...A549 cells stably expressing Cas9. A549 cells were transduced with lentiCas9-Blast 1 and then selected with...Figure 2: Cas9 expression in monoclonal cell lines generated from A549 cells transduced with lentiCas9-Blast... express Cas9 and are included as a control (WT). Vinculin is a loading control. 1 lentiCas9-Blast was...additional 3 weeks under blasticidin selection. Anti-Cas9 Western blotting was performed on whole-cell extracts...

...off-target effects” have included using a Cas9 nickase variant, lowering Cas9 expression, and truncating gRNA sequences... 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...specificity of the Cas9 nuclease with engineered variants: eSpCas9, SpCas9-HF1, & HypaCas9. The off-target...portions of Cas9 might be important for nuclease specificity. They found that eSpCas9(1.1) and SpCas9-HF1 each... and testing of eSpCas9 Looking at the structure of the Cas9 nuclease (PDB ID: 4OO8 and 4UN3), Slaymaker...your Cas9 homolog of choice and get improved on-target specificity. Design and testing of SpCas9-HF1 Kleinstiver...

... available with multiple Cas9 variants, including high fidelity Cas9s, Cas9s with various PAM requirements...strand SpCas9-HF1 - disrupt Cas9’s interactions with DNA phosphate backbone HypaCas9 - increase Cas9 proofreading...new tools. Figure 3: Overview of Cas9n Nickase and dead Cas9 The two Cas9 nuclease domains, RuvC and HNH...enhanced specificity by engineering Cas9 into high fidelity Cas9s (hfCas9) . There is currently no defined...application. Cas9 Orthologs While SpCas9 is the most commonly used Cas enzyme, additional Cas9 orthologs ...constructs with Cas9 fused to proteins that modulate HDR or NHEJ components, like miCas9 , Cas9-DN1S , or others...Plasmids: Purify Cas9 Alternatives for CRISPR Genome Engineering S. pyogenes Cas9 (SpCas9) is the most commonly...

...organism without a specific preferred mutation Cas9 (or Cas9 nickase) Single (or dual) gRNA targeting 5′ ...generating a point mutation or inserting a tag Cas9 (or Cas9 nickase); Base editor Single (or dual) gRNA ...concern, Cas9-gRNA ribonucleoprotein (RNP) complexes are advantageous due to the transient Cas9 expression... RNA delivery of Cas9 and gRNA In vitro transcription reactions generate mature Cas9 mRNA and gRNA, which...generate transgenic embryos Cas9-gRNA ribonucleoprotein (RNP) complexes Purified Cas9 protein and in vitro transcribed...and/or Cas9 expression or (2) a lack of efficient target cleavage in cells expressing both Cas9 and gRNA...research. Although we will use the example of CRISPR/Cas9 in mammalian cells, many of these principles apply...

...sequence, Cas9 will neither bind nor cut the target. Cas9 homologs from different organisms or Cas9 mutants...basic level, CRISPR/Cas9 genome editing systems use a non-specific endonuclease (Cas9 or closely related...the PAM sequence. Cas9 Species/Variants and PAM Sequences Species/Variant of Cas9 PAM Sequence ...genomic target for Cas9, and the tracrRNA which acts as a scaffold linking the crRNA to Cas9 and facilitates... to direct Cas9 to a specific genomic locus and the scaffolding sequence necessary for Cas9 binding. When...CRISPR/Cas9 for genome editing, researchers simply need to express a gRNA designed to direct Cas9 to their...tracrRNA and Cas9, bind to these transcripts and C) cleave them leaving mature crRNAs bound to the Cas9/tracrRNA...

...frequently used Cas9, spCas9 from Streptococcus pyogenes, this sequence is 3’-NGG. But Cas9s found in other...assessment of the off-target activity of Cas9. This can occur when Cas9 cleaves a site that is not a perfect...the PAM specificity of Cas9s isolated from different organisms and engineered Cas9 variants that were able...consideration when choosing a Cas9 is balancing specificity and sensitivity. Wild-type Cas9 exhibits high on-target... directed evolution to generate Sniper-Cas9 resulting in a Cas9 with improved specificity. They compared...on-site and off-site activity of Sniper-Cas9 to different Cas9 variants using gRNAs of different lengths...loci and gRNA lengths. Recent alternatives to Cas9 While Cas9 remains the most widely used CRISPR nuclease...

... can occur due to prolonged Cas9 expression     The requirement for Cas9 transcription and translation...consisting of the Cas9 protein in complex with a targeting gRNA, to your cells of interest. Cas9 RNPs are capable...pathways. This makes Cas9 RNPs useful for CRISPR applications where limited expression of Cas9 is required and...express and purify His-tagged Cas9 from E. coli using a plasmid such as pET-28b-Cas9-His from Alex Schier’s ... of Cas9-gRNA RNPs by specific cell types (Rouet et al. 2018). This was accomplished using a Cas9 mutant...for experiments where stable Cas9 expression is required, such as when a Cas9 fusion to a fluorescent protein...and stability of the Cas9 protein in the host. Indeed, research has shown that a Cas9 protein from Geobacillus...

... gRNA, Cas9 binds the target site, but does not cut either strand of DNA. Traditional Cas9 regulators... a potent Cas9-based transcriptional regulator without making any functional changes to Cas9. For instance...repression with nuclease-competent Cas9. When complexed with a truncated gRNA, Cas9 is still able to bind DNA ...tight control over Cas9 nuclease activity while eliminating the reliance on orthogonal Cas9 species. Within...capability, and this nuclease-null or “dead” Cas9 (dCas9) variant can then be fused to effector domains...importantly, most Cas9 orthologs are less well characterized than the conventionally used SpCas9 (derived from...nuclease-competent Cas9, obviating the need to generate new cell lines or transgenic animals expressing dCas9 as a ...

