We narrowed to 803 results for: Cre

...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...

..., 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 ...

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

...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 ...

...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...

... 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...

...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...

...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 ...

...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...

...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 ...

...genome at once! Knockout screening with CRISPR libraries In CRISPR knockout screening, sgRNAs guide Cas9 to...: Schematic of CRISPR knockout screening with a pooled library. Created with BioRender.com. The popular...libraries called Julianna, for mouse screening, and Jacquere, for human screening. These new libraries have improved...for more robust and accurate screens. Activation or inhibition screening with CRISPR libraries Libraries...activation or inhibition CRISPR screening are similar to those for knockout screens: sgRNAs to guide Cas9 to ...repressor to create a reversible effect. These approaches may also be less toxic than knockout screens. The Jonathan...Schematic of phage display screening with a pooled library. Surface display screening can also be conducted...

...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 ...

...genome-wide off-targets, since increasing PAM flexibility might also increase off-target activity. Surprisingly...gone one step further, using directed evolution to create xCas9, an enzyme recognizing a broad range of PAMs...PAMs like NG, GAA, and GAT, but also displaying increased editing specificity. We’re excited to learn more...easy, right? Yes and no. If you’re just trying to create a knockout, you might be happy targeting anywhere...precise edits or knock-ins. Editing efficiency decreases drastically once your cut site is more than 10...insertion site. Base editing, another method to create point mutations, also has a strict editing window... PAM sequences and/or broader PAM flexibility. Creating xCas9 through directed evolution Hu et al. used...

...nucleobase component of the nucleoside inosine. Created with BioRender.com. Although it was a huge ...it finds a PAM, Cas9 unwinds the adjacent DNA to create a region of separate strands called an R-loop (...of the enzyme. Figure 2: CRISPR R-loop. Created with BioRender.com. Base editors take advantage...activity window restricted to the stretch of ssDNA created by Cas9 binding. Pro tip! In CRISPR, the non-target...BER process was excising the incorrect U base, creating an “abasic,” or empty, DNA site, and then replacing...in the same journal on the same day.) This team created a similar construct that they called a Glycosylase...GBE, for its reliance on the glycosylase UNG to crease abasic sites (Zhao et al., 2021). Their GBE produced...

...be significantly increased through structural and functional optimization to increase the specificity ...efficiency! Screening mutant libraries After generating your mutant libraries, you have to screen thousands...and targeted delivery of treatments, ultimately increasing treatment effectiveness. How do you engineer ...single-domain antibody (often referred to as nanobodies). Created with BioRender.com.   Antibody directed evolution...generating the library of mutants and how they are screened. When conducting antibody evolution experiments...General process of antibody directed evolution. Created with BioRender.com.   Generating mutant libraries...insight, you can shrink your mutant library and increase the chances of finding beneficial mutations. In...

...ones? Here, I’ll cover some screening methods to identify them. Visual screening Regardless of the RNAi-trigger...endogenous miRNA precursors can be engineered to created artificial miRNAs. The processed miRNA or siRNAs...conclusion was based upon numerous phenotype discrepancies between gene silencing and knockout methods ...RNAi in the context of a high throughput survival screen found that either technology was equivalently precise... can be divided into three fundamental steps: creation of the RNAi trigger delivering the RNAi trigger...the first stage of an RNAi-based investigation. Creation of the RNAi trigger requires a few initial considerations...these constructs include pGAPi, which allows easy creation of long hpRNA with Gateway-based cloning and straightforward...

...modular nature, the Dueber Lab created a few versions of EvolvR. To increase the mutation rate of EvolvR,...bacteriophage T7 DNA polymerase increased the processivity of PolI3M, and thereby increasing the editing window of...forward genetics harnesses this at a large scale by screening diverse libraries of genetic variants. Several...nucleotides. Both the Bassik Lab and the Chang Lab have created methods that introduce diversification by targeting... integration of oligonucleotide libraries of a discrete size at the target site. The use of such libraries...occasionally introducing mutations (red nucleotide). Created with BioRender.com. The initial version of ...per generation of wild-type E. coli, while only increasing the standard mutation rate seen during DNA replication...

