...numerous applications, including transgenesis, gene-trap screens, and gene editing. How does the piggyBac...levels of your gene of interest, as needed. Controlling the expression levels of your gene may be particularly...Transposagen.   How does piggyBac™ enhance gene editing? Gene editing can now be accomplished in a more...(CRISPR/Cas9, TALENs, and ZFNs). However, several challenges still remain, such as making gene edits completely... How does the piggyBac™ gene editing system compare with other methods of gene editing? There are primarily... a variety of gene editing technologies including Zinc Fingers, TALENs, and CRISPR-Cas9 nucleases. Mathew...Decoding the Epigenetic Heterogeneity of Human Pluripotent Stem Cells with Seamless Gene Editing.,” Methods...

...Truncated gRNAs for Regulating Gene Expression and CRISPR 101: Epigenetics and Editing the Epigenome (plus...and CRISPR Antimicrobials Day 7: Multiplexing and Cas9 variants: CRISPR 101: Multiplex Expression of gRNAs...Joining and PITCHing MMEJ as an Alternative Route for Gene Editing Day 9: Precision editing: Single Base Editing...Genome-Wide Screening Using CRISPR/Cas9 and Plasmids 101: NGS Quality Control for Pooled Libraries Day 12: New... CRISPR Targeting Specificity with eSpCas9, SpCas9-HF1, & HypaCas9 Day 10: Transcriptional regulation:...Since the start of the CRISPR revolution, Addgene has distributed over 100,000 CRISPR plasmids. But that...flame for its adenine base editing capabilities. Epigenetics: CRISPR has made it possible to design programmable...

...70% of human genes have a counterpart in zebrafish. Understanding the function of these genes in fish will...unknown functions for genes, and lays the groundwork for understanding the gene networks that regulate...zebrafish-specific gene expression databases that provide information about when and where genes are active in...TALENs, and more recently CRISPR/Cas9. Another popular targeted gene deactivation method in zebrafish ...investigations. Genetic resources for zebrafish A common approach to unravel the gene networks that drive...model organism is its amenability to genetic manipulation. Tools for gene manipulation together with information...Additional mutagenesis projects have since been initiated and today thousands of mutations in genes affecting...

...found in the same gene. If we only have small, directed editing abilities, any gene editing treatment ...functional copy of the entire gene, allowing a one-size-fits-all approach to gene replacement therapy for most...programmable gene insertion with PASTE. The PASTEsystem involves insertion of landing sites via Cas9-directed...insertions, leaving a several kilobase-sized gap in the gene editing field. The AbuGoot lab has now developed...to work. (It was also found to be independent of gene repair.) This lowers the chance of the insert being...activity. They now had a system that could integrate genes up to 36 kb in length, with an integration rate ...bylanding site recognition and integration of cargo via Cas9-directed integrases.   Use PASTE can be delivered...

...-expressed Cas9 Cas9 System Selection PI 46968 pICSL01009::AtU6p aU6 BsaI none S. pyogenes Kamoun 52255...HDR). ID Plasmid Gene/Insert Promoter Selectable Marker PI Publication Prime Edit Cas9 H840A nickase fused...dead dCas9, or dCas9 fused to a transcriptional repressor peptide like KRAB, can knock down gene expression...ID Plasmid Gene/Insert Selectable Marker PI Publication Base Edit Catalytically dead dCas9 fused to a ...Plasmid Gene/Insert Promoter Selectable Marker PI Publication Activate Catalytically dead dCas9 fused to...transcription of a specific gene. Design your gRNA sequence to direct the dCas9-activator to promoter or ... plasmid to target the dCas9-activator to your specific locus. ID Plasmid Gene/Insert Promoter Selectable...

...’s first genetic engineering company, Genentech, was founded.  This commercialization of gene editing ...before the discovery of genes themselves.  How has scientific progress in genetic editing impacted the world... DNA sequences is not ideal. For the early generation gene editors, new amino acid recognition sites had...fused a catalytically impaired Cas9 protein to a DNA-editing enzyme to generate the prototypical base editor...followed by non-homologous end-joining that results in gene knockouts.   Star Trek: Into Darkness (2013) which...The major drawback of all the endonuclease-based gene editors (meganucleases, ZFNs, TALENs, and CRISPR-Cas...based on a prime editor which is a fusion between Cas9 and reverse transcriptase. Using an elongated template...

...individual. For example, with CRISPR-Cas9 gene editing, transformation may be identified by resistance ...resources on the Addgene blog  CRISPR 101: Engineering the Plant Genome Using CRISPR/Cas9 Tips for Arabidopsis...fluorescent proteins. Selection using resistance genes usually involves killing the untransformed seedlings...continued production of new alleles or off-target gene editing. Antibiotic selection would only then kill...requires specialized equipment. Reporters for studying gene expression have similar drawbacks, often requiring...Res. (2020).   I can do this by expressing a gene that rescues the mutations, using RUBY as a selection.... et al. A reporter for noninvasively monitoring gene expression and plant transformation. Hortic Res ...

