...the PAM sequence. Cas9 Species/Variants and PAM Sequences Species/Variant of Cas9 PAM Sequence ...sequence, Cas9 will neither bind nor cut the target. Cas9 homologs from different organisms or Cas9 mutants...' TTTV AsCpf1 RR variant 5' TYCV LbCpf1 RR variant 5' TYCV AsCpf1 RVR variant 5' TATV Campylobacter...3' NGCG SpCas9 EQR variant 3' NGAG SpCas9 VQR variant  3' NGAN or NGNG  xCas9 3' NG, GAA,...basic level, CRISPR/Cas9 genome editing systems use a non-specific endonuclease (Cas9 or closely related... general overview of CRISPR components, new Cas9 variants are being discovered all the time and the requirements...only CRISPR protein required is the Cas9 endonuclease or a variant thereof. This individual protein has...

...of novel S. pyogenes Cas9 variants with varying PAM sequences, 2) the use of Cas9 homologs derived from...reported the development of near-PAMless Cas9 variants. These variants, named SpG and SpRY, can target NGN ...identified four novel SpCas9 variants with altered PAM binding specificity: SpCas9 variant Mutations (relative...(relative to SpCas9) PAM sequence D1135E variant D1135E NGG VQR variant D1135V, R1335Q...VQR, EQR and VRER SpCas9 variants effectively double the targeting range of CRISPR/Cas9 within the human...as a binding signal for Cas9 and this sequence is a strict requirement for Cas9-mediated DNA cleavage. ...NGAN or NGNG EQR variant D1135E, R1335Q and T1337R NGAG VRER variant D1135V, G1218R, R1335E...

...off-target effects” have included using a Cas9 nickase variant, lowering Cas9 expression, and truncating gRNA sequences...specificity of the Cas9 nuclease with engineered variants: eSpCas9, SpCas9-HF1, & HypaCas9. The off-target... own hyper accurate Cas9 variant (HypaCas9) to the pool of enhanced CRISPR tools. In their work, Chen ... technology itself is constantly improving. Cas9 variants have been modified for genome editing, activating... 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...portions of Cas9 might be important for nuclease specificity. They found that eSpCas9(1.1) and SpCas9-HF1 each...

...PAM specificity of Cas9s isolated from different organisms and engineered Cas9 variants that were able to...on-site and off-site activity of Sniper-Cas9 to different Cas9 variants using gRNAs of different lengths (Lee...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...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...loci and gRNA lengths. Recent alternatives to Cas9 While Cas9 remains the most widely used CRISPR nuclease...

... domain of Cas9. After multiple rounds of engineering, they developed the SpRY Cas9 variant (Walton, et...several Cas9s that recognize NG PAMs. The Nureki lab generated SpCas9-NG, a rationally engineered Cas9 with...Requirement and Expanding Beyond Cas9 xCas9: Engineering a CRISPR Variant with PAM Flexibility History of...that don’t utilize NGG   There are plenty of Cas9 variants which utilize alternative PAMs. These alternative... at NGH PAMs (Zhang et al, 2021) NG Cas9 NG - SpG Cas9  NG  - SpRY nuclease  C to T Base Editor...targeting with near-PAMless engineered CRISPR-Cas9 variants. Science 368,290-296(2020). DOI:10.1126/science.aba8853...PAM compatibility and editing activities of Cas9 variants. Nucleic Acids Res. 2021 Sep 7;49(15):8785-8795...

...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...Further, this group also characterized a second Cas9 variant, SpRY, which cuts at NRN PAMs with high efficiency...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...

