...modular expression systems exist (eg. LexA/LexAop and Cre/Lox systems) in case you want to express one gene...Drosophila geneticists use the Gal4/UAS system. This incredibly useful, yet simple system allows you to systematically...simpler, this modular system saves you from having to create a new fly line every time you want to express YFG...

...Resources on the Addgene Blog Learn about Using Cre in Drosophila Read Other Genome Engineering Posts...

...modular system for packaging, secretion, and delivery of specific RNAs, such as Cre mRNA or Cas9 mRNA and sgRNA...for its small size. They were able to increase activity and decrease the gRNA size by intensively remodelling... engineered guide RNAs Yong-Sam Kim’s lab also created a miniature yet active Cas system starting with...

...dual virus and the experimental approach. (B) CR-Cre mice demonstrate pain response due to blue light ...microcircuits. “In the longer term, this screening platform may help us to create high-performance sensors to enable...mechanical allodynia, a condition where a light touch creates a painful sensation. The dorsal horn of the spinal...virus expression in CR neurons of the spinal cord. Created with BioRender.com. Image from Haoyi Qiu.   ...indicators. Tian developed a video-based high-throughput screen for directed evolution of biosensors based on their... to engineer many types of biosensors.   This screening method allowed Tian to optimize a red‑shifted ... the improved voltage indicator coming from the screen to understand mouse cortical and hippocampal neural...

...Fluorescent Proteins Control Protein Expression with Cre-lox Control Protein Activity with GFP Resources ...possible to create other photoswitchable kinases? The general design of psMEK1 also worked for creating three.... The Problem:  There is no common method for creating optically controlled kinases. The Solution: In...et al, the Lin lab present a generic design for creating photoswitchable kinases and use MEK1 as a model...often hard to decipher because it’s difficult to create short pulses of kinase activity. Zhou et al studied...feedback on endogenous MEK1/2 phosphorylation by creating a short pulse of psRaf1 activity. They did this...just 5 minutes post-Raf1 inactivation, and then decreases. Treatment of the cells with a PP1/PP2A inhibitor...

...delete the functional portion of the Nfix gene using Cre recombinase and to generate Nfix-negative neural ...burden of immune symptoms on CDG patients and to increase their quality of life by reducing their risks ...ligand binding sites in T4 lysozyme by deficiency-creating substitutions." Journal of molecular biology 277.2...

...expression levels of certain genes. A popular method uses Cre recombinase on transgenic flox animals to introduce...light or noise should be avoided as this will also increase stress levels and affect behavior. A fact to consider... also to minimize the damage of tissue due to increased pressure of the added liquid. The volume should...editing/deletion using CRISPR. In some cases, a decrease of the expression efficiency can be observed after...

... expression, and compatible with constitutive or Cre-dependent expression. These sensors have also been...binding sites per sensor (to decrease unnecessary calcium buffering and to increase sensitivity), and is a ...fundamental to muscle and nerve function, hormone secretion, blood clotting, and as we all know, a structural...illumination with violet light and (2) exposure to increased intracellular calcium. It has been applied to ...

..., lentiviral, retroviral, AAV, RNAi, luciferase, cre/lox, yeast expression, worm expression, insect expression...and Organization where your plasmids were first created. From there, we’ll use the information in the ...

...light-dependent Gal4 transcriptional activation and Cre recombination. Find PhoCl plasmids at Addgene. Zhang... for easy removal of the resistance cassette via Cre recombination. Each knockout includes unique barcodes...natural ancestors), and screened through libraries of new RFP variants for increased fluorescence, brightness...polycistronic cassettes and discovered reprogramming discrepancies, which they traced to a 9 amino acid N-terminal... did not lead to the observed reprogramming discrepancies. The polycistronic piggyBac transposon vectors... protein PhoCl expands optogenetics toolbox Increasingly popular Optogenetics tools use light to alter...PMID: 28241009 CRISPR coselection with ouabain increases editing efficiency Blue flame depositor Yannick...

...Engineering Guide CRISPR Mammalian Expression plasmids Cre-Lox plasmids References Prathipati, P., Rodriguez-Aguayo...representation of common injection routes in mice. Image created with biorender.com.   Needle techniques Intravenous... often used when injecting a cell suspension to create xenograft models. Restraint: back scruffing or ...

...targeted loci. The selectable marker can be removed by Cre-mediated excision for scarless tag insertion.   In...lies in the ability of the endonuclease Cas9 to create a DNA double stranded break (DSB) at a genomic ...user wants to carry out a high-throughput genetic screen, it is expensive and time consuming to individually...each insertion. Also, it remains challenging to screen for the genetically modified strains that contain...simultaneously introduce a selection marker for screening modified strains. With SapTrap, the user first...selectable marker (floxed Cbr-unc-119) for easy screening of the insertion event, and a variety of connector...hundreds of genes, generating powerful genetic screening libraries. Additional protein tagging systems ...

