...inhibit, activate, or knockout many genes at a time to identify ‘hits’ - genes that affect disease progression...progression or onset. These hits can reveal individual genes or sometimes even genetic pathways that are worth...exist a drug which targets some of the candidate genes! Disease model generation CRISPR has made the generation...both individual and combinatorial drugs on for effectiveness prior to patient treatment. Target validation... revolutionized the generation of knockout cell lines which has made this control step extremely accessible...your drug(s). If a drug is equally toxic to both lines, it probably has either high toxicity or high off...and Resources References Fellmann, C., et al., Cornerstones of CRISPR-Cas in drug development and therapy...

...neurotransmission. But with approximately 950 different human genes encoding GPCRs (Takeda S et al., 2002) and many ...can be measured using a technique known as Bioluminescence Resonance Energy Transfer (BRET). BRET is similar...BRET is that the energy transfer is between a luminescent donor and a fluorescent acceptor. TRUPATH uses...measures heterotrimeric G protein dissociation by bioluminescence resonance energy transfer 2, or BRET2. With... constructs. Cells are then treated with the luminescent enzyme substrate coelenterazine, followed by ...endogenous ligand or drug. In the inactive state, the luminescent chimeric protein Gα-Rluc8 and Gγ-GFP2 will be...transducerome on a single GPCR, showing the effectiveness of using TRUPATH as a screening technique. Lastly...

...the plant hormones strigolactones By Fabienne Furt Current methods used to detect strigolactones (SLs) are...editing in organoid Biosensors for detecting strigolactones The CRISPR Corner New ready-to-use viral preps...et al. 2021. The Organoid EasyTag workflow combines thoughtfully designed plasmid components with ...investigating genetic mechanisms related to morphogenesis, cell fate specification, and human disease....library from Alec Kimmelman targets mouse metabolic genes with ~6 gRNAs per gene.  CRISPR-Act3.0, developed...from Xiaole Shirley Liu's lab targets all mouse genes with 10 gRNAs per gene. The guides are optimized...

...deletion of one or several of these genes permits the expression of genes farther downstream in the construct...Brainbow uses a Cre/lox system to label neurons. Genes encoding the fluorescent proteins are organized ...developed, which differ in the number, stability, and brightness of the fluorescent proteins used in the system... which uses Cre to trigger the inversion of two genes aligned head-to-head. Another variation, Brainbow... to trigger the inversion of two tandem sets of genes aligned head-to-head and the deletion of one of ...located directly upstream of the fluorescent protein genes, Cre recombination is required before fluorescence...brainbow,” is a collection of transgenic zebrafish lines that allow for multicolor labeling of an entire ...

...during and after embryogenesis. Addgene has over 600 plasmids that contain Xenopus genes and a tool-kit ... concluded that nearly all disease-linked human genes have counterparts in the rat (Gibbs, et al., 2004...collection of plasmids containing Rattus norvegicus genes! Mexican axolotl (Ambystoma mexicanum) Ambystoma...entire axolotl genome, revealing an abundance of genes related to limb regeneration (Vieira et al., 2019...for use in CRISPR and Tol2 transposase induced transgenesis.  Xenopus Xenopus, or frogs, are one of the ... for scientists to study human disease-relevant genes in such a controlled environment displays how essential...molecular tools, such as gene manipulation and mutagenesis techniques, are available to the scientific community...

...the location of genes. With this knowledge, researchers were able to target specific genes for deletion ... at Harvard University. Just as computers, cell phones, and cars become more technologically advanced ...fluorescently labeled probes. Colorimetric and luminescence based detection methods are also commonly used...ddNTP, and then run these reactions out in separate lanes of a gel. Since the sequencing gel will separate...nucleotides were replaced with fluorescently labeled ones allowing for fluorometric-based detection. This ...generally considered first generation DNA sequencing machines (9). Today, similar technologies, sometimes referred...make techniques used to generate transgenic mouse lines, such as BAC transgenic or traditional knockins/...

... our purposes? In search of simple biological machines R bodies can repeatedly extend and contract...using R bodies: simple, force-generating protein machines that can extend and retract over many microns ...with proteins and remain functional, 2) break membranes outside of their natural context or 3) be modified...demonstrates that R bodies can function to break membranes of different organisms. And finally, we demonstrated...demonstrated that R bodies can be “tuned” with mutagenesis to extend at different pH values. Using a screen...in a particular region of the R-body. Further mutatgenesis of this region produced R-bodies that could ...could use them to lyse cells (and surrounding membranes) in keeping with their natural function (see below...

