...Furthermore, gene knockout strategies cannot be used to investigate essential genes. Historically, gene silencing...while gene knockouts have long been considered the “gold-standard” for LOF studies, the high gene copy ...two non-exclusive gene silencing pathways: transcriptional and post-transcriptional gene silencing (PTGS...modifications of complementary gene sequences (Figure 1). As a result, gene silencing can be easily induced...phenotype discrepancies between gene silencing and knockout methods of the same gene. However, recent work has...that is required dsRNA complementary to your gene or genes of interest to trigger RNAi activation. A typical...complementary to your gene target as inverted repeats (Figure 1). With this technique, multiple genes can be simultaneously...

...the env gene containing the rev response element (RRE), an internal promoter, and the desired gene (transfer...-1 genome containing all viral genes with the exception of the env gene (packaging plasmid), and 3) the...used to express your gene of interest either constitutively or inducibly. If your gene of interest is toxic...expression of your gene. The addition of Dox in the culture will then repress your gene of interest. This... Youichi Suzuki (2011). Gene Regulatable Lentiviral Vector System, Viral Gene Therapy, Dr. Ke Xu (Ed.)...harmful genes into a transfer vector. Lentiviral particles expressing proto-oncogenes or oncogenes for instance...safe use in a clinical setting. In this generation, the HIV Tat gene (previously used to drive expression...

...Entrez Gene matches to your insert. Select the appropriate gene if it appears in the Entrez Gene list, ...correct Entrez Gene does not appear in the auto-populated list, try entering the gene's Official Full ...known as" aliases in the Gene/Insert box. Once you have selected the appropriate gene from the list, save ...bacterial strain(s) through Addgene. Description – please indicate the gene cloned into your plasmid, any... for the gene or insert. Step 4: Growth and Distribution Please indicate which strain Addgene should propagate... Encodes insert(s) A plasmid with one or more genes, protein coding sequences, or other elements inserted...cloned into it. gRNAs are used for directing CRISPR/Cas9 to a targeted genomic location, while shRNAs are...

...The use of other technologies such as optogenetics and CRISPR/Cas9 will help immunologists create even ...molecule (see figure to the right). The excitation generated by these two-photons occurs only at a chosen focal...PubMed PMID: 18431356. Additional Resources on the Addgene Blog Get Tips on Choosing Your Fluorescent Protein...Monitor Protein-Protein Interactions Resources on Addgene.org Check out Our Fluorescent Protein Guide Pages...

...endogenous levels (Laflamme et al., 2019). CRISPR-Cas9 approaches are commonly used to produce KO cell ...procedure and application to the major ALS/FTD disease gene C9ORF72. eLife, 8, e48363. https://doi.org/10.7554...101: Validation Resources on addgene.org Addgene's Antibody Guide Addgene's Antibody Protocols... outlined below. Knockout cell line approach Genetic knockout-based validation can conclusively prove...you are working with cells that are amenable to genetic knockout, this represents a very robust approach...to start with a screening procedure in easily genetically modified cell types (top). The specific approach...10.1038/s41598-019-55133-7 More resources from the Addgene blog Introduction to Gating in Flow Cytometry Antibodies...

...downstream applications. Safety Warnings Lentivirus is generally considered biosafety level 2+. Please ensure that...adherent cells) 0.45 μm polyethersulfone filter, Nalgene, 565-0010 (for viral preps, if prep was not previously...transduced with a range of dilutions of pHAGE-TO-dCas9-3XmCherry . 72 h post transduction, cells were assayed...

...Use this protocol to generate lentivirus...downstream applications such as stable-cell line generation. Workflow Timeline Day 0: Seed 293T packaging...adherent cells) 0.45 μm polyethersulfone filter, Nalgene, 565-0010 Microcentrifuge tubes, Neptune 3745.X...per 10 cm dish). Using transfer plasmid pHAGE TRE dCas9-KRAB (total μg of plasmid DNA 27.8 μg), this would...

..., BD BD300912 0.45 μm polyethersulfone filter, Nalgene, 565-0010 (for viral preps, if prep was not previously...serial dilutions of the lentiviral vector pHAGE EF1α dCas9-KRAB . The following day, cells were treated with...

