...still separate the individual Cas components. Figure 1: Overview of Cascade-Cas3 mechanism. Created with BioRender.com... CRISPR-Cas and Cascade for large genomic deletions. CRISPR...comprised of a combination of Cas5, Cas6, Cas7, Cas8, and Cas11. Cascade is directed to target DNA using... CRISPR Cas3 CRISPR Plasmids: Cascade-Cas3 Browse ...recruits Cas3. After nicking the non-targeted strand, Cas3 starts unraveling DNA using its helicase properties...this, some CRISPR-Cas3 plasmids take an all-in-one approach and include Cas3 and Cascade on one single plasmid...Blog Posts Cas3 is a unique member of the CRISPR family. After nicking the target DNA, Cas3 initiates ...

...Nucleases (RFNs) CRISPR-Cas/RGN expression plasmids for use in human cells CRISPR-Cas/RGN expression plasmids...about RGNs and CRISPR-Cas systems can also be found at http://www.crispr-cas.org . Users are also encouraged...Joung Lab CRISPR-Cas/RNA-Guided Nuclease (RGN) Plasmids Genome...Plasmids Joung Lab CRISPR-Cas/RNA-Guided Nuclease (RGN) Plasmids You may also like... CRISPR Guide CRISPR...RGNs ), based on naturally occurring Type II CRISPR-Cas systems, are programmable endonucleases that can ... to additional pages describing Joung Lab CRISPR-Cas/RGN reagents: Cpf1 expression plasmids (AsCpf1 and...specifity of Staphylococcus aureus Cas9 (SaCas9 KKH variant) Engineered CRISPR-Cas9 nucleases with altered PAM...

...that do not co-express a Cas protein can be paired with a wide variety of Cas-containing plasmids. ID ...Drosophila and other insects. Cut Fully functional CRISPR/Cas enzymes will introduce a double-strand break (DSB... Gene/Insert Promoter PI Publication Nick CRISPR/Cas nickase mutants introduce gRNA-targeted single-strand...of the double-strand breaks created by wild type Cas enzymes. To use a nickase mutant, you will need two...When using CRISPR, you will need to express both a Cas protein and a target-specific gRNA in the same cell...time. Single plasmids containing both the gRNA and Cas protein act as all-in-one vectors, but their function...Enzyme(s) Delivery Resistance Co-expressed Cas9 Depositing lab Cas9 species = S. pyogenes (PAM = NGG) 49410...

...Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA, Smith HO. (2009). Enzymatic assembly of DNA molecules...opens in a new window) Gibson DG, Smith HO, Hutchison CA, Venter JC, Merryman C. (2010). Chemical synthesis...

...that do not co-express a Cas protein can be paired with a wide variety of Cas-containing plasmids. ID ...for use in zebrafish. Cut Fully functional CRISPR/Cas enzymes will introduce a double-strand break (DSB...When using CRISPR, you will need to express both a Cas protein and a target-specific gRNA in the same cell...time. Single plasmids containing both the gRNA and Cas protein act as all-in-one vectors, but their function...Enzyme(s) Delivery Resistance Co-expressed Cas9 Depositing lab Cas9 species = S. pyogenes (PAM = NGG) 46759...none, need Cas9 plasmid Chen and Wente 42250 DR274 T7 BsaI In vitro transcription none, need Cas9 plasmid...Editing Other Applications Purify Tag Visualize dCas9-FokI Screen Pooled Libraries gRNAs Empty gRNA Vectors...

...two main classes of CASTs are type I and type V-K. Type I systems use a type I Cas effector, composed ...composed of several separate Cas enzymes that form a complex called Cascade ( C RISPR- as sociated c omplex for...matters. Type V-K systems use a type V-K Cas effector, Cas12k, and three transposase components (TnsB,...transposases (Tns) with the precise DNA-targeting of CRISPR-Cas systems, allowing for the integration of large fragments...fragments of DNA at specific locations. The Cas effector lacks nuclease activity, instead recruiting the... CRISPR CASTs CRISPR Plasmids: CRISPR Transposases (CASTs) Browse...transposases (CASTs) for insertion of donor DNA. CRISPR...

