... 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...