...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...generating transgenic embryos. Cas9-gRNA ribonucleoprotein complexes Purified Cas9 protein and in vitro transcribed...need to deliver. Minimally, one or more sgRNAs and Cas9 are required for any application. If you want to...add a tag, you will also need to deliver a donor plasmid or single-stranded DNA oligonucleotide, so you ...Application Mammalian expression vector Promoter driving Cas9 expression can be constitutive or inducible. U6 ...stable cell lines. Transient or stable expression of Cas9 and/or gRNA in a mammalian cell line that can be...

...CRISPR plasmids available at Addgene. We will still periodically focus on specific CRISPR plasmid tools...CRISPR guide Find CRISPR plasmids by function Find recently deposited plasmids ...toolkit for epitope tagging Lentiviral capsid-based Cas9/gRNA ribonucleoprotein delivery Cytosine deaminase... a catalytically impaired Streptococcus pyogenes Cas9. They called this editor Target-ACE (adenine and...Read the paper in bioRxiv Find Target-ACE   CRISPR/Cas9-based toolkit for epitope tagging in neurons Harold... the paper in bioRxiv Find the ORANGE toolkit   Cas9/gRNA ribonucleoprotein delivery by lentiviral capsid-based...capsid-based bionanoparticle Transient expression of CRISPR/Cas9 in cells has advantages like reduced off-target ...

...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... 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...truncated Cas9. Unfortunately, certain drawbacks limit the utility of these systems: St1Cas9 requires ...

...CRISPR plasmids available at Addgene. We will still periodically focus on specific CRISPR plasmid tools...inducible CRISPR/Cas9 systems for transcriptional activation and genome editing by fusing Cas9 to a mutated...Nucleic Acids Research Find the drug inducible Cas9 plasmids E. coli genome-wide inhibition library In the...CRISPR guide Find CRISPR plasmids by function Find recently deposited plasmids ...vectors to create knock-ins CasX Drug inducible CRISPR/Cas9 activation E. coli genome-wide inhibition library...vectors GeneWeld from Jeffrey Essner’s lab uses CRISPR/Cas9 and a donor vector to create knock-ins of fluorescent..., or transcriptional regulators. In this system, Cas9 generates a double-stranded DNA break at the genomic...

... 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...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... guide sequence rather than an RNA guide as with Cas9. Third, it can be used to target “difficult” GC-...

...Designing the donor plasmid Figure 2: Donor plasmid design strategy. A donor plasmid contains 1 kb homology... areas of the plasmid to prevent the plasmid from being cut and destroyed by CRISPR/Cas9 during transfection...components of CRISPR/Cas9 (Cas9 protein and the crRNA-tracrRNA complex) and the donor plasmid into the cells...many labs. We’ve created and tested plasmids that use CRISPR/Cas9 to endogenously tag a wide variety of... the tag. Created with BioRender.com. Plasmids, plasmids everywhere To find out more about gene edited...regulatory control. But even with the advent of CRISPR/Cas9 technology, inserting large tags into a precise ... of genes with GFP. These plasmids are available through Addgene, and the stem cell lines we’ve made using...

...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...compared to DNA transfection of gRNA and Cas9 expression plasmids. These results are in line with previous...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 ...infiltrate cells. Options for CRISPR delivery include plasmid transfection, RNP electroporation, and viral transduction... leukemia virus (MLV)-like particles loaded with Cas9-gRNA ribonucleoproteins (RNPs). According to the...

...in Communications Biology Find the Cas9-PCV and PCV-Cas9 plasmids Selective curbing of unwanted RNA ...CRISPR plasmids available at Addgene. We will still periodically focus on specific CRISPR plasmid tools...CRISPR guide Find CRISPR plasmids by function Find recently deposited plasmids ...Read the paper in mSphere Find the dCas9 and dCas9-Mxi1 plasmids If you have a new CRISPR tool you’ve...Covalent tethering of DNA template to Cas9 SECURE base editors Nme2Cas9 CRISPR interference in Candida albicans... AAV delivery of Nme2Cas9 with gRNA cassette Erik Sontheimer’s lab identified a Cas9 protein from Neisseria...library Covalent tethering of DNA repair template to Cas9 Homology-directed repair (HDR) can be used to make...

...Postdoctoral Researcher in Jacob Corn’s lab. CRISPR-Cas9 (Cas9) is an RNA-guided nuclease that targets and cuts...replacement by HDR. Understanding Cas9 biophysics enhances HDR We first measured Cas9 dissociation rates and discovered...interaction with the Cas9-DNA duplex. Image from Richardson et al., 2016.   dCas9 enables sequence replacement...normally accompanies Cas9 cutting. We still don’t know the mechanism underlying dCas9 editing, but it could... cuts genomic DNA. The interplay between Cas9 (which causes the breaks) and host cell DNA repair factors...factors (which repair those breaks) makes Cas9 extremely effective as a genome editing reagent. This interplay... and thus, causes two types of editing outcomes: Cas9 breaks repaired by the non-homologous end-joining...

... target plasmid and pX330-sgRNA plasmid (Mashiko et al. 2013). The pCAG-EGxxFP target plasmid contains...pCAG-EGXXFP plasmid along with the individual gRNAs in their PX330 plasmids (or similar plasmids for gRNA...variants of the CRISPR/Cas9 system floating around, and many pre-designed plasmids containing these variants... between EGFP fragments. The pX330 plasmid contains humanized Cas9 expression cassette and sgRNA expression... (c) The pCAG-EGxxFP target plasmid was co-transfected with pX330 plasmids containing sgRNA sequences ...modification in vivo. Find Plasmids for Testing Your gRNAs in vitro at Addgene CRISPR/Cas9 gene editing in mice... encoding Cas9 into the oocyte. This original method was developed as the use of CRISPR/Cas9 in mammalian...