... Association for Creatine Deficiencies: Compensating for SLC6A8 mutations in Creatine Transporter Deficiency...SLC6A8 gene, also known as the creatine transporter. The Association for Creatine Deficiencies (ACD) is a parent-let...regeneration of ATP from ADP is creatine. In order to reach the brain, creatine needs to be transported from...Deficiency Creatine Transporter Deficiency (CTD) is a rare X-linked inherited neurometabolic condition and... and one of three Cerebral Creatine Deficiency Syndromes (CCDS) that present as global developmental delays... blood and cross the blood brain barrier using creatine transporters. However, mutations in the transporter...SLC6A8 impede this transport and result in global creatine deficiency, which is characterized by development...

...libraries here at Addgene, including cDNA, barcoding, screening, and gRNA libraries. Most of the pooled libraries...determined, larger lentiviral preps can be used for screening-scale transduction of cells, resulting in cell...library. These cells can then be used for genetic screening, followed by sequencing and further data analysis...some of your plasmids didn’t amplify after your screen is completed!  Producing Your Lentiviral Library... any issues.  Keep in mind that a low MOI will increase the chances of a cell being infected by only one... one viral particle, but it also increases the chances that a cell doesn’t get infected at all. To make...as seeding density and reagent concentration.  Screening time Phew! You are now (finally!) ready to use...

...pooled libraries have been created in lentiviral systems for mammalian screens, which enables researchers... as the screen readout. This retroviral mouse CRISPR pooled library can be used for screening of cells...pooled libraries are popular tools for genetic screens, enabling genome-wide interrogation of the genes...required for cellular processes. For a successful screen, it is necessary to identify the gRNAs that elicit...are poorly transduced by lentivirus, and thus screening with available CRISPR libraries was not possible...solve this issue, Sarah Teichmann and colleagues created a retroviral CRISPR knockout library using gRNAs... from mice that constitutively express Cas9 and screen for genes that affect Th2 differentiation and activation...

...goals and the resources. Instead of creating a start up budget, create a start up list.  In this post we...award by giving you step by step instructions on creating a budget, making decisions based on that budget...budget, and negotiating in regards to the budget. Creating a budget We start by using your research specific...budgetary needs from your experimental goals. When creating your budget, you should take the following steps... that timeline Avoid writing down prices as you create your list. This helps to ensure that all of the...”, “colony”, etc. This will help save time by screening out irrelevant candidates, and, more importantly...doesn’t have to be complicated, or difficult to create this list. Start BEFORE searching for a faculty...

...gene expression. By itself, increasing the copy number of a gene increases its expression. However, if...as plasmid copy number increases, the amount of repressor protein also increases thereby intensifying repression...When they increased concentrations of IPTG, a constitutive promoter upstream of GFP increased expression...that is responsive to a repressor protein, the increase in expression due to copy number is kept in check...origins of replication, the stabilized promoters created by Segall-Shapiro et al. showed nearly no position-dependent...stationary phase, the number of plasmids per cell increases 4-5 fold (for pUC plasmids) or ~2 fold (for p15a...compared to a constitutive promoter. To simulate the increase in plasmid copy number during the transition to...

... edited. Created with BioRender.com. Add silent mutations near point mutations to create 3-base (...target can increase the odds of incorrect editing outcomes, especially when MMR is inhibited. Created with ...edit. Figure 1: Prime Editing schematic. Created with BioRender.com. The pegRNA specifies both...PBS) and reverse transcriptase template (RTT). Created with BioRender.com. The first base of the ...to Cas9 with canonical sgRNA structure (right). Created with BioRender.com. What to edit The sequence... mismatched bases can often evade MMR (right). Created with BioRender.com. For specific prime editing...nicking sgRNAs ~50 bp from the prime editing nick. Created with BioRender.com. PE3b and PE5b: In this...

...activation. Created with BioRender.com. Tet1 demethylase Ronggui Hu’s lab has created pdCas9-Tet1-...targeted demethylation is associated with increased transcription. Created with BioRender.com. Tet1 initiates...the catalytic domain of p300 acetyltransferase increases levels of H3K27ac histone modification at specified...This plasmid is used with pcDNA3.1-MS2-Tet1-CD to decrease methylation and activate transcription. A lentiviral...DNA oxidation and repair factors that allow for increased gene activation at the targeted site compared ... of transduced cells. Grant Challen’s lab also created constitutive (pCMV-dCas9-D3A) and Tet-dependent...is associated with transcriptional repression. Created with BioRender.com. DNA Methyltransferase MQ1...

