cas9C-VPR for targeted gene activation...

.... pyogenes Cas9 in the ABE complex with PAM-variant Cas9 proteins: the S. pyogenes NG-Cas9 (PAM: NG) to...constructs using a catalytically “dead” S. pyogenes Cas9 (dC9-ABE8.x-m/d). The dC9-ABE8.x-m/d constructs ...frequency more than 90% compared to ABE7.10. Choosing Cas9 proteins to increase the targeting scope of adenine... base editors The most commonly used S. pyogenes Cas9 requires the target site in the genomic DNA to contain... to create NG-ABE8.xm/d and the S. aureus Cas9 (PAM: NNGRRT) to create Sa-ABE8.x-m/d. With these variants...frequency over ABE7.10. The ability to use non-standard Cas9 in the ABE8 complexes enables a broader targeting...and 𝛽-thalassemia, can be mitigated with the expression of fetal hemoglobin. Previous studies have shown...

...lines, with an efficiency similar to that of Cas9. Like Cas9, NgAgo is also able to mediate homology-directed...template is supplied. Find Plasmids from the NgAgo Publication at Addgene Cas9 and NgAgo head to head NgAgo...features compared to Cas9. These include: More targeting flexibility Unlike Cas9 and the other CRISPR ...interest. In contrast, RNA guides must be expressed from a plasmid or in vitro transcribed, and changes in...the Addgene Blog Read About Cas9 Homolog Cpf1 Check Out High Fidelty Cas9 Mutants Browse All Genome Engineering...for short. Addgene has already distributed this plasmid all over the world, and the question on everyone... why you might choose NgAgo over CRISPR proteins Cas9 or Cpf1 - keep reading to learn more! Background...

...pTargetF system consists of one plasmid that expresses the S. pyogenes Cas9, a temperature-sensitive replicon...inducing expression of the sgRNA that targets the pMB1 replicon, resulting in cutting of the plasmid. At this...serious leaky expression in BL21(DE3), which we presume caused pTargetF to be cut by Cas9. This would result...beneficial for plasmid clearance. It is more convenient to update the spacer(20nt) on pEcgRNA plasmid as only...protein expression) (Zhang et al., 2020). Multicopy chromosomal integration for gene expression is an alternative... alternative versus plasmid-based expression which can be unstable and lack precise control over copy ...left end) pTnsABC expresses the transposases TnsA, TnsB, and TnsC pQCascade expresses the transposase TniQ...

.... Find CRISPR plasmids at Addgene! Knock-in: Introducing tags or stop signals Once Cas9 generates double-strand...powerful tools. CRISPRi uses a nuclease-dead Cas9 protein (dCas9) and a sgRNA to temporarily block the transcription...protein-coding genes thanks to systems like CRISPR/Cas9 that allow them to manipulate full genes and observe...system that can be tweaked to target ncRNA. CRISPR/Cas9 techniques and targeting methods Knock-out: Targeting...Targeting short vs. long ncRNAs The CRISPR/Cas9 system is often used to knock-out, or permanently disable...insertions in the DNA sequence can completely disrupt expression of the transcribed ncRNA (Jiang et al. 2014)....a lncRNA on nearby genes and its role in gene expression, though this may still unexpectedly affect nearby...