We narrowed to 304 results for: cas9 genes

...Figure 2: Cas9 expression in monoclonal cell lines generated from A549 cells transduced with lentiCas9-Blast...A549 cells stably expressing Cas9. A549 cells were transduced with lentiCas9-Blast 1 and then selected with... express Cas9 and are included as a control (WT). Vinculin is a loading control. 1 lentiCas9-Blast was...additional 3 weeks under blasticidin selection. Anti-Cas9 Western blotting was performed on whole-cell extracts...Blog Posts Addgene Protocols Viral Service Introduction This protocol describes how to generate a monoclonal...Selective pressure on this heterogeneous cell pool could lead to reduced transgene expression over time, as...more likely to retain stable transgene expression. Other methods of generating a monoclonal cell line include...

...constantly improving. Cas9 variants have been modified for genome editing, activating gene expression, visualizing...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...non-target strand and Cas9, Kleinstiver et al disrupted interactions between Cas9 and the phophate backbone...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...from this publication at Addgene. Resources on the Addgene Blog Read about Cas9 Homolog Cpf1 Read How ...

...sequence, Cas9 will neither bind nor cut the target. Cas9 homologs from different organisms or Cas9 mutants...basic level, CRISPR/Cas9 genome editing systems use a non-specific endonuclease (Cas9 or closely related...the PAM sequence. Cas9 Species/Variants and PAM Sequences Species/Variant of Cas9 PAM Sequence ...genomic target for Cas9, and the tracrRNA which acts as a scaffold linking the crRNA to Cas9 and facilitates... to direct Cas9 to a specific genomic locus and the scaffolding sequence necessary for Cas9 binding. When...CRISPR/Cas9 for genome editing, researchers simply need to express a gRNA designed to direct Cas9 to their...tracrRNA and Cas9, bind to these transcripts and C) cleave them leaving mature crRNAs bound to the Cas9/tracrRNA...

... sites for S. pyogenes Cas9, S. aureus Cas9, S. thermophilus Cas9, N. meningitidis Cas9, or Cas12a from...vectors using ssDNA oligos p201G Cas9 ; p201B Cas9 ; p201H Cas9 ; p201N Cas9 ; pUC gRNA Shuttle PDF, 500 KB...CRISPR RNA array: Cas9 (pX260) or Cas9 D10A (pX334) ; tracrRNA: Cas9 (pX330) or Cas9 D10A (pX335) PDF,... window) CRISPR/Cas9 reagent generation; gRNA design; HDR design; Deep sequencing Cas9: pMLM3613 dsDNA...CRISPR/Cas9 Knockout (MAGeCK). This tool can identify positively or negatively selected sgRNAs, genes, or...118 KB Church gRNA design and cloning for Cas9 orthologs Cas9 plasmids PDF, 107 KB Chen and Wente Zebrafish...vitro transcription, injection gRNA core ; Cas9 ; optimized Cas9 PDF, 66.8 KB Fujii gRNA design and cloning...

...Return to top Cas9 SAM transcriptional activation plasmids and screening library CRISPR/Cas9 Synergistic... of endogenous genes. It consists of three components: A nucleolytically inactive Cas9-VP64 fusion A sgRNA...of cells and nuclei. Return to top AAV - Cas9 mouse CRISPR-Cas9 is a versatile genome editing technology...constitutively expressing Cas9 knockin mice (Platt et al ., Cell 2014). In these mice the CRISPR-Cas9 system can be...constitutively Cas9-expressing mouse. Described here are AAV vectors that can be combined with Cas9 in a wide...are below. The Cas9 knockin mouse can be purchased from Jackson Labs: Cre-dependent Cas9 mouse Constitutively...cloning information: CRISPR-Cas9 mouse toolbox protocol 637.8 KB CRISPR-Cas9 Cre expression vectors for...