...stem cells By Jennifer Tsang CRISPR gene interference (CRISPRi) and gene activation (CRISPRa) are great ways...the plasmids at Addgene! A new tool for synthetic biology uses CRISPRi to control gene expression. This...nucleus and modulate transcription of the mCitrine gene. mCherry is constitutively expressed. Cells are ...selectivity and found that the best motif was from the Lck gene. For marking microtubules of mammalian cells, the... ways to modulate the expression of nearly any gene via specific guide RNA (gRNA) sequences. But these...-IgG1-Fc, Addgene #140700) pMAK463 (NBαMouse-IgK, Addgene #140701) pMAK464 (NBαEGFP, Addgene #140702) ...and the all-in-one dCas9-KRAB and dCas9-VPR targeting plasmids can be found at Addgene. Check out these ...

...going to bind on the chromosome. When dCas9 binds to a gene, it blocks the progression of the RNA polymerase...Tuning dCas9’s ability to block transcription enables robust, noiseless knockdown of bacterial genes. Molecular...of two genes without cross‐talk. (A) Schematic of the strain expressing two reporters and dCas9 under ...simultaneously. By fusing sfGFP and/or mCherry to genes of interest, it is thus possible to study the effect... protein dCas9 is expressed along a CRISPR array that contains 20 bp "addresses", where dCas9 is going...for quick integration of the aTc-inducible pTet-dCas9 in the attachment site of the phage 186. Detailed...

... ID Plasmid Gene/Insert Promoter Selectable Marker PI Publication Bacteria ID Plasmid Gene/Insert Promoter...Drosophila ID Plasmid Gene/Insert Promoter PI Publication Plant ID Plasmid Gene/Insert Promoter Selectable...C. elegans ID Plasmid Gene/Insert Promoter PI Publication Yeast ID Plasmid Gene/Insert Promoter Selectable...transcription of a specific gene. Design your gRNA sequence to direct the dCas9-activator to promoter or ...CRISPR plasmids for activating or upregulating gene expression in various model organisms. CRISPR...or regulatory regions of your gene of interest. If the plasmid that you choose does not also express a ...Transcriptional Regulation Activate Interfere Epigenetics RNA Targeting RNA Targeting RNA Editing Other...

... ID Plasmid Gene/Insert Promoter Selectable Marker PI Publication Bacteria ID Plasmid Gene/Insert Promoter...Plant ID Plasmid Gene/Insert Promoter Selectable Marker PI Publication Yeast ID Plasmid Gene/Insert Promoter...dead dCas9, or dCas9 fused to a transcriptional repressor peptide like KRAB, can knock down gene expression...plasmids for transcriptional repression of target genes. CRISPR...with transcription. Design your gRNA to target your gene of interest’s promoter/enhancer or the beginning...Transcriptional Regulation Activate Interfere Epigenetics RNA Targeting RNA Targeting RNA Editing Other...Other Applications Purify Tag Visualize dCas9-FokI Screen Pooled Libraries gRNAs Empty gRNA Vectors gRNAs...

...amplification kit. In this case, regions of gene S and Orf1ab gene are amplified. RPA reactions also contain...amplification by primers that target the E and the N2 gene of SARS-CoV-2. If the Cas12a-sgRNA complex binds...Approach: FnCas9 Editor Linked Uniform Detection Assay (FELUDA) is a technique that uses FnCas9, a protein...sensitivity of FnCas9, this is also a good way to diagnose single nucleotide variations. Also, FnCas9 cleaves...like CRISPR. Figure 1: An overview of the general schematic of CRISPR/Cas based COVID-19 testing ...protocol for Cas13-based detection of SARS-CoV-2 genetic material (CREST) Sensitivity: 10 copies of RNA ...field-deployable nucleobase detection and identification using FnCas9 (FELUDA) Sensitivity: – 110 femtomolar  Time: Not...

...transcription. Epigenetics dCas9 fused to an epigenetic modifier alters chromatin structure/gene expression...Regulation Activate dCas9 fused to an activator peptide activates gene expression. Interfere dCas9 alone, or fused...factors. gRNAs gRNA plasmids to target specific genes....CRISPR? Experts cover CRISPR topics on Addgene's blog. Addgene's Viral Service : Request ready-to-use viral...Cas enzymes generate double strand breaks (DSBs). Nick Mutated Cas nickase enzymes generate single-strand...CRISPR plasmids available at Addgene. Search by species and CRISPR function. CRISPR...technologies are continously developing and expanding. Addgene works with leading scientists in the field to ensure...