... disease, cholesterol gallstone disease, senescent illnesses, and the metabolic syndrome. Coregulators...functional groups of genes involved in the execution of a specific regulatory regime (such as genes involved in...ligand-dependent sensors for a diverse set of fat-soluble hormones, vitamins, and dietary lipids. Included in this...remain unknown. Together, NRs govern expression of genes involved in a broad range of reproductive, developmental...For example, the phosphorylation status of SRC-3 defines its association with other members of the complex...as in important physiological states such as adipogenesis and macrophage activation. Our mass spectrometry-affinity...

...2014). Microbial rhodopsins are encoded by opsin genes, and include bacteriorhodopsins, halorhodopsins,...control the flow of electric charges across cell membranes and maintain membrane potential in response to...light-sensitive, so the selective expression of opsin genes in targeted cells makes it possible to control neuronal..., 2014). This could be overcome by fusing opsin genes to cell-type specific promoters; however, those ...those usually drive weak expression of downstream genes. Strong cell-specific promoters, on the other side... circuitry behind the mechanisms of sleep and anesthesia. In Cre transgenic mice, they targeted the dopamine... previously subjected to steady-state general anesthesia (Taylor et al., 2016). These very same neurons...

.... These could be genes, specific polymorphisms, including antibiotic resistance genes. This approach successfully...Genome editing tools for bacteria CRMAGE system combines CRISPR and recombineering-based MAGE The Nielsen...CRMAGE system is a fast, multiplexable method that combines CRISPR and recombineering-based MAGE (Multiplex...Yang’s lab describes a two-plasmid system that combines recombineering with CRISPR to create a system ...effectively silenced transcription of one or more genes in E. coli. and in a mammalian cell line (Qi et ... repressed or activated transcription of select genes in E. coli. Furthermore, to demonstrate wider applicability... the Husson lab used CRISPRi to study essential genes in M. tuberculosis. dCas9 and a gRNA are expressed...

... AdVs are stripped of regulatory genes E1 and E3. Without these genes, AdVs cannot replicate on their ...produced at high titers, can accommodate large transgenes, transduce quiescent and dividing cells, and ..., or high-capacity (HC-Ad__ are devoid of viral genes and therefore can accommodate up to 36 kb. Gutless...can be produced in E1-containing mammalian cell lines such as HEK293 cells. 1st generation AdV cloning...immunogenicity. In addition to E1/E3, non-structural genes E2 and E4 are deleted in 2nd generation AdVs, thereby...cloned) and pAdEasy™ which contains the adenoviral genes necessary for virus production. For detailed instructions...therapies in the new era of personalized medicine." Genes & diseases 4.2 (2017): 43-63. PubMed PMID: 28944281...

... and bleed dates and even clonal hybridoma cell lines can produce more than one mAb (Bradbury et al., ...Hybridomas can lose gene expression of the mAb-encoding genes or fail to revive after cryopreservation (Bradbury...antibodies that are generated in vitro using synthetic genes that are typically expressed from a plasmid or from... integrated sequence in a stable cell line. The genes encode the heavy chain and light chain for the antibody...Figure 2: (Left) The light chain and heavy chain genes of the antibody are encoded in a plasmid. (Right...better and better at identifying and cloning the genes for antibody expression. If starting from a protein...sequences of the antibodies and then synthesize the genes that encode those amino acids (Tran et al., 2016...

...Specially constructed plasmids can be used to replace genes in yeast, mice, or Drosophila through homologous...techniques and the plasmids used to inactivate specific genes in mammalian cells. Despite the popularity of CRISPR-based... Neomycin, puromycin, and hygromycin resistance genes are commonly used for positive selection. While ...methods have been used to create many knockout cell lines and animal models, their efficiency is very low,...easier to use and are more efficient at inactivating genes. CRISPR can target a genomic sequence and create...perfected, the production of cells and mouse knockout lines should increase. New genome engineering tools also...have tools for creating knockout animals or cell lines, please consider sharing them by depositing with...

... Cells Transformed cell lines Transformed cell lines Transformed cell lines Cargo Nucleic acids or ...cells or a selection marker to generate stable cell lines. Transient or stable expression of Cas9 and/or gRNA...contains a selection marker to generate stable cell lines. Packaging and Envelope plasmids provide the necessary...and/or gRNA in a wide variety of mammalian cell lines. Useful for difficult to transfect cell types and... cell.   Delivery methods for mammalian cell lines As mentioned above, the expression system you choose...Delivery of these components into mammalian cell lines is quite broad and includes several different methods...