... available with multiple Cas9 variants, including high fidelity Cas9s, Cas9s with various PAM requirements...dual nickases to generate a knockout or gene edit; or using Cas9 to generate large genomic deletions or ...strand SpCas9-HF1 — disrupt Cas9’s interactions with DNA phosphate backbone HypaCas9 — increase Cas9 proofreading...Plasmids: Purify Cas9 Alternatives for CRISPR Genome Engineering S. pyogenes Cas9 (SpCas9) is the most commonly...more Cas9 fusion tools later in the guide. Figure 3: Overview of Cas9n. Increasing fidelity of Cas9 Researchers...enhanced specificity by engineering Cas9 into high fidelity Cas9s (hfCas9) . There is currently no defined...application. Cas9 Orthologs While SpCas9 is the most commonly used Cas enzyme, additional Cas9 orthologs ...

...Figure 3: Gene repression with nuclease-competent Cas9. When complexed with a truncated gRNA, Cas9 is still... site (TSS) of a gene of interest, it induces potent gene expression with no genetic alterations observed...efficiently transcribing the gene. This results in gene silencing, with no observed genetic alterations at the ... gRNA, Cas9 binds the target site, but does not cut either strand of DNA. Traditional Cas9 regulators... modifications to DNA, Cas9 can be reprogrammed to serve as a regulator of gene expression. Its catalytic...promoter of a gene of interest, they have been shown to dramatically alter levels of expression. Cas9’s ability... of modulating gene expression makes it an invaluable tool in the interrogation of gene function. However...

...including: generating single or multi-gene knockouts in a wide variety of cell types, gene editing using... can occur due to prolonged Cas9 expression     The requirement for Cas9 transcription and translation...consisting of the Cas9 protein in complex with a targeting gRNA, to your cells of interest. Cas9 RNPs are capable...pathways. This makes Cas9 RNPs useful for CRISPR applications where limited expression of Cas9 is required and...express and purify His-tagged Cas9 from E. coli using a plasmid such as pET-28b-Cas9-His from Alex Schier’s ... of Cas9-gRNA RNPs by specific cell types (Rouet et al. 2018). This was accomplished using a Cas9 mutant...for experiments where stable Cas9 expression is required, such as when a Cas9 fusion to a fluorescent protein...

... Split Cas9, Split Cas9 nickase, Split dCas9 Genome editing by NHEJ and HDR, repression of gene expression...Photoswitchable Cas9 Richter et al., 2016 tsRC9 RsLOV from R. sphaeroides Cas9, dCas9 Gene editing Zhou...oligonucleotides Cas9, photocaged-gRNA Gene editing Manna et al., 2019   NPPOC-caged trimethoprim (TMP) Cas9 fused...domains (Cas9-DHFR) Gene editing, transcription Zhou et al., 2020   NPOM-caged nucleobases Cas9, photocaged-gRNA...showing that this genetically encoded, photocaged Cas9 system could silence endogenous gene expression - demonstrating...Zhou et al., 2018   pdDronpa Cas9, dCas9 Gene editing, transcription Light-Dependent Anti-CRISPR Bubeck...-CRISPR protein, co-expressed with Cas9 or dCas9 Repression of gene editing and transcription. Label genomic...

...frequently used Cas9, spCas9 from Streptococcus pyogenes, this sequence is 3’-NGG. But Cas9s found in other...Lab used directed evolution to generate Sniper-Cas9 resulting in a Cas9 with improved specificity. They...assessment of the off-target activity of Cas9. This can occur when Cas9 cleaves a site that is not a perfect...the PAM specificity of Cas9s isolated from different organisms and engineered Cas9 variants that were able...consideration when choosing a Cas9 is balancing specificity and sensitivity. Wild-type Cas9 exhibits high on-target...on-site and off-site activity of Sniper-Cas9 to different Cas9 variants using gRNAs of different lengths...loci and gRNA lengths. Recent alternatives to Cas9 While Cas9 remains the most widely used CRISPR nuclease...