... In cases where Cas9 won’t work, there are other options. Commonly used Cpf1 variants, with a  5’ TTTV...broadest-targeting variants. For the final round of evolution, they used a no-G PAM library to enrich for Cas9 variants...References Hu, Johnny H., et al. "Evolved Cas9 variants with broad PAM compatibility and high DNA specificity... the Addgene Blog Learn about Engineered Cas9 PAM Variants Read about CRISPR Base Editing Learn about...flexibility of xCas9 3.7. Base editor variants with xCas9 are similarly flexible. Like the xCas9 3.7 nuclease...and keeping that dCas9 sequence in the pool. According to this logic, ω-dCas9 variants recognizing multiple...In order to bind DNA, Cas9 and other CRISPR enzymes require a short PAM sequence adjacent to the targeted...

..., a base editor containing high fidelity Cas9 variant HF-Cas9. They subsequently tested the original BE3...Plasmid ID Plasmid Name Cas9 Variant Speacial Features 85169  pJL-SaBE3  SaCas9 (NNGRRT)    85170  pJL-SaKKH-BE3...Campbell’s lab added two new mCherry variants to the repository. These variants can be used as tools to learn...editing. Kim et al. used natural and engineered Cas9 variants to develop five new base editors with distinct...-BE3  EQR-Cas9 (NGAG)  Lower off-target activity than BE3  85173  pBK-VRER-BE3  VRER-Cas9 (NGCG)   ...The popular CRISPR base editing technique uses dCas9/Cas9 nickase fused to a cytidine deaminase for targeted... pJL-SaKKH-BE3  SaCas9 (NNGRRT)    85171  pBK-VQR-BE3  VQR-Cas9 (NGA)  Lower off-target activity than...

...capability, and this nuclease-null or “dead” Cas9 (dCas9) variant can then be fused to effector domains such... gRNA, Cas9 binds the target site, but does not cut either strand of DNA. Traditional Cas9 regulators...limitations to a system that relies on two distinct Cas9 variants—nuclease-competent or nuclease-null—in order...both the nuclease-positive and nuclease-null variants of Cas9 along with the necessary gRNAs, but it would...control which gRNA becomes complexed with which variant of Cas9. To circumvent this problem, previous studies... 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 ...

..., a base editor containing high fidelity Cas9 variant HF-Cas9 (Rees et al., 2017). HF-BE3 showed 37-fold...The Liu lab also used natural and engineered Cas9 variants to develop five new base editors with distinct...other methods. In a head-to-head comparison with Cas9, Cas9 modified 9/12 known off-targets with a 14% indel... genome. So the development of near-PAMless Cas9 variants significantly expands the library of accessible...Kim et al., 2017 SaBE3, BE3 PAM variants, BE3 editing window variants   Greatly expands the number of ...targeting with near-PAMless engineered CRISPR-Cas9 variants. Science (New York, N.Y.), 368(6488), 290–296...individual Cas9. They also mutagenized the cytidine deaminase portion of the base editor to create SpCas9 base...

...library of AAV9 variants (Chuapoco et al., 2023). They found the best-performing variant, CAP-Mac, was ...genome editing for primary cells Monomeric StayGold variants AAV.CAP-Mac for systemic gene transfer in primate...peptide” that facilitates endosomal escape. Combining Cas9-PAGE with lentiviral sgRNA expression yielded editing...lines and primary cells. Although cell entry of a Cas9 ribonucleoprotein (RNP) was limited (likely due ...multiplexed guides. Figure 1: A) Cell-penetrating Cas9, fused to HIV TAT, Myc and SV40 Nuclear Localization...Localization Signals, and GFP. B) Workflow to assess Cas9-PAGE editing with viral sgRNA targeting mCherry reporter... Breaking up is hard to do: Monomeric StayGold variants By Mike Lacy In 2022, Atsushi Miyawaki and colleagues...

.... pyogenes Cas9 in the ABE complex with PAM-variant Cas9 proteins: the S. pyogenes NG-Cas9 (PAM: NG) to... and the S. aureus Cas9 (PAM: NNGRRT) to create Sa-ABE8.x-m/d. With these variants, ABE8 could target ... evolved base editor: ABE8e Read about xCas9, a CRISPR variant with PAM flexibility Resources on Addgene.org...modified ABE7.10 accordingly which resulted in variants named “ABE8.x”: “8” stands for the eighth round...x-d.  This resulted altogether in 40 new ABE8 variants. When comparing on-target DNA editing efficiency...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...