...PA-FPs by performing several rounds of random mutagenesis screening for enhanced RFP variants. PA-mCherry... Color Contrast (Fold Change in Fluorescence) Brightness Excitation/ Emission (nm) PA-GFP Monomer UV-Violet...negative” photoswitching. In 2008, scientists used mutagenesis screens to find a version of Dronpa that doesn't...background fluorescence, but their single molecule brightness and ability to switch from a dark state to a ... Color Contrast (Fold Change in Fluorescence) Brightness Excitation/ Emission (nm) Dronpa Monomer UV-Violet...there are several parameters to consider, such as brightness, photostability, pH stability, chromophore maturation... is the contrast ratio- a comparison of PA-FP brightness before and after photoactivation. A high contrast...

...binding. Formaldehyde cross-links the protein (red lines), which maintains structural epitopes (orange region...proteins that need a little help crossing cell membranes as they are not able to diffuse across natively...Permeabilization essentially punches holes in the cell membranes of your sample to allow antibodies better access...example, saponin interacts with cholesterol in cell membranes and leaves many membrane-associated proteins in...detergents that interact with lipids and proteins in membranes and permeabilize non-selectively. This non-selectivity...helpful when trying to pass tough-to-permeabilize membranes and these detergents avoid some of the negative...but it is better at permeabilizing organellar membranes. Created with BioRender.   How do I choose...

...substantially enhance the brightness and photostability of the classic rhodamines and their analogs without... a greater burden on the photon budget, which determines how long and precise we can track individual ...selectivity of the fluorophore attachment, the brightness of the resulting conjugate, and the complexity... classic small molecule fluorophores, such as rhodamines (Figure 2a), were discovered in the 19th century...due to its established chemistry, small size, brightness, and cell permeability. In past decades, further... the Alexa Fluor and ATTO dyes, with improved brightness, photostability, and spectral range. Nevertheless...PMCID: PMC4344395. 3. Lavis, Luke D., and Ronald T. Raines. "Bright ideas for chemical biology."ACS chemical...

...varies across targets, they tested two genes in mice. For both genes, they saw indel formation and phenotypic...certain tissues. However, packaging Streptococcus pyogenes (SpCas9) and a gRNA together (~4.2 kb) into an...al. successfully used CjCas9 with AAV to target genes in mouse muscle and eye tissue. Neisseria meningitidis...prime editors. While using AAV to deliver large transgenes might seem daunting, it’s actually a challenge...Trans-spliced Cas9 allows cleavage of HBB and CCR5 genes in human cells using compact expression cassettes...ncomms14500  Levy JM, Yeh W-H, Pendse N, Davis JR, Hennessey E, Butcher R, Koblan LW, Comander J, Liu Q, Liu...https://doi.org/10.1002/biot.201400046  Strecker J, Jones S, Koopal B, Schmid-Burgk J, Zetsche B, Gao L, Makarova...

...sequences While PAM sequences for the commonly used S. pyogenes Cas9 (3'-NGG) are abundant throughout the human...circumvent this limitation: 1) the use of novel S. pyogenes Cas9 variants with varying PAM sequences, 2) the...Cas9 homologs derived from species other than S. pyogenes, and 3) the use of non-Cas9 enzymes. (For more...check out this video from IGI!) Synthetic S. pyogenes Cas9s with novel PAM recognition In 2015, Keith... screens in bacteria to identify mutants of S. pyogenes Cas9 that were able to cleave target DNA sequences...variant is far more selective for the canonical S. pyogenes PAM sequence NGG compared to wild-type SpCas9,...Cas9 species PAM sequence ’) Streptococcus pyogenes (Sp) 3' NGG Staphylococcus aureus (Sa) ...

...PHP.B.  A Ly6a variant is linked to PHP.B CNS permissiveness Both the Deverman and Wilson labs determined...as non-permissive to PHP.B CNS transduction. Two genes were associated with the PHP.B permissive phenotype... suggesting it was a likely player in PHP.B permissiveness. The Wilson Lab also looked for the gene variant...mice to map the inheritance pattern of PHP.B permissiveness. The first generation of outbred mice (F1) ...~4.5 Mbp region of the mouse chromosome. Of the genes located in this stretch of DNA, Ly6a seemed the ...cells: galactose and the AAV receptor (AAVR). Cell lines lacking galactose were still transduced by PHP.B...to the CNS. The bottom line of viral vector permissiveness While PHP.B has impressive CNS transduction...