...introducing Cas9-mediated frameshift mutations in the gene of interest. The genomic target for Cas9 and the...using a Cas9 variant with higher specificity (eSpCas9 and HypaCas9) instead.  Choosing a Cas9 for site-specific...target genes. Repressing target genes using dCas9-based repressors Early experiments using dCas9 in bacteria...frequently fused to Cas9 for this purpose. dCas9-KRAB plasmids for repressing target genes in a variety of...Next CRISPR Experiment Cas9 expression systems Addgene carries a variety of Cas9 containing plasmids that... CRISPR/Cas9 has made it easier than ever to make precise, targeted genome modifications. Cas9 has been... repress, and even image your favorite gene. With so many Cas9 reagents available, it can be difficult...

... context of the gene, Cas9 activators efficiently solve these problems.   Which Cas9 activator should...prevent your gene from being up-regulated. For instance, generally, the more highly expressed a gene is under...with CRISPR/Cas9 Learn How CRISPR Has Been Used with AAV Resources on Addgene.org Find Cas9 Activators...CRISPR/Cas9 is an enormously plastic tool and has taken the scientific world by storm. While Cas9 has been...the upregulation of essentially any gene by fusing mutants of Cas9 deficient in DNA cutting activity to... research CRISPRa for specific gene targets CRISPR activation for genes with known functions has the potential.... Your gene of interest might already be hitting an upper bound of activation that current Cas9 systems...

...CRISPR/Cas9 genome editing varies across targets, they tested two genes in mice. For both genes, they ...one AAV, Cas9 must be made smaller. Previous attempts to “shrink” Cas9 include the use of St1Cas9 (~3.3 ...amino acids in length, Cas9 from Campylobacter jejuni (CjCas9) is the smallest Cas9 ortholog characterized...used CjCas9 with AAV to target genes in mouse muscle and eye tissue. Neisseria meningitidis Cas9 (NmeCas9...editing, but in vivo gene editing approaches have been limited by problems with Cas9 delivery. Adeno-associated...vehicle for Cas9. However, to allow for more room for regulatory sequences while still fitting Cas9 and its...equally potent Cas9 protein, the Zhang lab took a different approach. They analyzed over 600 Cas9 orthologs...

...the basic parts of the Cas9 enzyme.   Figure 1: A cartoon depiction of Cas9’s two major lobes, REC...   Unbound Cas9 Now that we know what we’re looking at, let’s peek at unbound—“apo”—Cas9 from Streptococcus...on these regions.   Cas9’s transition from weak, random binding to precision gene editing requires three...Figure 8: Comparison of Cas9:RNA crystal structure (left, PDB ID 4ZT0) with Cas9:RNA:DNA electron microscopy...Streptococcus pyogenes.   Figure 2:  Crystal structure of S. pyogenes Cas9 in the apo state (PDB ID...   Figure 3: Crystal structure of S. pyogenes Cas9 bound to guide RNA (PDB ID 4ZT0), shown opaque...isn’t in place yet.     Figure 4: S. pyogenes Cas9 in the apo state (PDB ID 4CMP, top) compared...

...Additional Gene Silencing Resources A modular plasmid assembly kit for multigene expression, gene silencing...plant-optimized plasmids for cloning, gene expression, reporter assays, genome editing, gene silencing, and more. Plasmid...expressing different functional variants of Cas9. A CRISPR/Cas9 toolkit for multiplex genome editing in variety...sgRNA and Cas9 in rice . Includes a protocol (Link opens in a new window) for targeted gene mutation. ... a new window) — Tools for gene and protein expression and mapping of gene expression in plants. Grassius...Resources Cloning & SynBio Genome Editing Gene Silencing Reporters Addgene’s repository contains many plasmid ...from genome engineering and gene expression to reporter systems. Addgene’s collection includes plasmids...