... 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...selection. Using an inducible Cas9 expression and removing the sgRNA plasmid after identifying successful... 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...

...applications. This plasmid was the top requested plasmid of 2017. Find pMD2.G. pSpCas9(BB)-2A-GFP (PX458...and a gRNA. But instead of Cas9, this plasmid expresses Cas9n (a D10A nickase mutant) to nick, rather than...and an envelope expressing plasmid, such as our second most requested plasmid of all time, pMD2.G. Find...PX458) - This plasmid from the Zhang lab expresses Cas9 from Streptococcus pyogenes with an invariant sgRNA...deliver Cas9 and your gRNA of interest into mammalian cells. This vector was the top requested plasmid of ...CRISPR plasmids are from the Zhang lab. In fact, the Zhang lab has shared over 40,000 CRISPR-Cas9 components...the end of this plasmid which indicates that there was a prior version of this plasmid. Indeed the first...

...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...epigenetic modulation. While catalytically inactive cas9 (e.g. dCas9) has been fused to a variety of protein modules...

... smaller size of the plasmids. The Zhang lab, on the other hand, uses a 1 plasmid, single infection system...It’s clear that CRISPR-Cas9 technology has really changed the game for anyone looking to quickly and ... thousands of targets at once. That’s what our plasmids allow.” Wang’s library is designed to target a...Lander library uses sequential infection first of Cas9, then with the sgRNAs, and can result in a higher...that have previously been stably transfected for Cas9 expression. The Yusa lab has used their library ...application offer proof-of-principle that CRISPR-Cas9 can be used to target any sequence in the genome... Genetic Screens in Human Cells Using the CRISPR/Cas9 System. Wang T, Wei JJ, Sabatini DM, Lander ES. ...

...available from Addgene consists of two plasmids: pCas9: carries Cas9 and chloramphenicol resistance pCRISPR.... 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 ...targeting either a catalytically dead Cas9 (dCas9) alone or a dCas9 fused to the omega subunit of RNA polymerase... lab M. tuberculosis CRISPRi plasmids Noting that the more common spCas9 system has generally low efficiencies.... They identified the Cas9 orthologue from Streptococcus thermophilus, Cas9sth1 as a robust and highly...

...CRISPR/Cas9 shows great potential for multiplexed genome editing. Interested in using PTG plasmids in your...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...construct size, it requires coexpression of Csy4 with Cas9 and the polycistronic transcript. As nucleases may... (15% and 9% of target sites.) How many loci can Cas9 modify? To test the application of this system to...-20% frequency at the four loci, indicating that Cas9 can be guided to at least 8 distinct loci. They ...common to prokaryotes and eukaryotes. Though the plasmids made by Xie et. al are specifically designed for...

...or apoptosis with Cas9/gRNA expression. A pooled analysis found no evidence of Cas9 cleavage at predicted...were located near the Cas9 cut site, leading Wang et al. to conclude that some Cas9-derived indels may not...CRISPR/Cas9 escape occurs in an animal model. If these escape mutations are frequent, the dCas9-mediated...destroy infected cells, the other using wild-type Cas9 to remove the HIV-1 genome from infected cells. Read ...realistic with the advent of CRISPR. Using CRISPR/Cas9 SAM to "shock and kill" To “shock and kill” the...targeted approach, Zhang et al. turned to the CRISPR/Cas9 Synergistic Activation Mediators (SAM) system available... a latent microglial line, treatment with CRISPR/Cas9 SAM increased the percentage of cells with activated...

...Abudayyeh et al., 2016). In the endogenous CRISPR/Cas9 system, Cas9 uses both a tracrRNA and crRNA to facilitate... 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...conformational changes in the Cas13a molecule. Like Cas9, Cas13a tolerates single mismatches between the ...

...alternative Cas9, from Streptococcus aureus, is as effective at editing mouse embryos as Cas9 from S. pyogenes...create a mouse model, the gRNA, Cas9, and donor oligonucleotide or plasmid components are brought together... Find CRISPR Plasmids for use in Mammals Check out Our CRISPR Guide Pages Find Plasmids for Mouse Targeting...guest bloggers, Wenning Qin and Haoyi Wang. CRISPR/Cas9 is revolutionizing the mouse gene-targeting field...ever to manipulate the mouse genome using CRISPR/Cas9. Similar to the human genome, the mouse genome ...robust in performance. In its natural setting, CRISPR-Cas9 is an acquired immune system in bacteria and archaea...) of about 125 nt that specifies the target, the Cas9 endonuclease that creates the DNA double-strand ...

... than Cas9, confirming that this protein is well suited for genome editing.  Find Cpf1 Plasmids at Addgene...overcome some of Cas9’s shortcomings. Cpf1’s preferred PAM is 5’-TTN, differing from that of Cas9 (3’-NGG) in...than that of the widely used SpCas9 and comparable to SaCas9. As seen with Cas9 orthologs, Cpf1 efficiency...rate is similar to that of high fidelity Cas9s eSpCas9 and SpCas9-HF1. Both AsCpf1 and LbCpf1 ribonucleoproteins...two Cpf1 nucleases, distant cousins of well-known Cas9. Cpf1 cleaves DNA in a staggered pattern and requires...rather than the two (tracrRNA and crRNA) needed by Cas9 for cleavage. Now, two new studies show that Cpf1...advantages of Cpf1 Type II CRISPR systems based on Cas9 were thought to be the simplest CRISPR systems and...