...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 ...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...light-activated split-Cas9 (FAST) system constitutively expresses the C-terminal fragment of Cas9 while transcription...p65 and VP64 which in turn transcribes N-Cas9. The N- and C-Cas9 fragments then dimerize to form a functional...Off Genome Editing Single-chain Cas9 photoswitches Photoactivation of Cas9 can also be achieved using single-chain...

...the basic parts of the Cas9 enzyme.   Figure 1: A cartoon depiction of Cas9’s two major lobes, REC...   Unbound Cas9 Now that we know what we’re looking at, let’s peek at unbound—“apo”—Cas9 from Streptococcus...Figure 8: Comparison of Cas9:RNA crystal structure (left, PDB ID 4ZT0) with Cas9:RNA:DNA electron microscopy...Components of CRISPR  xCas9: Engineering a CRISPR Variant with PAM Flexibility Cas9 vs the Other Cases Further... with a nearby protospacer-adjacent motif (PAM)? Cas9 is becoming an ever more ubiquitous tool for genome... bridge helix. Created with BioRender.com.   Cas9 has two major lobes, called NUC (for nuclease) and...system (Nishimasu et al., 2014). Table 1. Summary of Cas9 domains, the lobe each domain belongs to, and their...

...using a Cas9 variant with higher specificity (eSpCas9 and HypaCas9) instead.  Choosing a Cas9 for site-specific... 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...targeted to the promoter region. Cas9 activators include Cas9 fused to VP64, VP64 along with other SAM factors...Next CRISPR Experiment Cas9 expression systems Addgene carries a variety of Cas9 containing plasmids that...target two Cas9-nickases (Cas9n) to the target locus, enhancing specificity over a single SpCas9. The drawback...

...CRISPR/Cas9 is an enormously plastic tool and has taken the scientific world by storm. While Cas9 has been...required for Cas9-based activation. CRISPRa for genome-wide studies One of the best uses for Cas9 activators...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...there has also been significant work on constructing Cas9 transcriptional activators. These constructs allow...upregulation of essentially any gene by fusing mutants of Cas9 deficient in DNA cutting activity to a transcriptional...

...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...truncated Cas9. Unfortunately, certain drawbacks limit the utility of these systems: St1Cas9 requires ...used CjCas9 with AAV to target genes in mouse muscle and eye tissue. Neisseria meningitidis Cas9 (NmeCas9...

...They went on to show that their CRISPR/Cas9-nuclease and CRISPR/Cas9-nickase vectors work successfully to...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...offers ease and efficiency for all-in-one CRISPR/Cas9 vectors expressing up to seven guide RNAs (gRNAs...Figure 1: Schematic overview of the all-in-one CRISPR/Cas9 vector construction system for multiplex genome ... based on earlier evidence showing that a CRISPR/Cas9 plasmid could be injected directly into mice for...

...cells stably expressing Cas9 via transduction with Cas9 lentiviral vector, lentiCas9-Blast (52962-LV, Figure... 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...with too many Cas9 integrations per cell, and my stable cell pool had wildly variable Cas9 expression (... variable Cas9-expression was across the different cells (Figure 3).   Figure 2: Cas9 expression...to assay Cas9 activity in my stable A549 cell pools. Using this virus, it’s expected that Cas9 activity...gene. So, cells with no Cas9 should appear green and cells with functional Cas9 should appear less green...

...as a binding signal for Cas9 and this sequence is a strict requirement for Cas9-mediated DNA cleavage. ...of novel S. pyogenes Cas9 variants with varying PAM sequences, 2) the use of Cas9 homologs derived from...2020). Characterization of Cas9 from additional bacterial species Many more Cas9 orthologs have been isolated...them more useful than SpCas9. For example, Cas9 from Staphylococcus aureus (SaCas9) is about 1 kilobase... in length, Cas9 from Campylobacter jejuni (CjCas9) is even smaller than SaCas9 and is also compatible...VQR, EQR and VRER SpCas9 variants effectively double the targeting range of CRISPR/Cas9 within the human... ~20 additional Cas9 species PAM sequence may not be characterized Non-SpCas9's bind a variety ...

...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...

...CRISPR/Cas9 system to precisely edit specific genes in monkeys. "Our study shows that the CRISPR/Cas9 system...for CRISPR Plasmids? CRISPR-Cas9 Plasmids available at Addgene CRISPR-Cas9 References, Protocols, and ...advances in the understanding and application of CRISPR/Cas9 technology are coming along at a fast and furious...Beisel and Rodolphe Barrangou, had applied the CRISPR/Cas9 system to developing an antibiotic that selectively...important details in their understanding of how CRISPR/Cas9s actually work. Last month, the Lawrence Berkeley... Nogales presented the first detailed picture of Cas9's three-dimensional shape via x-ray crystallography...Institute and MIT published another report showing Cas9's structural details. "Understanding this structure...

...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...Genome Editing of Pten by CRISPR/Cas9 in Mouse Liver in Spite of Cas9-Specific Immune Responses. Human... out the plasmids at Addgene: pAdSh.PGK.Cas9 (expresses S. pyogenes Cas9 from the PGK promoter) and U6...left) and targeted DNA knock-in (right) by CRISPR/Cas9. Adenoviral CRISPR vectors (left) or CRISPR vectors...transduced to target cells. Within target cells, Cas9 machinery is transiently expressed and binds to ...; made with BioRender.   Delivering gRNA and Cas9 using adenoviral vectors As compared to other viral...

...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...