...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...