...Specific Recombination in Drosophila Learn about Cre-Lox Resources on Addgene.org Find Drosophila Plamids...

...expression, shRNA-mediated knockdown, Tet and Cre-regulated expression, CRISPR genome editing, and more...requirement for the HIV Tat protein, thus decreasing the probability of creating replication-competent lentivirus...an extensive collection of lentiviral plasmids created for a variety of applications including cDNA expression... very common due to its broad host range, and increased particle stability; however, the protein is cytotoxic...replication-deficient meaning they can be used to create virus that is capable of infecting target cells...our eBook: Plasmids 101 Learn about genome-wide screening with CRISPR and Lentivirus Read about the ClonTracr...

... The authors have deposited AAV plasmids for Cre-dependent and -independent versions that are trafficked...O’Neill The Lin Lab and collaborators recently created ASAP5, a faster and more responsive genetically-encoded...for its improved kinetics and responsivity in screening ASAP3 variants. B) Electrical and optical recordings...

...PMCID: PMC5529245. Deverman, Benjamin E., et al. "Cre-dependent selection yields AAV variants for widespread...determined LY6A was a receptor used by PHP.B viruses. Creating PHP.B, a neurotropic AAV9 variant PHP.B’s specificity...the Gradinaru labs engineered PHP.B. PHP.B was created by first generating a diverse library of AAV9 mutants... the AAV9 capsid. This mutant library was then screened in vivo in C57BL/6J mice strains to identify variants...injection. Compared to AAV9, PHP.B has a >40-fold increase in CNS transduction and transduces the majority...Ly6a in primary C57BL/6J brain endothelial cells decreased transduction rates while overexpression of the...

... approach proved a challenge. The old standby of CRE/flp didn’t work – it turns out these tyrosine recombinases...likely do it using Prime editing alongside some creative (and significant!) protein engineering. They combined...insert being “dropped” in the wrong place, while increasing the number of potential applications. But they...

...floxed neoR gene is removed via infection with adeno-cre. AAV-based gene editing compared to CRISPR editing... repair template. It is this application - the creation of sequence-specific knockins - for which the ...Asuri et al, for example, used zinc fingers to increase the efficiency of AAV mediated genome editing ... and encourage to deposit any new AAV tools you create. Many thanks to our guest blogger, Todd Waldman...

...antibiotic resistance markers, and other enzymes such as Cre-recombinase drastically expanding the base-editing...Base editors create specific point mutations in the genome, but they’re inefficient compared to CRISPR...experiments. In the past few years, scientists have created an array of base editing reporters that can help...Cas9-mediated editing in real time The Harris lab created ACE, a reporter that monitors APOBEC (a family ...and a downstream constitutively active eGFP. To create the inactive mCherry, the lab introduced a 43 base-pair...DSB in their previous reporter, the Harris Lab created a panel of eGFP reporters to quantify the on-target...complexes in real-time (St. Martin et al., 2019). To create the eGFP reporters the Harris lab individually ...

...loxP-stop-loxP cassette into the Rosa26 locus, allowing for Cre-dependent expression of a GMAP inserted gene. The...of a good screening experiment can be found on our blog post entitled “Genome-wide Screening Using CRISPR...Receptor-ome Expression and Screening via Transcriptional Output-TANGO (PRESTO-TANGO) to screen the NCC-1 library...and later create stable cell lines expressing it. The Church lab fixed this issue by creating a new dCas9...  CRISPR pooled libraries enable researchers to screen the entire genome for genes that regulate a wide...CRISPR knockout library and used this library to screen for genes involved in West Nile Virus (WNV) induced... gRNAs that confer protection to WNV. From this screen the authors were able to identify 7 previously ...

...Wet transfer Increase transfer time Increase voltage Increase SDS/decrease alcohol in transfer...Semi-dry transfer Decrease voltage Decrease transfer time Decrease SDS/increase alcohol in transfer...over-transferring, increase the amount of alcohol and decrease the amount of SDS, with a decreased transfer time...under-transferring, try increasing the amount of SDS while decreasing the amount of alcohol. The increased SDS will ... density of the bands, which increase as protein concentration increases, show the protein concentrations..., you’ll see the signal increase as the concentration of the protein increases (Figure 3A).  This step...linear range (i.e., expression decreases as protein concentration decreases).     Figure 3: Testing...

...upon these findings by screening for additional activation domains that increase the expression of target...Synchronization of secretory protein traffic in mammalian cells The journey of secretory proteins, from their... mutagenized and screened for speed and voltage sensitivity. This hierarchical screen led to the generation...has modified the RNA-guided CRISPR-Cas9 system to create a tool that is quick, versatile, and robust. Dubbed...adopt basic biological concepts and employ them in creative and innovative ways. The principle of protein ...molecule imaging and inducible gene expression by creating the SunTag system. The SunTag system, named after...cells without affecting protein function, thereby creating a single-molecule reporter of intracellular processes...

