...for using iSPI, rEcoli XPS, and pSerOTS-C1* (V70) (ca. 2022) from the Rinehart lab: Methods for iSPI, rEcoli...PubMed (Link opens in a new window) Improved Reagents (ca. 2015) described in: A flexible codon in genomically...window) Protocol for using the Improved Reagents (ca. 2015) from the Rinehart lab: Users Guide for Improved...Phosphoprotein Reagents (DOCX, 525.9 KB) Reagents (ca. 2014) described in: Enhanced phosphoserine insertion...Link opens in a new window) Protocol for Reagents (ca. 2014) from the Rinehart lab: Users Guide for 2014...Phosphoprotein Reagents (PDF, 691.3 KB) Original Kit (ca. 2011) described in: Expanding the genetic code of...

...more information on the wide variety of Cas enzymes? CasPEDIA is an encyclopedia of Class 2 CRISPR systems... Tools CRISPR Blog Posts Fully functional CRISPR/Cas enzymes will introduce a double-strand break (DSB...Editing Other Applications Purify Tag Visualize dCas9-FokI Screen Pooled Libraries gRNAs Empty gRNA Vectors...

...more information on the wide variety of Cas enzymes? CasPEDIA is an encyclopedia of Class 2 CRISPR systems...the fusions, modifying the linker region between Cas variant and deaminase to adjust the editing window...are created by fusing Cas9 nickase or catalytically inactive “dead” Cas9 (dCas9) to a cytidine deaminase...editors are available with multiple Cas9 variants including high fidelity Cas9’s. Further advancements have ...Editing Other Applications Purify Tag Visualize dCas9-FokI Screen Pooled Libraries gRNAs Empty gRNA Vectors...base editors, the evolved TadA domain is fused to a Cas9 protein to create the adenine base editor. Both ... lab and CRISPR-X from the Bassik lab , in which Cas9 is fused to activation-induced cytidine deaminase...

...Yinong Yang. The MoClo CRISPR/Cas Toolkit for Plants includes CRISPR/Cas nucleases, base editors, gRNA...MoClo-compatible zCas9i Cloning Kit includes a high efficiency intron-optimized SpCas9-coding gene, zCas9i, and an...assembly of one or more gRNA expression cassettes - Qi-Jun Chen. Cas9 and gRNA backbone for use in rice. ... an intronized Cas9 series of vectors and DNA modules for assembly of CRISPR/Cas9 constructs - Sylvestre...CRISPR/Cas9 assembly for fast generation of multiple Arabidopsis mutants - Niko Geldner. CRISPR-Cas9 vector...development. The TapRoot - Podcast (Link opens in a new window) - The Taproot is a podcast to understand how scientific...plasmids expressing different functional variants of Cas9 and empty gRNA backbones. A few examples of useful...

...more information on the wide variety of Cas enzymes? CasPEDIA is an encyclopedia of Class 2 CRISPR systems...Editing Other Applications Purify Tag Visualize dCas9-FokI Screen Pooled Libraries gRNAs Empty gRNA Vectors...modifications, researchers have fused catalytically dead dCas9 to epigenetic modifiers. Design your gRNA to target...

...target the dCas9-repressor to your specific locus. Want more information on the wide variety of Cas enzymes...Tools CRISPR Blog Posts Catalytically dead dCas9, or dCas9 fused to a transcriptional repressor peptide...Editing Other Applications Purify Tag Visualize dCas9-FokI Screen Pooled Libraries gRNAs Empty gRNA Vectors...enzymes? CasPEDIA is an encyclopedia of Class 2 CRISPR systems with wiki entries describing enzyme activity...

...more information on the wide variety of Cas enzymes? CasPEDIA is an encyclopedia of CRISPR systems with...VI CRISPR systems, including the enzymes Cas13a/C2c2 and Cas13b, target RNA rather than DNA. Fusing the...CRISPR plasmids based on Cas13 designed to precisely edit RNA targets. CRISPR...Editing Other Applications Purify Tag Visualize dCas9-FokI Screen Pooled Libraries gRNAs Empty gRNA Vectors...E488Q) adenosine deaminase to catalytically dead Cas13b creates a programmable RNA editor that converts...guanosine, the result is an A->G change in RNA. dPspCas13b does not require a Protospacer Flanking Sequence...