...Cas protein variants may also be useful for targeting ncRNAs. For example, the Cas12a variant can cleave...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...

...uncover new gene variants associated with conditions of interest, then studying those variants in model systems... kit to CRISPR/Cas9? When would you choose one or the other? Musunuru: The CRISPR/Cas9 system is easier...makes possible, and how it compares to his CRISPR/Cas9 system. Addgene: For people unfamiliar with this...implicated in disease or, in cases where particular variants or mutations have been reported in people, we .... You could use the kit to insert all of those variants into stem cells with the same genetic background...pieces you need to assemble. Our system for CRISPR/Cas9 (also available from Addgene) only has two plasmids...more efficient. You might often start with CRISPR/Cas9, but TALENs do have more flexibility in terms of...

...For more information on Cas9 variants, check out our blog post on choosing a Cas9 enzyme. Fast facts – ...similarities and differences to Cas9. DNA targeting Cas enzymes Cas9 – The genome engineering workhorse...CRISPR’ without shortly thereafter hearing ‘Cas9’. The Cas9 nuclease is a genome engineering powerhouse.... Dozens of variants exist with unique cutting features and evolved PAM sites, making Cas9 an adaptable...same mechanism as traditional spCas9. If you are looking to modify RNA, Cas9 just won’t cut it – literally...We are all oh so familiar with the nuclease Cas9, but how does it stack up to the new Cas enzymes on ...proceed to generate a blunt DNA double strand break. Cas9 has been studied for genome engineering purposes...

...single base editing by APOBEC-Cas9 Though the ACE reporter quantifies APOBEC-Cas9 efficiency in real-time, ...by infecting HEK-293 cells with the BFP variant, a BE4am-Cas9 base editor, and sgRNA vectors for the BFP-to-GFP...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 ...genome, but they’re inefficient compared to CRISPR/Cas9 edits that rely on double strand DNA breaks. Due...can help you do just that. Monitoring APOBEC and Cas9-mediated editing in real time The Harris lab created...APOBEC (a family of cytidine deaminase proteins) and Cas9-mediated editing in real time (St. Martin et al....

... when compared to the most commonly used Cas9 variant, SpCas9. These properties, combined with other benefits...Generating Mouse Models Using CRISPR/Cas9 Cpf1 Update: Comparison to Cas9 and NgAgo Special Delivery: Fluorophore...generate mouse models.   Cpf1 Update: Comparison to Cas9 and NgAgo As you might expect given the excitement...in the post, may make Cpf1 the preferred CRISPR variant going forward.   Special Delivery Fluorophore Targeting...

...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...with an evolved Cas9 domain from PE6e-g produced additive improvements. These variants are worth testing... ~ ~ ✓ ✓ ✓ R221K N394K Cas9 mutations Improve Cas9 nuclease activity ~ ~ ~ ~ ...Over 75,000 pathogenic genetic variants have been identified in humans and cataloged in the ClinVar database... potential to correct only a minority of those variants in most cell types. But prime editing, a CRISPR...homology to the genomic DNA locus. After nicking by Cas9, the homologous pegRNA sequence hybridizes to the...

...summarize, CRISPRa uses a nuclease-dead variant of Cas9 (dCas9) and engineered combinations of transcriptional...upregulation of the targeted variants without inducing expression of the non-targeted variant. Moreover, isoform-specific...with neuron-specific expression of dCas9-VPR (Figure 2), a popular Cas9 activator developed by the Church...primary rat neurons with dCas9-VPR and sgRNAs targeting either transcript variant led to robust and specific...limited to in vitro settings due to the large size of Cas9 activators, which largely precludes delivery by ...mouse expresses a modified version of the “Suntag” Cas9 activator that recruits up to twenty copies of the...here. Neuron-optimized viral vectors Alternatively, Cas9 activators can be delivered to the CNS by lentivirus...