...closer to an innovative resolution. Creative negotiation can also decrease or entirely eliminate the feeling...and everyone leaves unhappy.   Negotiation as a creative problem-solving process Although at first glance...negotiating, what if we instead approached it as a creative problem-solving exercise? Asking questions like...involved and be used as a guide to point you toward a creative solution where everyone wins. What are the underlying...but still be beneficial to both.  Coming up with creative solutions where everyone wins can initially feel...person. It can especially be difficult to think creatively when you are in the middle of a frustrating situation...practice will make negotiating feel more natural. Creative negotiation skills will help you strengthen relationships...

...engineer specific DNA edits and to perform functional screens to identify genes involved in a phenotype of interest...sequence preferences must largely be ignored. Created with BioRender.com. The hammer: Gene knockout...terminus of the protein, to maximize the chances of creating a non-functional allele. For a 1 kilobase gene...primary importance (more on this below). Created with BioRender.com. The jigsaw: Editing by ...the need for single cell cloning and subsequent screening for successful edits. This is a very time consuming...constrained by the desired location of edit; efficiency decreases dramatically when the cut site is >30nt from the...options that can greatly expand gRNA options. Created with BioRender.com. Two newer technologies ...

...of themselves. This increases the number of copies of the TE while also increasing the size of their host...mutagenesis screens that detect loss-of-function or gain-of-function mutations. In these screens, the transposons...transposon for a mutagenesis screen. For example, piggyBac and Tol2 are best for screening promoters and enhancers...Then they can be inserted into new target sites. Created in BioRender.com.   Class II TEs: DNA transposons...which produces a target site duplication (TSD). Created in BioRender.com.   Autonomous vs. non-autonomous...capacity >100 kB, although integration efficiency decreases with cargo size. Sleeping Beauty has a close-to-random...can deliver 10-11 kB to mammalian cells without decreased efficiency, with a maximum cargo capacity of ~...

...round, due to the decrease in ligation efficiency. To combat this, you can increase the volume of competent...other independently regulated transcription units. Created with BioRender.com.   Planning makes perfect... to any tags or fluorescent markers? If you’re creating a multigene construct, in what position do you...stop codon; TAG, C-terminal tag; TER, terminator. Created with BioRender.com. The Sainsbury Laboratory...as well as the restriction enzymes, and it will create the resulting plasmid map and identify any mismatches... new bacterial cultures from your kit plate and create your own glycerol stocks for more frequent use....the MoClo assembly levels, you will be using an increasing number of parts in your one-pot one-step reactions...

...during your in vivo work is to screen you gRNA in vitro first. In vitro screening is a simple and fast method...Researchers are now using this system in ever more creative ways, (Wang et al., 2013, Cho et al., 2014). There...has been quite successfully adapted for use in screening experiments as well). Therefore, if you’ve started...previously validated gRNAs at Addgene. In vitro screening for the best gRNA Figure 1: Validation ...gRNAs to Target Mouse Genes To begin the in vitro screening process, you should first develop primers to the...same time, and choose the most effective ones to increase your chances of gene modification in vivo. Find...embryos to develop. Once they are born, it’s time to screen them for your desired edit. Good news! Remember...

...compose the secretory pathway, endocytic vesicles, mitochondria, lysosomes or they are secreted into the ...your cloning strategy and decide whether you will create an N- or C-terminal fusion based on your experimental...residues form inappropriate disulfide bonds in the secretory pathway (Figure 1) [4,6–8]. Additionally, FPs ...and a sequence intended to localize them to the secretory pathway, a dark pool that is mislocalized to the...misfolded, blocked from proceeding through the secretory pathway, and retained in the ER. FPs have been...recent publication illustrated the quantitative increase in fluorescent signal achieved when utilizing ...optimized moxFP as opposed to standard FPs. The increase in signal is not due to a brighter FP, but rather...

...the process that was used to create this important collection.  Creating the Plasmids To begin constructing...were able to greatly increase the number of functional constructs that were created. This greatly limited...construction and validation of some of these tools created by the Trimmer Lab that will be the first antibodies...that had already been thoroughly validated. The creativity in employing a variety of cloning techniques ...from cryopreserved hybridomas, immortalized cells created by fusing an activated B cell that produces the...VH) chain sequences. Fusion PCR was performed to create an amplicon that  was cloned into a plasmid backbone...plasmid to express the target antigen could be screened in a high-throughput manner. Cells were labeled...