...out to make my first CRISPR/Cas9 gene edit. I wanted to inactivate the gene BRAF (a kinase implicated in...virally-delivered GFP gene. So, cells with no Cas9 should appear green and cells with functional Cas9 should appear...analyzing a gene edit using Addgene’s lentiviral CRISPR tools. Schematic overview of CRISPR/Cas9 gene...experiments, we generated A549 cells stably expressing Cas9 via transduction with Cas9 lentiviral vector... Figure 1: Cas9 expression in A549 cells transduced with lentiCas9-blast. Anti-Cas9 Western blotting... Cas9 delivery Overview: Before any editing can begin we need our A549 cells to express Cas9. In these...with too many Cas9 integrations per cell, and my stable cell pool had wildly variable Cas9 expression (...

...High fidelity Cas9s: Three high fidelity Cas9 variants , SpCas9-HF, eSpCas9, and HypaCas9 display very ...III and Cas9 enzymes, which together separate the individual crRNAs. (3) The tracrRNA and Cas9 nuclease... anti-CRISPR genes in bacteriophages infecting Pseudomonas aeruginosa . Anti-CRISPR genes employ varied...Staphylococcus aureus (SaCas9) is smaller than SpCas9 and more easily packaged in AAV. Cas9 orthologs also have...especially amenable to genetic screens. Pooled gRNA libraries can be used to identify genes that are important...beyond SpCas9, moving us closer to being able to target every locus in every genome. Multiple Cas9 orthologs...capability of Cas9 to make other types of modifications at specific loci. Activate or Repress : dCas9 fused to...

...They went on to show that their CRISPR/Cas9-nuclease and CRISPR/Cas9-nickase vectors work successfully to...versions of Cas9, nuclease and nickase, but we are now adopting dCas9, dCas9-VP64, and dCas9-KRAB in our...24954249 Find the Mulitplex CRISPR/Cas9 Assembly System Kit at Addgene here. Other Popular CRISPR Posts...all-in-one vector construction system for CRISPR/Cas9-mediated multiplex genome engineering is now available...one of the most advantageous properties of CRISPR/Cas9 compared to ZFNs and TALENs,” said Tetsushi Sakuma...offers ease and efficiency for all-in-one CRISPR/Cas9 vectors expressing up to seven guide RNAs (gRNAs...Figure 1: Schematic overview of the all-in-one CRISPR/Cas9 vector construction system for multiplex genome ...

...backbone. Zhang Cas9 and Accessories for Activating Gene Expression Catalytically-dead Cas9 (dCas9) can be fused...mouse genes along with 1000 non-targeting controls Puromycin Doench , Root Cas9 Lentivirus Cas9 viruses... 52962 lentiCas9-Blast Sp Cas9 Cut Blasticidin Expresses human codon-optimized S. pyogenes Cas9 protein...page . Cas9 for Editing Genomic Sequences To perform genome edits, Cas9 nuclease is used to generate double-stranded...backbone contains SpCas9. SpCas9 and 76,441 unique sgRNAs targeting 19,114 human genes along with 1000 ...Lentivirus Pooled CRISPR Libraries Cas9 Pooled Barcoding Libraries Control Addgene's lentiviruses are made from...backbone does not contain SpCas9. 76,441 unique sgRNAs targeting 19,114 human genes along with 1000 non-targeting...

...use of novel S. pyogenes Cas9 variants with varying PAM sequences, 2) the use of Cas9 homologs derived...as a binding signal for Cas9 and this sequence is a strict requirement for Cas9-mediated DNA cleavage. ...when trying to edit a gene using homology directed repair, since HDR-mediated gene editing is most efficient...2020). Characterization of Cas9 from additional bacterial species Many more Cas9 orthologs have been isolated...them more useful than SpCas9. For example, Cas9 from Staphylococcus aureus (SaCas9) is about 1 kilobase... in length, Cas9 from Campylobacter jejuni (CjCas9) is even smaller than SaCas9 and is also compatible...While PAM sequences for the commonly used S. pyogenes Cas9 (3'-NGG) are abundant throughout the human genome...