...–C, with the beloved SpCas9 (from Streptococcus pyogenes) belonging in II-A. Cas9 was the first to be ...first time. The discovery and application of CRISPR-Cas9 kicked off over a decade of frenzied interest in... simple hierarchy: class to type to subtype to variant. Despite the seemingly complicated steps to arrive...on main function and composition; subtypes and variants are typically based on species of origin. There...currently two classes, six types, 33 subtypes, and 17 variants. Figure 1: Overview of the CRISPR classification...hierarchy flows from Class to type to subtype to variant (shown in italics). Created with BioRender.com....while Class 2 use single protein Cas effectors, like Cas9. The Cascade complex doesn’t usually carry out the...

... un-tagged versions of SpCas9 and further variants replace SpCas9 with SpCas9n (nickase), which nicks ...plasmid expresses Streptococcus pyogenes (Sp) Cas9 from a Cbh promoter. Cbh is a modified version of CAG promoter...has tested in a variety of mammalian cell lines. Cas9, of course, is the key component of most CRISPR ...cleaving" peptide causes the ribosome to translate Cas9-2A and the Puromycin resistance gene as separate...homology directed repair. You can find all of these variants, as well as links to the Zhang Lab's Nature Protocols...Ann, et al. "Genome engineering using the CRISPR-Cas9 system." Nature protocols 8.11 (2013): 2281-2308... express both SpCas9 and your gRNA in a variety of mammalian cell types. Purpose  SpCas9 This plasmid ...

...are 24 variants of the cas14 gene that cluster into 3 subgroups (cas14a-c). All these variants share the...many others are still unknown. Identifying Cas14 variants In this quest to discover new and maybe more efficient...primitive version of the larger and more complex Cas9 and Cas12 proteins. Cas14 cleaves ssDNA But what... and cleave a targeted sequence of ssDNA. Unlike Cas9, Cas14 does not require a PAM sequence. In addition...

...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 ..., if these variants are fused to the MLV Gag in place of SpCas9. gRNA A gRNA targeting a DNA sequence ... leukemia virus (MLV)-like particles loaded with Cas9-gRNA ribonucleoproteins (RNPs). According to the...generation. Key components of Nanoblades Nanoblades are a Cas9 RNP delivery vehicle that’s based on mouse leukemia...

...encoding the gRNAs and Cas9. As with the Gersbach lab plasmids, multiple Cas9 variants are available: wt humanized...clone your own entry vectors containing other Cas9 variants to use with the MuLE system. Multiplexing from...subsequently inserted into a Cas9-containing destination vector based on pCAMBIA. Cas9 is plant-optimized, and...Topic Page Get Advice on Choosing The Proper Cas9 Variant for Your Experiment Learn About Multiplexing...humanized Cas9, D10A nickase mutant (Cas9n), dCas9 (transcriptional repression), and Fok1-dCas9 (dimeric...options include humanized wt Cas9, dCas9 (transcriptional repressor), and dCas9-VP64 (transcriptional activator... then digested using BsmBI and ligated into Cas9 or dCas9-containing destination vectors. Destination ...

... active variant, the phage is not infectious. The editing speed dictates which phage variants can propagate...compatible with all Cas9 and Cas12 homologs that the researchers tested, including SpCas9, SaCas9, LbCas12a, enAsCas12a...the DNA. ABEs are somewhat slow and need a strong Cas9 domain to “hold on” long enough to the DNA to induce...complete ABE protein. If the phage delivers an active variant of the base editor, a functional gene III product...stringency PACE, resulted in the evolution of TadA variants with much higher base editing activity compared... TadA-7.10 enzyme. After testing numerous TadA variants, they identified TadA-8e, which edits 590-fold... enAsCas12a, SpCas9-NG, SaCas9-KKH, CP1028-SpCas9, and CP1041-SpCas9. The resulting base editor was named...