...editing, this epigenetic strategy doesn’t induce DNA damage or permanently alter a gene product; it simply...on Prime-editing-Assisted Site-Specific Integrase Gene Editing (PASSIGE), their previously-reported technique...). Efficient site-specific integration of large genes in mammalian cells via continuously evolved recombinases...CRISPRoff system — a programmable epigenetic editor consisting of dCas9 fused to a DNA methyltransferase... is generalizable to a variety of DNA-targeting proteins, so the team created versions using dCas9, zinc...optimizations for prime editing CHARM: A compact epigenetic silencer Recombinant antibodies for marking astrocytes...magnetic beads for scarce macromolecules in heterogeneous samples. bioRxiv 2024.01.21.576499; doi: https...

...tool for large inserts.  DAP arrays for multiplex gene editing: Guest blogger Qichen Yuan tells us about...Sapozhnikov tells us how to use dCas9 (with no modifications!) as a CRISPR/dCas9-based tool for DNA demethylation...ancestral bias in CRISPR guide library design.  dCas9-directed DNA Demethylation: Guest blogger Daniel... share on our blog, please reach out to news@addgene.org - we'd love to hear from you!       ...

...plasmids for epigenetic modification in mammalian or plant systems. Mammalian ID Plasmid Gene/Insert Promoter... target a specific promoter or enhancer for your gene of interest. Available modifications include: histone...Selectable Marker PI Publication Plant ID Plasmid Gene/Insert Promoter Selectable Marker PI Publication...targeted epigenetic modifications, researchers have fused catalytically dead dCas9 to epigenetic modifiers... CRISPR Epigenetics CRISPR Plasmids: Epigenetics Browse CRISPR Plasmids By Function...CRISPR plasmids for targeted epigenetic modification, including histone acetylation/demethylation, and...Transcriptional Regulation Activate Interfere Epigenetics RNA Targeting RNA Targeting RNA Editing Other...

...antibiotic resistance genes. Genes that are targets of antibiotics are often essential genes which creates a.... ICEBs1 carries the genes needed for conjugation, but can also carry cargo genes, like Mobile-CRISPRi...if that gene is a target of an antibiotic. For example, let’s take a look at the essential gene folA which...targeted essential and non-essential genes with two guides per gene. The library was grown in glucose minimal...essential genes were less represented while bacteria containing guides targeting non-essential genes remained...does not disrupt gene function and is stable and active for greater than 50 generations. Figure ...tools scientists can use to study them. For example, gene knockdown with CRISPR interference (CRISPRi) has...

...protein (FP) tags. Information on genes and associated neurodegenerative diseases was curated from several...sgHTT1-7sk-sgCas9 HTT U6, 7sk Huntington's Nicole Deglon 190901 pAAVss-U6-sgHTT51-7sk-Cas9 HTT U6, 7sk...but not exhaustive. If you do not see a particular gene of interest, please search our full plasmid collection... Huntington's Disease Parkinson's Disease Others Gene Type Wildtype Mutant Partial Sequence Codon-Optimized... T7 polH GAL Other Clear Filters ID Plasmid Name Gene Tags Promoter Disease PI 1177 p416 25Q GPD HTT GFP...Parkinson's Frank Soldner 180437 Px330-EGFP-LRRK2-CRISPR/Cas9 LRRK2 U6 Parkinson's Frank Soldner 180439 STR-KDEL_PGRN-SBP-mCherry... Neurodegeneration Neurodegeneration Plasmids Neurodegeneration Plasmid Collection Addgene has a ...

... ID Plasmid Gene/Insert Promoter Selectable Marker PI Publication Bacteria ID Plasmid Gene/Insert Promoter...Drosophila ID Plasmid Gene/Insert Promoter Selectable Marker PI Publication Plant ID Plasmid Gene/Insert Promoter...elegans ID Plasmid Gene/Insert Promoter Selectable Marker PI Publication Yeast ID Plasmid Gene/Insert Promoter... ID Plasmid Gene/Insert Promoter Selectable Marker PI Publication Xenopus ID Plasmid Gene/Insert Promoter...Selectable Marker PI Publication Parasites ID Plasmid Gene/Insert Promoter Selectable Marker PI Publication...Transcriptional Regulation Activate Interfere Epigenetics RNA Targeting RNA Targeting RNA Editing Other...Other Applications Purify Tag Visualize dCas9-FokI Screen Pooled Libraries gRNAs Empty gRNA Vectors gRNAs...