...target the dCas9-activator to your specific locus. Want more information on the wide variety of Cas enzymes...Editing Other Applications Purify Tag Visualize dCas9-FokI Screen Pooled Libraries gRNAs Empty gRNA Vectors...Design Tools CRISPR Blog Posts Catalytically dead dCas9 fused to a transcriptional activator peptide can...specific gene. Design your gRNA sequence to direct the dCas9-activator to promoter or regulatory regions of your...enzymes? CasPEDIA is an encyclopedia of Class 2 CRISPR systems with wiki entries describing enzyme activity...

...Sensor for Ex Vivo and In Vivo Imaging of Ca Dynamics in High-Ca Organelles. Cell Chem Biol. 2016 Jun 1....-6273(20)30398-6. Edward Boyden Calcium GCaMP6f/s Ca sensors and separate nuclear localized dTomato fluorophore...membrane targeted GCaMP6s calcium sensor A polarized Ca(2+), diacylglycerol and STIM1 signalling system regulates...Calcium erGAP3 (GFP-Aequorin Protein) for imaging of Ca+ dynamics in endoplasmic reticulum GFP-Aequorin Protein...green, red or blue/green) Imaging intraorganellar Ca(2+) at subcellular resolution using CEPIA. Nat Commun...fluorescent CEPIA indicators for visualization of Ca(2+) dynamics in mitochondria. Sci Rep. 2020 Feb 18...CEPIA1er ER-mitochondria tethering by PDZD8 regulates Ca(2+) dynamics in mammalian neurons. Science. 2017 ...

...that of the Cas protein present on the plasmid. gRNA plasmids that do not co-express a Cas protein require...more information on the wide variety of Cas enzymes? CasPEDIA is an encyclopedia of Class 2 CRISPR systems... CRISPR requires that you have to express both a Cas protein and a target-specific gRNA in the same cell.... Single plasmids containing both the gRNA and a Cas protein act as an all-in-one vector, but their function... plasmid contains Cas9 and if so, which function of Cas9. We have plasmids with Cas9 that can cut, activate... guide RNA expression cassettes and a Cas9 nuclease/nickase expression cassette using the Golden Gate ...accessory pack is available for dCas9 and FokI-dCas9. Yamamoto Multiplex CRISPR/Cas9-based genome engineering...

...more information on the wide variety of Cas enzymes? CasPEDIA is an encyclopedia of CRISPR systems with...sequences, which include the enzymes C2c2/Cas13a and Cas13b. CRISPR...VI CRISPR systems, including the enzymes Cas13a/C2c2 and Cas13b, target RNA rather than DNA. In bacteria...Editing Other Applications Purify Tag Visualize dCas9-FokI Screen Pooled Libraries gRNAs Empty gRNA Vectors...crRNA. This activity provides a stark contrast to Cas9 and Cpf1, which require that each DNA target have...to attentuate RNA levels. In mammalian systems, Cas13a does not exhibit the collateral RNA degradation...

...CRISPR/Cas plasmids for labeling and visualization of genomic loci.... inactive Cas9 (dCas9) fused to a fluorescent marker like GFP, researchers have turned dCas9 into a customizable...Editing Other Applications Purify Tag Visualize dCas9-FokI Screen Pooled Libraries gRNAs Empty gRNA Vectors...

...help of Scott Lindner . Cut Fully functional CRISPR/Cas enzymes will introduce a double-strand break (DSB...Editing Other Applications Purify Tag Visualize dCas9-FokI Screen Pooled Libraries gRNAs Empty gRNA Vectors...research for decades. Recent developments with CRISPR-Cas9 gene editing have propelled both reverse and forward...forward genetics of these parasites. Moreover, CRISPR-dCas9 gene regulation has also been established for some...

...more information on the wide variety of Cas enzymes? CasPEDIA is an encyclopedia of Class 2 CRISPR systems...species or PAM binding variant of Cas9. For instance, wild-type SpCas9 must be used with targets that are...Editing Other Applications Purify Tag Visualize dCas9-FokI Screen Pooled Libraries gRNAs Empty gRNA Vectors...increases off-target effect. Which species or variant of Cas9 ( S. pyogenes , S. aureus etc.) was this gRNA sequence...

