...for doing gene targeting, reporter generation, controllable gene knockouts, and inducible gene inactivation...Alec Kimmelman targets mouse metabolic genes with ~6 gRNAs per gene.  CRISPR-Act3.0, developed by Yiping... Shirley Liu's lab targets all mouse genes with 10 gRNAs per gene. The guides are optimized to maximize... to develop CRISPR-based approaches for studying gene function in these systems. As a part of this effort...Yiping Qi's lab, allows simultaneous gene activation in plants.  FnCas12a, unlike other Cas12a nucleases identified...opportunities for investigating genetic mechanisms related to morphogenesis, cell fate specification, and...Lab have compiled a new toolkit for performing genetics studies in organoids. The Organoid EasyTag System...

...multiple terminators can serve to interrupt genes (or gene clusters) in metabolic engineering. So far,...pTargetF with the ccdB gene containing BsaI restriction enzyme sites at its ends to generate pEcgRNA. The ccdB...to successfully delete genes of interest in BL21(DE3) using the newly generated pEcCas/pEcgRNA system....used MUCICAT to optimize the gene dosage of glucose dehydrogenase and generated a strain that could achieve...self-curing, an arabinose inducible lambda-red gene, and an IPTG-inducible sgRNA sequence targeting the...non-temperature-sensitive replicon, and 3) added the sacB gene for counterselection to yield pEcCas. In pTargetF... of its E. coli host, and the addition of a sacB gene renders it beneficial for plasmid clearance. It ...

...2020. . “Advances in CRISPR/Cas-based Gene Therapy in Human Genetic Diseases.” Theranostics. https://doi.org...treat AD using CRISPR/Cas9. They are targeting the genes responsible for both early onset and late onset ...Sciences found that mice with a dominant mutation in a gene that causes cataracts could be rescued by coinjection...dominant cataract disorder caused by a single copy of a gene known as Crygc. When all was said and done, the ...able to demonstrate functional correction of the gene in clonally expanded organoids. “CRISPR in adult...fibrosis may not be the most likely candidate for gene therapy, however, because the disease affects multiple...proof-of-principle for use of CRISPR to correct single-gene conditions. Remaining hurdles in CRISPR biology...

...reporter gene (e.g., mCherry) to easily detect opsin-expressing cells. The resulting fusion gene is inserted...downside in optogenetics (Belzung et al., 2014). This could be overcome by fusing opsin genes to cell-type...Krook-Magnuson, 2018). Optogenetic FLEx vectors contain a strong promoter upstream of an opsin gene (e.g., channelrhodopsin...2014). Microbial rhodopsins are encoded by opsin genes, and include bacteriorhodopsins, halorhodopsins,...light-sensitive, so the selective expression of opsin genes in targeted cells makes it possible to control neuronal...viruses (rAAVs) have become increasingly popular for gene delivery in the central nervous system due to their...those usually drive weak expression of downstream genes. Strong cell-specific promoters, on the other side...

... on a break from expressing some genes of interest).   Blugene is so excited they can’t stop dancing...GIFs, feel free to let us know on Twitter @addgene! Addgene is always looking for new ways to help you...We’re making some moves over here at Addgene...and they’re looping? What’s the deal with looping...coworkers, labmates, and friends as well!   Addgene is sharing so much more than plasmids these days...GIPHY   You can see for yourself at giphy.com/addgene. But that’s not all! You’ll be able to use these...Twitter, Instagram, TikTok and more. Just type “addgene” into the GIF search bar to find that perfect response....   This could be you after watching Addgene’s protocol videos. via GIPHY   If you’re also...

...either side of the cargo (ex: fluorescent protein gene) on the donor vector. Therefore, targeting requires...developed one of these systems further for simultaneous gene activation and editing. Read the paper in Nucleic... with an NGG PAM motif, averaging 19 targets per gene. The lab has previously used the library with SpdCas9...perform pooled CRISPRi screens to identify essential genes and phage host factors, and identify sequence-specific...Knockout Library is a lentiviral library targeting 26 genes chosen from an analysis of human metastasis cohorts...metastasis CRISPR screen. The Chen Mouse Tumor Suppressor Gene CRISPR Knockout Library is an AAV CRISPR knockout...knockout library targeting mouse tumor suppressor genes (TSG). Find the Mouse Metastasis CRISPR-Cpf1 Double...

