...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...Cpf1 displays lower off-target editing than Cas9, confirming that this protein is well suited for genome...advantages of Cpf1 Type II CRISPR systems based on Cas9 were thought to be the simplest CRISPR systems and...

...lenti-Cas9-VQR plasmid page. Despite their differences, both of these PID formats for lenti-Cas9-VQR contain...available through Addgene. Let’s take a look at lenti-Cas9-VQR-Blast, which has the Addgene ID #87155. The ...The resolvable compact identifier format for lenti-Cas9-VQR is the URL “http://n2t.net/addgene:87155” where...n2t.net web address. For the RRID format, lenti-Cas9-VQR is represented as “RRID:Addgene_87155” and the...formats. Let’s go back to our example plasmid, lenti-Cas9-VQR-Blast. If you scroll down to the bottom of this...semi-colons. For our example plasmid it will read “lenti-Cas9-VQR-Blast was a gift from Daniel Bauer (Addgene ... a resolvable compact identifier and RRID “lenti-Cas9-VQR-Blast was a gift from Daniel Bauer (Addgene ...

... Figure 2: Cas9 RNP complexed with 2-part cr:tracrRNA tags genes more effectively than Cas9 complexed ...Enhanced CRISPR/Cas9-mediated precise genome editing by improved design and delivery of gRNA, Cas9 nuclease,...target site. With the widespread adoption of CRISPR/Cas9 technology and rapid improvements in the field, ...set out to develop a hassle-free, highly efficient Cas9 protein-based method for epitope tagging in mammalian...genes at a time using Custom Synthetic cr:tracrRNA/Cas9 Ribonucleoprotein (csRNP) delivery Epitope tagging...delivery of three reagents to cells: a guide RNA, Cas9 protein, and a donor DNA that contains the tag sequence...annealed cr:tracrRNA is then mixed with recombinant Cas9 protein to form the ribonucleoprotein complex (csRNP...

...alternative Cas9, from Streptococcus aureus, is as effective at editing mouse embryos as Cas9 from S. pyogenes...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 ...model basics To create a mouse model, the gRNA, Cas9, and donor oligonucleotide or plasmid components...AAV can also be used as a vector to deliver CRISPR/Cas9 either through injection into either an oocyte or...

...surprise then that stability of the RNA used in CRISPR-Cas9 experiments – the “gRNA” – suffers from the same...still effectively serving their function of guiding Cas9 to generate a targeted break. These advances in ...modifications The recognition sequence for CRISPR-Cas9 targeting is led by a ~20 bp gRNA which is part ...trans-activating crRNA (tracrRNA) which interacts with the Cas9 protein. These RNA molecules are vulnerable to attack...partially DNA gRNA is surprisingly well-tolerated by the Cas9 protein and can increase on-target efficiency. In...modifications listed above were developed before CRISPR-Cas9, for use in applications like siRNA and antisense...On-off switches for gRNAs A major drawback of CRISPR-Cas9 technology is that most expression systems are constantly...

...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... Knock-in: Introducing tags or stop signals Once Cas9 generates double-strand breaks, a new DNA sequence... Figure 1: A schematic illustration of CRISPR/Cas9-based approaches to study ncRNAs. (A) Transcription...CRISPR systems for targeting ncRNAs While CRISPR/Cas9 has proven to be an extremely useful system for ...

... a colorful Cas9 toolbox, Ma et al. turned to SpCas9 and its orthologs NmCas9 and St1Cas9. Each ortholog...states. dCas9 gets a colorful makeover Even without its nuclease activity, catalytically dead Cas9 (dCas9... colorful system that uses fluorescently labeled Cas9 to label multiple genomic loci in live cells. While...hybridization (FISH) or fluorescently labeled TALEs, CRISPR/Cas9’s ease of use and ability to label live cells make... chromatin organization. This fluorescent CRISPR/Cas9-based method is a great step forward in live cell...As this study joins the pantheon of useful CRISPR/Cas9 techniques, we at Addgene are excited to see what... crispr/cas9...

...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 ...off-target activity and decreased immune response to Cas9. Recently, Baisong Lu’s lab developed a lentiviral capsid-based...capsid-based bionanoparticle system for packaging Cas9/gRNA ribonucleoproteins for transient expression...

...co-conversion markers. Expanding beyond Cas9 Since the development of CRISPR/Cas9 editing methods, other Cas proteins...University of California, Santa Cruz. Emerging CRISPR/Cas9 editing technologies have transformed the palette...regulation during developmental processes, CRISPR/Cas9 allows us to delete sequences and introduce mutations...remain in the edited animal. Development of CRISPR/Cas9 editing strategies in C. elegans As brilliantly ...reported in 2017 uses the direct injection of a CRISPR/Cas9 ribonucleoprotein complex with linear DNA as repair...determined optimal PAM orientation for editing using two Cas9 targets. An orientation where the 5’ ends of the...efficiency suggesting that sperm are more permissive to Cas9 editing (Farboud et al., 2019). Work from the Mello...

...strands bluntly with WT Cas9 and one gRNA, you can create a staggered cut using a Cas9 nickase and two gRNAs...CRISPR nickase experiment Overview of Cas9 nickase We’ll use SpCas9 nickases as examples for this post. ...By mutating one of two Cas9 nuclease domains, researchers created the CRISPR nickase. Nickases create...targeting range: using an individual gRNA with WT Cas9, repair levels decrease rapidly 10 bp from the cut...The HDR efficiency compared favorably to using WT Cas9 with the left gRNA across the window. In a subsequent... this problem. D10A nickase outperformed both WT Cas9 sites tested by a wide margin, exhibiting >20% repair...adapted from an IDT webinar: Optimized methods to use Cas9 nickases in genome editing. For more information...