...Lab General Cloning Protocol 237.2 KB SpCas9 Plasmids The CRISPR/Cas system can be implemented in mammalian...Return to top References SpCas9 plasmids Multiplex Genome Engineering using CRISPR/Cas Systems. Cong L, Ran...hosts contain a combination of CRISPR-associated (Cas) genes as well as non-coding RNA elements capable...efficiency. SpCas9 and SpCas9n with 2a-Puro and 2a-EGFP are also available. 2. SpCas9 (or SpCas9n, D10A nickase...Protocols 2013). SpCas9 Cas9: The PX551 plasmid (AAV-SpCas9, #60957) contains a single cassette expressing ...plasmid expressing SpCas9 alone without sgRNA. Full references are below. 1. SpCas9 (or SpCas9n, D10A nickase...plasmids contain two expression cassettes, a human codon-optimized SpCas9 or SpCas9n, and the single guide RNA...

...type I Cas effector, composed of several separate Cas enzymes that form a complex called Cascade (see Cas3...Base Editing with CRISPR CasPEDIA: A Functional Classification of Cas Enzymes Cas Enzymes and PAM Sequences...application. Cas9 Orthologs While SpCas9 is the most commonly used Cas enzyme, additional Cas9 orthologs ...editing with Cas3 Browse Plasmids: Cascade-Cas3 Want more information on the wide variety of Cas enzymes? ...from. Many variations of Cas9 exist, along with other Cas enzymes like Cas12a, which are available for...paired with the appropriate Cas enzyme. Specific enzymes, such as Cas12a or Cas13 , can improve multiplexing...Other natural Cas orthologs include Hsp1Cas9 (from Helicobacter sp. MIT 11-5569 ) and Nme2Cas9 (from Neisseria...

...Species of Cas enzyme and gRNA Species of promoter and expression pattern of promoter for Cas enzyme and...compatible with smaller Cas enzymes) Transient or stable expression of SaCas9 and/or gRNA Infects dividing...sequence, you may want to consider alternative Cas enzymes ( see Cas9 variants and PAM sequences ). Once possible... exon or essential protein domains High-fidelity Cas enzymes increase specificity. Dual-nickase approach...of System Application Mammalian expression vector Cas enzyme promoter can be constitutive (CMV, EF1alpha...specificity, you can also consider using a high-fidelity Cas enzyme. In addition to off-target activity , it is...Once you have successfully delivered the gRNA and Cas enzyme to your target cells, it is time to validate...

...possibilities for Cas proteins With their enhanced specificity, eSpCas9, SpCas9-HF1, and HypaCas9 should enable...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...sites tested when compared to eSpCas9(1.1) and SpCas9-HF1. The HypaCas9 plasmids are available from Addgene...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... and testing of eSpCas9 Looking at the structure of the Cas9 nuclease (PDB ID: 4OO8 and 4UN3), Slaymaker...

...puromycin resistance cassette, such as PX459 ). After you co-transfect the CRISPR/Cas system with your homologous...that can be inserted into the genome using CRISPR/Cas for Homologous Recombination (HR)? How long should...the Cas9 nickase doesn't have the ability to induce double-stranded breaks like the wildtype Cas9 does... engineering experiments, consider that wildtype Cas9 with optimized chimeric gRNA has high efficiency...effect). The double nickase system is based on the Cas9 D10A nickase described in Figure 4 of the Cong, ...express two spacers and use PX335 to express the Cas9n (nickase). The concept of the double nickase system...can express two different chimeric gRNAs with the Cas9 nickase which will together introduce cleavage of...

... A multitasking Cas Most Cas effectors have one job — Cas see DNA, Cas chop DNA. Cas3, however, has two...CRISPR 101: Cas9 vs. The Other Cas(s) CRISPR 101: Anti-CRISPR Proteins Switch Off CRISPR-Cas Systems INTEGRATE...CRISPR has brought many other Cas effectors to light. One lesser-known Cas is waiting for its chance in...and Cas11. Type I-E Cascades contains all five of these Cas proteins, while type I-C Cascades do not contain... the spotlight — Cas3. Cas3 can induce genomic alterations on a scale that other Cas effectors can’t, ...a multi-Cas complex called Cascade (CRISPR-associated complex for antiviral defense). Cascade is brought...complex” system Cas3 is part of the Class 1 CRISPR family, which means it requires multiple Cas effectors to...