...simple example with the goal is to generate mice that have two modified genes in their genome, and assuming...your targeting vector or gene of interest. 2. Breed your parental mice to generate F1 Once you’ve performed...other will generate approximately 1 in 16 mice that harbor homozygous alleles at both of your genes of interest...your mice. Because their generation involves the integration of a novel gene randomly into their genome...your parental mice were all homozygous for your genes of interest, your F1 mice will be carriers at those... all of your desired genes. Remember Punnett squares from high school biology? They will come in handy...expresses Cre with a strain that expresses a floxed gene of interest. Cre may be expressed globally (in every...

...antibiotic resistance genes, as well as non-selectable DNA fragments, such as gene replacements and tags...repressor gene, CI857, is used.  This mutant repressor prevents expression of the recombination genes at low...the red genes needed for recombineering, but also leads to expression of the int and xis genes which are...Substrate DNA design and generation Expression of lambda red recombination genes. Electroporation of substrate... a different genetic background via P1 transduction. Plasmid Expressing lambda red genes from a plasmid...recombinant clones are generated.  A simple way to do this is to express lambda red genes from a plasmid with...using Lambda Red recombineering system to replace a gene of interest with an antibiotic resistance cassette...

...number of positive droplets for the gene of interest and reference gene, copy number can be determined through...your experiment?  Complexity of the gene of interest Some gene sequences are harder to amplify and quantify...is a well known fact if a multicopy variant of a gene exists, there must be assays that researchers can... ubiquitously expressed, one-copy ‘housekeeping’ gene), and quantified relative to a 'normal' sample with...to use a reference sample with two copies of the gene (Ma, 2014.) As with all qPCR assays, samples, standard...between 0.80-1.20) would mean you have one copy of the gene; an RQ value of 2 means you have two copies, and... assays, allowing the reference sequence and the gene of interest to be amplified in the same reaction...

...disease in which disruption of the smn1 gene results in the degeneration of spinal cord motor neurons. This...Schiavi will use Addgene plasmids to characterize smn1 interactions with other genes. He will use fluorescent...to identify candidate genes as drivers of peritoneal mesothelioma tumorigenesis. They plan to use CRISPR...assess their tumorigenesis. These experiments will serve to validate these candidate genes and allow Nabavi...19,000 live births (1).  While disruption of the smn1 gene is known to cause spinal muscular atrophy, the molecular...fluorescent proteins to monitor the localization of these genes, CRISPR to disrupt their function, and C. elegans... Di Schiavi will be able to better choose which genes and pathways to target in future small molecule ...

...interspaced palindromic repeats) Cas (CRISPR associated genes) system. This popular genome engineering tool offers... be incorporated into the bacterial genome. Cas genes, which code for polymerases, nucleases, and helicases...adaptive immune system that utilized various cas genes not only to store a record of invading phages but...conserved CRISPR subtypes comprised of more than 40 cas gene families have been characterized. To date, 83% of...Rising to the challenge - Evolution of anti-CRISPR genes in phages The CRISPR/Cas adaptive immune system ...nucleases from re-identifying it (3). Surprisingly, few genes have been identified that neutralize CRISPR/Cas....Earlier this year, expression of 1 of 5 anti-CRISPR genes were found to inactivate the Type I-F CRISPR/Cas...

...plasmid in the host bacterium that expresses gene III. Gene III is under the control of a T7 RNA polymerase... 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...Cas domains beyond SpCas9. In order to expand the gene-editing applications and targeting scope of ABEs...bacteria links the expression of gene III to base editor activity and editing speed and consists of the... active variant of the base editor, a functional gene III product is produced and can infect bacterial...

...variants in a gene of poorly classified function and a well established cellular signaling gene, thus providing...above to demonstrate how gene expression profiling, systematic tumorigenesis assays, drug epistasis assays... be used as generalizable strategies to map the function of rare cancer variants and genes: High-throughput...bottleneck is that most recurrently mutated cancer genes are rare and most of the individual variants found...well-annotated cellular pathways). It wasn’t clear whether gene-agnostic bioassays could accurately test for molecular...numbers of cancer variants across a large number of genes as systematically as possible. In developing these...assays were designed to complement the one assay per gene approach that has been the historical standard. ...