... out this post. Learn how to Choose the Best Cas9 Variant for Your Next Experiment Read more about Optogenetic...et al., 2016). While Streptococcus pyogenes Cas9 (SpyCas9) is the most used and well studied CRISPR system... CRISPR-Cas technology is constantly evolving. Variants of Cas proteins can be used for genome editing... 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... crRNA loadingExample: AcrIIC2 interactions with Cas9 interferes and prevents the correct assembly of ...

... even distinguish between protein variants when used with a variant-specific antibody. TRIM21, an E3 ubiquitin...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 modulation. While catalytically inactive cas9 (e.g. dCas9) has been fused to a variety of protein modules...Communications. 2017. PubMed PMID: 28924139. FIRE-Cas9 for inducible interrogation of the epigenome Article...type. By varying which sgRNAs are used with FIRE-Cas9, experiments can be designed to target multiple ... epigenetic memory at nearly any locus. The FIRE-Cas9 plasmids can be found here. Braun et al. Nat Communications...

...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...

...dual-nuclease Cas9-Cas9 chimeras Scot Wolfe’s lab has developed dual-nuclease Cas9-Cas9 chimeras with ...compared to monomeric Cas9’s. Read the paper in Nature Communications Find the Cas9-Cas9 chimera plasmids...optogenetic control of CRISPR-Cas9. By combining AcrIIA4, a Streptococcus pyogenes Cas9 inhibitor, with the LOV2...specificity of multiple wild-type and engineered variants of Cas nucleases. In doing so, they also created...Anti-CRISPR proteins for optogenetic control of CRISPR-Cas9 Anti-CRISPR proteins that inhibit type II CRISPR...

...inhibitor CASANOVA for “CRISPR-Cas9 activity switching via a novel optogenetic variant of AcrIIA4.” CASANOVA responds... It works with the S. pyogenes Cas9, dCas9-effector fusions, and xCas9, and requires no additional chemicals...episode of Hot Plasmids! Optogenetic control of CRISPR-Cas9 by CASANOVA Article contributed by Cary Valley  ...CRISPR systems including the popular S. pyogenes Cas9 (Rauch et al., 2017, Hynes et al., 2017). However..., they have developed a system to control CRISPR-Cas9 activity using blue light (Bubeck et al., 2018)...., inhibiting function of Acr. This allows CRISPR-Cas9 to function on its target.  They name their most... robotic picking approach to select cells with variant characteristics they wanted, the team identified...

...nicking Cas9. A guide directs Cas9 to a site of interest, where it nicks its target DNA (left). Cas9 dissociates...error prone PolI3M that’s fused to a nicking Cas9 (nCas9). Similar to mutagenesis with error prone PCR...scale by screening diverse libraries of genetic variants. Several methods for generating mutant libraries... a user-defined genomic region. EvolvR, a CRISPR-Cas9 based targeted mutagenesis method developed by the...provides a new approach for generating novel genetic variants in bacteria. Read on to learn about the key components...enhance nCas9 (enCas9) EvolvR with a targeted mutagenesis rate ~9-fold higher than the original nCas9, while...used to direct the PolI3M-nCas9 complex to a DNA site of interest, which nCas9 nicks and then dissociates...

...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 ... in human cells. Thus, they engineered two CBE variants with mutations in the rat APOBEC1 that decreases...After testing dCas9 fusions with transcriptional repressors, they found that fusing dCas9 to an Mxi1 repressor...

...Cho et al., 2014). There are several variants of the CRISPR/Cas9 system floating around, and many pre-... encoding Cas9 into the oocyte. This original method was developed as the use of CRISPR/Cas9 in mammalian... guest blogger Samantha Young. The use of CRISPR/Cas9 for gene editing has expanded since its adaptation...pre-designed plasmids containing these variants ready for purchase. But what is the easiest and fastest ...as soon as possible! Selecting your target CRISPR/Cas9 is often used to study the biology surrounding a...the target sequence was digested by sgRNA guided Cas9 endonuclease, homology dependent repair (HR, homologous... fragments. The pX330 plasmid contains humanized Cas9 expression cassette and sgRNA expression cassette...