...the neomycin resistance gene 64325 3xFLAG-dCas9/p-bacteria Expresses 3xFLAG-dCas9 in E. coli in a tetracycline-dependent...consisting of a catalytically inactive form of Cas9 (dCas9) plus guide RNA (gRNA) for biochemical purification...CRISPR Cas9 gRNA plasmids from Dr. Hodaka Fujii's lab. Genome... expression vector with the puromycin resistance gene 63591 3xFN-Tel-TAL/pMXs-neo Expresses a 3xFLAG-tagged...(gRNA) to target human IRF-1 promoter for the Sa-Cas9 system. 105283 hIRF-1 gRNA #351 (Sa) Expresses a...(gRNA) to target human IRF-1 promoter for the Sa-Cas9 system. References An enChIP system for the analysis...Tanigawa N, Yuno M, Ikawa M, Taniuchi I, Fujii H. Genes Cells. 2018 Apr;23(4):318-325. doi: 10.1111/gtc.12573...

...REVeRT, for gene delivery using reconstitution via mRNA trans-splicing. By splitting a large gene into smaller...the introduction of extraneous genetic material to reconstitute the gene of interest, reducing the immunogenicity...Blainey’s lab has developed a multiplexing approach to Cas9-based pooled CRISPR screens called CROPseq-multi...Anzalone et al., 2019, including modifications to the Cas9 or reverse transcriptase enzymes, inhibition of ...end of the pegRNA, which is not well protected by Cas9. This is similar to the proposed mechanism for engineered...hIBA1a, 466 bp) that drives high microglia-specific gene expression when incorporated in an AAV vector (Figure...doi.org/10.1101/2023.12.12.571321.  Revolutionizing Gene Therapy: The REVeRT Dual AAV Vector System By Vaibhav...

...effectively targets your gene/region of interest is a key step for CRISPR/Cas9 gene editing. Dr. Masahito...plasmids available! Interested in purifying Cas9? Check out pET-28b-Cas9-His from Alex Schier's lab, designed...sequence or gene of interest into the pCoofy vectors, select the appropriate pair of vector and gene primers...vector and gene primer for SLIC cloning. Amplify the pCoofy vector and the sequence of gene of interest... can be used to generate random mutagenesis libraries or to insert heterologous genes, reporters, or other...Updated mini-transposon vector for bacterial mutagenesis or gene targeting Victor de Lorenzo's lab has...approach to genetic engineering which drastically reduces the time and effort needed to insert new genes into...

...Mammalian ID Plasmid Gene/Insert Promoter PI Publication Bacteria ID Plasmid Gene/Insert Promoter PI Publication... genomic deletions. Compared to deletions generated by Cas9, large deletions created using Cas3 have fewer...underutilized compared to their Class 2 counterparts like Cas9. This is attributed in part to the difficulties ... Publication Plant ID Plasmid Gene/Insert Promoter PI Publication Last reviewed on: January 30, 2025 Do...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...out our Suggest a Plasmid form or e-mail help@addgene.org to help us improve this resource!...

...off-target effect. Which species or variant of Cas9 ( S. pyogenes , S. aureus etc.) was this gRNA sequence ...species or PAM binding variant of Cas9. For instance, wild-type SpCas9 must be used with targets that are... lists gRNA plasmids designed to target various genes or genomic regions. It is highly recommended that...the experimental use of these plasmids. ID Plasmid Gene/Insert Vector Type PI Publication Do you have suggestions...gRNA expression vectors available from Addgene. CRISPR...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...

...relies on the sacB gene product of B. subtilis (Hmelo et al., 2015). The sacB gene product is a sucrase... the CRISPR/Cas9 system with lambda red recombineering enables simplified chromosomal gene replacement... in the chromosome is to disrupt expression of a gene with an antibiotic resistance cassette. This leaves...sometimes interfering with expression of surrounding genes. However, there are ways to create scarless mutations...(Thomason et al., 2014). In recent years, CRISPR-Cas9 technology has also aided in the creation of scarless...Red that contained one of eight bacterial toxin genes isolated from E. coli or Psuedomonas aeruginosa....toxins, and phage holins. They hypothesized that genes that had evolved specifically for toxicity would...