...them together. Gene expression The reason for most viral experiments is to deliver a genetic cargo, typically...primary plasmid with the delivered cargo (often a gene) is called the transfer plasmid while the other ...component as well. On both sides of this promoter-gene combo are two long terminal repeats (LTRs). These...infiltrate the host cell and deposit viral cargo. Gene expression While AAV often does not integrate into...(more on this below), it’s still used to deliver gene expression constructs. Sandwiched between two inverted... AAV also requires some back up from adenovirus genes – E4, E2a, and VA – which are supplied on a helper...viral replication. More information on these viral genes is available in the adenovirus section below!   ...

...the ccdB gene is replaced by the gene of interest. As a result, cells that retain the ccdB gene die off...tool. The plasmid pKSCTMF contains the mazF gene and a gene for Zeocin resistance (Figure 2). This MazF-ZeoR...antisense RNA regulates hok gene expression indirectly through the overlapping mok gene." Journal of molecular...associated genes. These systems better ensure that plasmids are passed down through generations (Figure ...application of CcdB is GatewayTM cloning, where the ccdB gene serves as a counterselectable marker in the destination...death. The team used this cassette to knock out genes, knock in a GFP cassette, and introduced site-directed...toxin-antitoxin system to construct a stable synthetic gene circuit that can be used over prolonged periods....

...Express a gene of interest fused to the C-terminus of SCFP3A Amber N1 Yellow Mammalian Express a gene of interest...Express a gene of interest fused to the C-terminus of Amber mVenus N1 Yellow Mammalian Express a gene of interest...your choice of fluorescent fusion protein with your gene of interest: Plasmid Color Expression Description...Aquamarine mCerulean N1 Cyan Mammalian Express a gene of interest fused to the N-terminus of monomeric...Cerulean mCerulean C1 Cyan Mammalian Express a gene of interest fused to the C-terminus of monomeric...monomeric Venus mVenus C1 Yellow Mammalian Express a gene of interest fused to the C-terminus of monomeric...C-terminal His tag SYFP2 Yellow Mammalian Express a gene of interest fused to the C-terminus of SYFP2 Clover...

... this gene. Should you cut it at the beginning of the coding sequence to delete the entire gene? Perhaps... in vitro, and using it in mouse embryos to generate gene modified mouse lines. Hopefully this post will...they have been generated (more on that later). Find Validated gRNAs to Target Mouse Genes To begin the ...chances of gene modification in vivo. Find Plasmids for Testing Your gRNAs in vitro at Addgene CRISPR/Cas9...decide to do a gene modification experiment in mice to the day you have your F0 generation (barring life...Haoyi, et al. "One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated ...blogger Samantha Young. The use of CRISPR/Cas9 for gene editing has expanded since its adaptation for use...

...activators and repressors to target genes. Repressing target genes using dCas9-based repressors Early ...of the gene of interest. dCas9-based repressors are useful when knocking out a particular gene isn’t feasible...repressing target genes in a variety of species and cell types can be found on Addgene’s website. Activating...activating target genes in a variety of species and cell types can be found on Addgene’s website. ...Designing or selecting a sgRNA Addgene may carry a validated sgRNA for your target gene.  Validated gRNAs are ... activate, repress, and even image your favorite gene. With so many Cas9 reagents available, it can be...introducing Cas9-mediated frameshift mutations in the gene of interest. The genomic target for Cas9 and the...

...knockout, activate or repress target genes in plants using the same general experimental design principles ...Resistance by CRISPR/Cas9-Targeted Mutagenesis of the ERF Transcription Factor Gene OsERF922. PLoS ONE 11:e0154027...for targeted modification of known or suspected genes that regulate a desired phenotype. In fact, CRISPR... can also be used to activate or repress target genes in plant cells, respectively. Cas9 expression is...regulate expression of longer RNAs (such as mRNAs for gene expression). Examples of commonly used RNA pol II..., activation and repression of target genes in plants and many of the aforementioned empty gRNA backbones...Agrobacterium tumefaciens as a vehicle to deliver your gene of interest into a target cell line or organism ...

..."Future of rAAV gene therapy: Platform for RNAi, Gene Editing and Beyond." Human Gene Therapy ja (2017...required for the onset of gene expression. However, it has yet to be determined what gene expression looks like...Additional Resources on the Addgene Blog AAV: A Versatile Viral Tool for Gene Expression in Mammals A Match...transport desirable or not desirable? If diffuse gene expression is tolerated, then axonal transport is...Otherwise, using a specific system for controlling gene expression such as Cre-lox, may be sufficient to...tool to use. However, we are starting to see that gene expression really does not take much time at all... normally require second strand synthesis before gene expression begins. ScAAV avoid this rate limiting...