...inducible CRISPR/Cas9 systems for transcriptional activation and genome editing by fusing Cas9 to a mutated...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...Nucleic Acids Research Find the drug inducible Cas9 plasmids E. coli genome-wide inhibition library...factors, and identify sequence-specific toxicity of Cas9. Read the paper in Nature Communications Find.... The lab has previously used the library with SpdCas9 to perform pooled CRISPRi screens to identify essential...

...combining specific sgRNAs with Cas9 or Cas9 derivatives. Unmodified Cas9 causes double stranded DNA breaks... loss of function while nuclease-deactivated Cas9 (dCas9) can be tethered to a repressor (CRISPRi) or ... issues. CRISPRi libraries utilize an engineered Cas9, which contains multiple point mutations in the ...resistance in A375 cells. The SAM system (CRISPR/Cas9 Synergistic Activation Mediator) uses an improved...improved activator design that consists of inactive Cas9-VP64, a sgRNA with two MS2 RNA aptamers, and the  ...Rational design of highly active sgRNAs for CRISPR-Cas9–mediated gene inactivation." Nature biotechnology...activity and minimize off-target effects of CRISPR-Cas9." Nature biotechnology 34.2 (2016): 184. PubMed ...

...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 ...

...shifts. This backbone model of Cas9 is based on Fuguo Jiang’s structure of Cas9 – primed for cleavage. This...contacted us in 2014 to ask if we could make a model of Cas9 based on the structure published by Martin Jinek...other aspects of the structure. While this first Cas9 model is certainly useful, it is also wrong in at... at least one serious way. We represented the Cas9 protein in a surface format made of rock-hard, inflexible... the amazing conformational flexibility that the Cas9 protein must have in order to scan along the double-stranded...model of a protein. We now have another model of Cas9 – a flexible alpha-carbon backbone model made of...reduce off-target hits in a rationally engineered Cas9 with improved specificity. Hold this flexible model...

...populations that already express Cas9 from an unlinked locus, while a Cas9+sgRNA drive targeting a synthetic...School. Scientists making transgenic organisms with Cas9 should be aware of the potential hazards of creating...caution. Why does making transgenic organisms with Cas9 risk creating a gene drive? The simplest way to ...last year2, delivering a DNA cassette encoding the cas9 gene and single guide RNAs (sgRNAs) with appropriate...easiest: don't use a DNA vector that encodes both Cas9 and sgRNA. What if I want to work with gene drives...sequence (save for the residual sgRNAs and possibly cas9 gene). An immunizing reversal drive is a variant...underlying advance enabling RNA-guided gene drives is Cas9 genome editing. Finally, gene drives are an inherently...

...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 ...standard pol III-gRNA, which will facilitate CRISPR/Cas9 multiplexing in systems with limited packaging capacity...afforded by PTGs will improve the utility of CRISPR/Cas9 in new avenues of research. The use of multiple ...

...would be compatible with CRISPR/Cas9 genome editing. Although CRISPR/Cas9 is all the rage, they had another... method and its potential applications to CRISPR/Cas9 genome editing.   Protein-only transduction? Niels...expression. Image courtesy of Diego D'Astolfo. CRISPR/Cas9: a potential application of iTOP Having tested iTOP...expression has a long-lasting effect, such as Cre- or Cas9-mediated recombination. Both primary hESCs and immortalized...cells treated with iTOP efficiently took up both Cas9 protein and a gRNA (yes, iTOP can also be used for... indicating that iTOP may be valuable for CRISPR/Cas9 stem cell editing. The small molecule NSDB... elements of iTOP. iTOP can be used to transduce Cas9 and a gRNA, which subsequently enter the nucleus...

... CHIME works through transduction of Cas9-expressing (Rosa26-Cas9) hematopoietic stem cells with a lentiviral...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...Haining, W. N., & Sharpe, A. H. (2019). A CRISPR-Cas9 delivery system for in vivo screening of genes in...

...paper in Communications Biology Find the Cas9-PCV and PCV-Cas9 plasmids Selective curbing of unwanted ...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...oligodeoxynucleotide HDR template covalently attached to the Cas9-gRNA RNP complex via the fused HUH endonuclease ...After testing dCas9 fusions with transcriptional repressors, they found that fusing dCas9 to an Mxi1 repressor...gene. Read the paper in mSphere Find the dCas9 and dCas9-Mxi1 plasmids If you have a new CRISPR tool...

...effects of gRNA and Cas9 expression, they fail to account for the effects of Cas9-induced dsDNA breaks... either stably expressed Cas9 or that had been lentiviral infected with Cas9. Validating the Library The...Genome-wide CRISPR/Cas9 screens are a high-throughput systematic approach for identifying genes involved.... Unanswered questions about genome-wide CRISPR/Cas9 screen design Are non-targeting guides an appropriate...single-guide studies? Many strategies exist for reducing Cas9 off-target effects, but most studies have focused...previously identified essential genes while previous Cas9 and shRNA library screens only identified  60% of...screen. This toxicity was used a proxy for detecting Cas9 cutting. Safe targeting guides better control for...