...Semi-dry transfer Decreased efficiency Electroblot transfer Increased cost; decreased efficiency ...transfer Increased time Handcast gel Increased time; have to make as-needed Increase specificity...film imaging Increased time; increased consumables cost Decrease background Direct detection method... Wet transfer Increased time Specialty chemical-based buffers Increased cost X-ray film...low-fluorescence PVDF membranes Increased fragility (nitrocellulose) or increased cost (specialty PVDF) ...expressed genes Not as accurate; increased antibody costs; increased optimization Normalizing to total... total protein loading Increased time; increased reagents Good luck and happy blotting! Many thanks...

...uracil DNA glycosylase. Created with BioRender.com. The BE3 system increased editing frequency to above...editing efficiency decreasing off-target effects In 2016, Akihiko Kondo’s lab created the Target-AID base... offers a 2.3 fold decrease in C→G and C→A products as well as a 2.3 fold decrease in indel formation....nucleobase component of the nucleoside inosine. Created with BioRender.com. How does base editing work...editing Cytosine base editing The beginnings Komor created the first cytosine base editor by coupling a cytidine... which would change the U back to a C base. To increase base editing efficiency, Komor and team wanted...cytidine deaminase portion of the base editor to create SpCas9 base editors with editing windows as small...

...contain many sgRNAs per gene to increase confidence in the genetic screen. However, there are benefits to...to conduct genome-wide gain of function screens. These screens can help reveal the function of lowly expressed...large-scale functional screens Read this blog post about genome-wide screening using CRISPR Resources...allowed scientists to easily perform genome-wide screens to effectively and efficiently investigate gene...resources and are particularly useful when trying to screen a limited number of cells like primary cells or... they conducted genome-wide negative selection screens with both A375 (melanoma) and AT29 (colon cancer...metrics to assess library performance in viability screens. Based on these metrics the Brunello library was...

...expanded the genetic screening toolkit by optimizing a Cas12a-based method for screens. Cas12a’s ability ...underutilized in genetic screens because of its lowered editing rate. This research aimed to increase the potential...parallel. Lastly, this Cas12a-based screening method was used to create a genome-wide library capable of ...single cassette provides increased simplicity for combinatorial genetic screens, and this optimized toolkit...imaging of cellular targets Optimized genetic screening technique using Cas12a for multiplexed gene perturbation...10.1038/s41467-019-10846-1.   Optimized genetic screening technique using Cas12a for multiplexed gene perturbation...Gabrielle Clouse While many CRISPR-based genetic screening methods have primarily used Cas9 to knock out ...

...Library Screening Why the kinome? While properly designed perturbations – in the case of screens using ...at the Broad Institute, John Doench. A genetic screening project can be a tremendous undertaking, producing...technology, gRNAs to knockout genes – are essential screening components, the model system and the assay are...is to do it huge, there are times when a more incremental approach is prudent. What's in the CRISPR kinome... in two sets, each with 4 gRNAs per kinase, so screens can be performed with either 4 or 8 gRNAs per gene...found in genome-wide libraries, and thus can be screened in pooled format at a more-typical scale of tissue.... The individual plasmids will be useful when screening for phenotypes, such as cell-non-autonomous effects...

... target strand and the gRNA. Created with BioRender.com. To decrease the electropositivity of the ...unwanted changes at non-target sites. Means to decrease these so-called “off-target effects” have included...lower on-target efficiency, and sometimes even increase off-target effects, respectively. Recognizing ...School, and the Doudna Lab at Berkeley set out to decrease the off target effects of CRISPR/Cas9 by altering...al. hypothesized that Cas9 cutting efficiency increases when target DNA strand separation is stabilized...higher propensity to reform double helices and decrease Cas9 cutting efficiency. However, this is not ...being cut. Slaymaker et al reasoned that, if they decreased the positive charge in the HNH/RuvC groove, then...

...are ready to begin the screening process. Want to set yourself up for a screening success? Before you start...need to screen hundreds of clones looking for your edit of interest. For all of the screening strategies...phase of your experiment for a screening strategy. Restriction enzyme screening is sensitive to single base...needle in a haystack before going on to generate and screen individual clones. If a clonal line isn’t your ... isn’t a multiple of three will do. A popular screening method for knockouts is Tracking of Indels by ... the minimum number of clones you would have to screen to identify a knockout clone. For example, if your...interest. Thus, to find a null clone, you should screen, at the very least, 4 clones. Did you use a dual...

...specific markers. The Screening Criteria While choosing which antibodies to screen, we kept in mind our...We therefore use both rAb and mAB in our screening. To screen the antibodies, we have developed a criterion...and his lab members (like me!) are dedicated to creating a public and accessible database for low-cost,...Initiative Cell Census Network, the Wu lab will be screening over 3,000 antibodies for use in mice and human...batch-to-batch variability found in polyclonal antibodies, creating a potentially unlimited supply of identical antibodies...antibodies; Addgene collaborates with these companies to create and provide general availability of their recombinant...to see the reproducibility and accuracy while screening. We also compare different clones of the same ...