...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 ...