... the Addgene blog CRISPR 101: Which Cas9 do I choose for my CRISPR experiment? CRISPR 101: Cas9 The ...X, Gao F, Guo Y. Off-target effects in CRISPR/Cas9 gene editing. Front Bioeng Biotechnol. 2023 Mar 9;11...confounding mutations, the risk is high. You are generating a gene therapy product with CRISPR to (hopefully)...not to cleave DNA and instead activates/represses gene expression by various mechanisms. The RNA guide ...which involves making specific knock-out edits in a gene(s) of interest and has an average off-target frequency...involves making a large sgRNA library to thousands of genes and targeting and sequencing millions of cells. ...confound an experiment due to their location (e.g. in a gene in a similar pathway, a growth factor, etc.) this...

...Gibson assembly, imagine that you want to insert your gene of interest into a vector with a large tag at the...by using Gibson assembly, you can insert both the gene of interest and the tag sequences into the vector...restriction enzyme or PCR to make linearized vector, the Cas9 enzyme along with a specific gRNA was used to cleave...Li K, Wang B, Jin S, Reiser M, Lockey RF. CRISPR/Cas9 nuclease cleavage combined with Gibson assembly ... Zhao X, Gabrieli T, Lou C, Ebenstein Y, Zhu TF. Cas9-Assisted Targeting of CHromosome segments CATCH ...CATCH enables one-step targeted cloning of large gene clusters. Nat Commun. 2015; Sep 1;6:8101. PubMed PMID...the J. Craig Venter Institute in 2009. Here at Addgene we added this option to our drop down menu of common...

...fluorescent protein to various Cas9 variants, SpCas9, NmCas9, and St1Cas9. These fusions localized to specific...The catalytically dead Cas9 protein (dCas9) is well known for its ability to bind DNA targets without... observing low-copy genomic loci or single copy genes. If you want to visualize genomic loci brightly,...inhibition, among others. Over the past few years, dCas9 has also become a robust visualization tool to study...University of Massachusetts Medical School developed a dCas9-based multicolor labeling system to visualize chromosomal...PP7, or boxB sequences inserted into the sgRNA generates blue, green, or red colors for labeling. A combination... PP7, or boxB sequences in the same sgRNA will generate additional colors. Image from Ma et al., 2016 ...

... applications Gene expression control Researchers are always wanting to manipulate gene expression – turn... Viral CRISPR-Cas9 tools have been engineered to introduce frameshift mutations in genes of interest, ...viral vector for your gene of interest – an shRNA sequence specific to your gene’s mRNA or a sgRNA targeting... Connie Cepko Discuss Gene Therapy with AAV Adenoviral Delivery of CRISPR/Cas9 Aims to Expand Genome ...Additional Resources on the Addgene Blog AAV: A Versatile Viral tool for Gene Expression in Mammals Listen...from basic science research all the way to clinical gene therapy. But how can you use these amazing tools... knock it out, etc. It’s how we understand what genes do! Viruses have several options to allow you to...

...bacterium expressing dCas9 A plasmid in the host bacterium that expresses gene III. Gene III is under the ...compatible with all Cas9 and Cas12 homologs that the researchers tested, including SpCas9, SaCas9, LbCas12a, enAsCas12a...compatible with many Cas domains beyond SpCas9. In order to expand the gene-editing applications and targeting... the expression of gene III to create infectious particles. A plasmid-based genetic circuit in the bacteria...activity. A plasmid-based genetic circuit in the bacteria links the expression of gene III to base editor activity... 1: David Liu gives a talk at the Cell Symposia: Gene- and Cell-based Therapies: CRISPR, Stem Cells, and...the DNA. ABEs are somewhat slow and need a strong Cas9 domain to “hold on” long enough to the DNA to induce...

...design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation." Nature biotechnology (2014... tools yet. As a genome editing tool, the CRISPR/Cas9 technology has been surrounded by a whirlwind of... parasite Plasmodium falciparum using the CRISPR-Cas9 system." Nature biotechnology(2014). PubMed PMID... et al. "DNA targeting specificity of RNA-guided Cas9 nucleases." Nature biotechnology 31.9 (2013): 827...to select a tool based on your project and not general opinion. I could spend a lot of time running through... Online tools dominate the software space but generally rely on less powerful algorithms to detect off-targets...tools available mostly work on any computer but generally require working knowledge of the command line ...

...inactive dCas9 and microbial SSAPs (dCas9-SSAP) which insert genes in a cleavage free manner. The dCas9-SSAP...cleavage-free dCas9-based gene editor using microbial SSAPs. Image from Wang, et al, 2022.  Find dCas9-SSAP...of up to 20%, comparable to commonly used CRISPR/Cas9 methods for large inserts. However, this system ...in comparison to the 62-90% error rate seen with Cas9 editors. The lab believes that this system could...reduces the need to generate “single-use” transgenic lines. Instead, researchers can generate and maintain transgenic...Cre-mediated AAV Targeted Evolution method (M-CREATE) dCas9-SSAP with reduced error rate for long sequence knock-ins...each protein of interest and, for in vivo work, generate a dedicated transgenic line. Using zebrafish as...