...that do not co-express a Cas protein can be paired with a wide variety of Cas-containing plasmids. ID ...designed for use in yeast. Cut Fully functional CRISPR/Cas enzymes will introduce a double-strand break (DSB...Promoter Selectable Marker PI Publication Nick CRISPR/Cas nickase mutants introduce gRNA-targeted single-strand...of the double-strand breaks created by wild type Cas enzymes. To use a nickase mutant, you will need two...When using CRISPR, you will need to express both a Cas protein and a target-specific gRNA in the same cell...time. Single plasmids containing both the gRNA and Cas protein act as all-in-one vectors, but their function...PI Publication Interfere Catalytically dead dCas9, or dCas9 fused to a transcriptional repressor peptide...

...prevent the CRISPR-Cas complex from binding to DNA. Others inhibit the recruitment of Cas proteins to the...allowing multiple Cas9s to be used together. Researchers have engineered mutant Cas enzymes with alternative... Fu Y, Sander JD, Reyon D, Cascio VM, Joung JK. 2014. Improving CRISPR-Cas nuclease specificity using ...immune system - utilizing various CRISPR-associated (Cas) genes to not only store a record of invading phages...upon re-exposure. More specifically, specialized Cas proteins snip foreign DNA into small fragments approximately...stretches of DNA known as CRISPR arrays. Separate Cas proteins then express and process the CRISPR loci... Through sequence homology, these crRNAs guide a Cas nuclease to the specified exogenous genetic material...

...that do not co-express a Cas protein can be paired with a wide variety of Cas-containing plasmids. ID ...for use in C. elegans. Cut Fully functional CRISPR/Cas enzymes will introduce a double-strand break (DSB...When using CRISPR, you will need to express both a Cas protein and a target-specific gRNA in the same cell...time. Single plasmids containing both the gRNA and Cas protein act as all-in-one vectors, but their function...Co-expressed Cas9 Depositing lab Cas9 species = S. pyogenes (PAM = NGG) 47549 pDD162 (Peft-3::Cas9 + Empty ...need Cas9 plasmid Joung 46170 PU6::klp-12_sgRNA cU6 PCR (see paper) Transfection none, need Cas9 plasmid... none, need Cas9 plasmid Boxem 47941 pMB60 T7 BsaI In vitro transcription none, need Cas9 plasmid Boxem...

...that do not co-express a Cas protein can be paired with a wide variety of Cas-containing plasmids. ID ...for use in bacteria. Cut Fully functional CRISPR/Cas enzymes will introduce a double-strand break (DSB.... ID Plasmid Promoter PI Publication Nick CRISPR/Cas nickase mutants introduce gRNA-targeted single-strand...of the double-strand breaks created by wild type Cas enzymes. To use a nickase mutant, you will need two...When using CRISPR, you will need to express both a Cas protein and a target-specific gRNA in the same cell...time. Single plasmids containing both the gRNA and Cas protein act as all-in-one vectors, but their function...PI Publication Interfere Catalytically dead dCas9, or dCas9 fused to a transcriptional repressor peptide...

...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...

...that do not co-express a Cas protein can be paired with a wide variety of Cas-containing plasmids. Do ... expression systems. Cut Fully functional CRISPR/Cas enzymes will introduce a double-strand break (DSB...Promoter Selectable Marker PI Publication Nick CRISPR/Cas nickase mutants introduce gRNA-targeted single-strand...of the double-strand breaks created by wild type Cas enzymes. To use a nickase mutant, you will need two...When using CRISPR, you will need to express both a Cas protein and a target-specific gRNA in the same cell...time. Single plasmids containing both the gRNA and Cas protein act as all-in-one vectors, but their function...PI Publication Interfere Catalytically dead dCas9, or dCas9 fused to a transcriptional repressor peptide...

...that do not co-express a Cas protein can be paired with a wide variety of Cas-containing plasmids. ID ...When using CRISPR, you will need to express both a Cas protein and a target-specific gRNA in the same cell...time. Single plasmids containing both the gRNA and Cas protein act as all-in-one vectors, but their function...Enzyme(s) Delivery Resistance Co-expressed Cas9 Depositing lab Cas9 species = S. pyogenes (PAM = NGG) 51306...Editing Other Applications Purify Tag Visualize dCas9-FokI Screen Pooled Libraries gRNAs Empty gRNA Vectors...designed for use in Xenopus. Cut Fully functional Cas9 enzymes designed to introduce a double-strand break...surrounding the DSB is introduced along with the Cas9 and gRNA plasmids, the cell may instead repair the...