...toolkit for epitope tagging Lentiviral capsid-based Cas9/gRNA ribonucleoprotein delivery Cytosine deaminase... they’ve engineered SECURE adenine base editor variants using a protein-truncation strategy with reduced... 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...

...using a nickase version of Cas9, but were also successful using a dCas9 variant as well. Furthermore, they...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... from Addgene consists of two plasmids: pCas9: carries Cas9 and chloramphenicol resistance pCRISPR: carries.... They identified the Cas9 orthologue from Streptococcus thermophilus, Cas9sth1 as a robust and highly...bacteria. The authors were able to engineer a dCas9 variant fused to bacterial enhancer binding protein ...

...expressing variants of Yellow Fluorescent Protein (YFP). The team identified a new YFP variant, mGold. mGold...Kivanc Birsoy. Find the library here A new SaCas9 ABE variant (microABEI744) has improved on-target editing...footprint compared to current N-terminal linked SaCas9 ABE variants. It's also one of the smallest AAV-deliverable... ~70kDa, with a molecular weight roughly half of Cas9 or Cas12a. This is in part due to the fact that ...CRISPR Plasmids and Resources page.  CDDR (CRISPR–Cas9-based Dual-fluorescent DSB Repair) is a double-strand...

...expression of S. pyogenes Cas9 or Cas9 variants (hereafter referred to as Cas9) and a single stranded guide...to the nucleus, and several codon optimized Cas9 variants have been created in an effort to increase translation... gRNA backbones also contain Cas9, which enables expression of both Cas9 and the gRNA off of the same ...Nuclease dead Cas9 (dCas9) based activators (such as dCas9-VP64) or repressors (dCas9-KRAB or dCas9-SRDX) can...get more information on multiplexing gRNAs here). Cas9 is commonly tagged with a nuclear localization sequence...repress target genes in plant cells, respectively. Cas9 expression is commonly driven by plant-derived RNA...Examples of commonly used RNA pol II promoters for Cas9 expression include the ubiquitously expressing cauliflower...

... 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...the other orthologs. Ma et al. tested their dCas9 variants using gRNAs specific for telomeric sequences... 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...

...are numerous GFP variants. Here I use the name ‘GFP’, which refers to mEGFP. This variant was made by introducing...nuclease-deficient Cas9 can bind loci in the genome that are defined by a gRNA. Since Cas9 is a protein, a...jellyfish GFP (AvGFP) to create enhanced GFP (EGFP), a variant with improved brightness (Tsien, 1998). Another...necessary to generate the strictly monomeric EGFP variant, mEGFP (Zacharias et al., 2002). GFP needs oxygen...Cranfill et al., 2016 and FPbase.org). A number of variants remain fluorescent under acidic conditions, with...although it still is worthwhile to generate several variants and check the functionality of the fusion (Mastop...

... CRISPR 101: Which Cas9 do I choose for my CRISPR experiment? CRISPR 101: Cas9 The Other Cas(s) The...field has exploded with dozens of Cas enzymes and variants available. Without a comprehensive resource, it...Addgene. So whether you're looking for the exact Cas9 plasmid used by Jinek et al. 2012, or browsing for...CRISPR–Cas systems: a burst of class 2 and derived variants. Nature Reviews Microbiology, 18(2), 67–83. https...Once upon a time, not so long ago, spCas9 was the only Cas enzyme widely available and applied by researchers...and trans activity of each entry. For example, SpyCas9a (one of the most frequently used Cas enzymes) ...CasPEDIA Cas ID display information for a given Cas (SpyCas9a) from CasPEDIA.   Helpful resources and information...