...that do not co-express a Cas protein can be paired with a wide variety of Cas-containing plasmids. ID ...designed for use in plants. Cut Fully functional CRISPR/Cas enzymes will introduce a double-strand break (DSB...Promoter Selectable Marker PI Publication Nick CRISPR/Cas nickase mutants introduce gRNA-targeted single-strand...of the double-strand breaks created by wild type Cas enzymes. To use a nickase mutant, you will need two...When using CRISPR, you will need to express both a Cas protein and a target-specific gRNA in the same cell...time. Single plasmids containing both the gRNA and Cas protein act as all-in-one vectors, but their function...PI Publication Interfere Catalytically dead dCas9, or dCas9 fused to a transcriptional repressor peptide...

...RNA-guided endonuclease) Tools: Cas-OFFinder & Cas-Designer From the Kim Lab , Cas-OFFinder identifies gRNA ...of off-target effects. Cas-Designer integrates information from the Kim Lab's Cas-OFFinder and Microhomology... sativa), tomato, corn, monkey (macaca mulatta). Cas-Designer searches for targets that maximize knockout...tropicalis ), and zebrafish. Off-Spotter: tool for CRISPR/Cas design Program for designing optimal gRNAs. Provides... potential DNA target sites for TALEs and CRISPR/Cas. CRISPRdirect From the Database Center for Life Science...vectors using ssDNA oligos p201G Cas9 ; p201B Cas9 ; p201H Cas9 ; p201N Cas9 ; pUC gRNA Shuttle PDF 488.6 ...CRISPR RNA array: Cas9 (pX260) or Cas9 D10A (pX334) ; tracrRNA: Cas9 (pX330) or Cas9 D10A (pX335) ; PDF...

...more information on the wide variety of Cas enzymes? CasPEDIA is an encyclopedia of Class 2 CRISPR systems...Protocols gRNA Design Tools CRISPR Blog Posts CRISPR/Cas nickase mutants introduce gRNA-targeted single-strand...of the double-strand breaks created by wild type Cas enzymes. To use a nickase mutant, you will need two...Editing Other Applications Purify Tag Visualize dCas9-FokI Screen Pooled Libraries gRNAs Empty gRNA Vectors...

...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...

...Engineering Cut Wild-type Cas enzymes generate double strand breaks (DSBs). Nick Mutated Cas nickase enzymes generate... generate single-strand DNA breaks. Prime Edit Cas nickase enzymes fused to reverse transcriptase install... without promoting DSB formation. Delete NEW Cascade-Cas3 generates large genomic deletions. Insert NEW...Regulation Activate dCas9 fused to an activator peptide activates gene expression. Interfere dCas9 alone, or fused...repressor peptide, inhibits transcription. Epigenetics dCas9 fused to an epigenetic modifier alters chromatin..., not DNA, across multiple systems. RNA Editing Cas13 enzymes fused to deaminases can edit bases in RNA...powerful tool for genome-wide screening. Purify dCas9 fused to an epitope tag can be used to isolate a...

...proteins. This CRISPR/Cas plasmid-system consists of two components: A vector containing Cas9 and a validated...marker. The first deposited plasmids in this CRISPR-Cas tagging system were tested by tagging transcription...into human FBL locus Jorgensen Lab SapTrap CRISPR/Cas Toolkit SapTrap is a modular toolkit from Erik Jorgensen's...GFP targeting vector. Jorgensen Lab SapTrap CRISPR/Cas Toolkit Kanemaki Lab Auxin-Inducible Degron Tagging...Kanemaki's lab has developed a simple and scalable CRISPR/Cas-based method to tag endogenous proteins by using ...Auxin-inducible degron (AID) tagging with CRISPR/Cas to generate conditional alleles of essential nuclear...Doyon lab, eSpCas9(1.1)_No_FLAG_AAVS1_T2 (Addgene #79888) , which expresses an untagged Cas9 AND a gRNA...

... CRISPR dCas9-FokI CRISPR: dCas9-FokI...Tools CRISPR Blog Posts A catalytically inactive Cas9 (dCas9) is fused to FokI nuclease. When FokI dimerizes...with dCas9 fused to FokI for creating double-strand breaks (DSBs). CRISPR...Editing Other Applications Purify Tag Visualize dCas9-FokI Screen Pooled Libraries gRNAs Empty gRNA Vectors...gRNAs, binding ~15-25 bp apart, are required for dCas9-FokI to dimerize at a given region of the genome...