...you can be incredibly frustrating. Clarifying the words —> decreasing frustration —> increasing the happiness...in meetings and 2) creating a pictorial guide for our engineers (Fig. 2). Creating this guide was relatively... an engineer that you want the LIMS software to create a barcode for your plate, you will get in trouble...happening. Our solution for this problem was the creation of an Addgene lexicon. The base lexicon was and...received formal definitions in our lexicon. We created documents and presentations, gave real life examples... field or technology in a consistent way. Just creating a lexicon was not enough. We also had to enforce...the terminology of a new field. Annotate: When creating important documentation, including SOPs or project-related...

...about pooled library screening, read our recent blog post on Genome-wide Screening Using CRISPR/Cas9. If...metalloproteinase-9 secretion, and identifying lysine methyltransferase substrates. We’re incredibly happy that...interest to mammalian cells. It was designed to have increased lentiviral titer as compared to previous lentiviral... population of cells as part of pooled library screens (1,2). The human and mouse GeCKO libraries, targeting...lentiCRISPR v2 is key to its efficient use in screening experiments; large amounts of virus are required...you get full library coverage in pooled library screens. Availability and Use LentiCRISPR v2 is available...Ophir, et al. "Genome-scale CRISPR-Cas9 knockout screening in human cells." Science 343.6166 (2014): 84-87...

...where DNA is being created, such as PCR amplification or bacterial growth. Created with BioRender.com....effort into our quality control (QC) processes to screen for and reduce these errors. These processes allow...  Figure 2. Addgene’s basic QC process. Created with BioRender.com. Once Addgene receives deposited...process ensures that any potential sequence discrepancies can be caught prior to distribution. Catching...issue? At Addgene, a QC issue is defined as a discrepancy that we believe may affect the function of the...decisions when analyzing the wide variety of discrepancies encountered in plasmids deposited to Addgene...resolve. Sequencing and assembly issues Sequence discrepancies are not always a result of mutations in the ...

...NG-Cas9 (PAM: NG) to create NG-ABE8.xm/d and the S. aureus Cas9 (PAM: NNGRRT) to create Sa-ABE8.x-m/d. With....xm/d showed a 1.6x median increase and Sa-ABE8.x-m/d a 2x median increase in editing frequency over ABE7.10...combination with a synthetic library of TadA sequences to create the eighth generation of adenine base editors, ...selection. As a result, 8 mutations in TadA could create all three edits and were thus more efficient. Informed...behavior to ABE8.x-d. Overall, ABE8s showed a median increase of 1.94x editing activity compared to ABE7.10....ABE8.17-m/d, ABE8.20-m/d) to evaluate further.  The creation of accidental insertions and deletions (indel)...ABE7.10 can be a problem. Therefore, the authors created ABE8 constructs using a catalytically “dead” S....

... type of primer pair is also great for screening clones created with the same backbone but that contain...it's in the plasmid Can be used to screen clones that were created with the same backbone because primers...Molecular cloning requires some method of screening colonies for the presence of an insert. Traditionally...is, allowing you to use the same primer pair to screen for the presence of many different inserts. The...one primer anneals to the insert. A simple way to create this type of primer pair is to mix-and-match insert-specific...test your colony PCR primers before using them to screen colonies. The best way to do this is by using your... background, the more colonies you will need to screen.   Saving clones for later culture: At this point...

...ve learned that increased accessibility is achieved through interconnected incremental changes. It was...When Addgene opened in 2004, we focused on creating a repository that made it easy to share plasmids,...identified and offered a number of solutions that increased accessibility, such as using couriers that refill...In some cases, the distributors broadly helped increase access in the country, and we can see a growth...re continuing to use our data and resources to increase accessibility to researchers around the globe....the materials in our repository. We started to increase accessibility to our repository by making it easier...Bacterial Transformation How to Design a Primer Creating Bacterial Glycerol Stocks Inoculating a Liquid...

...article.) Modifying AraC to create BLADE The team modified AraC to create BLADE by replacing its dimerization...images created by the team (Fig. 2). These images are based on the concept of bacteriographs created about...bacteriograph of Michelangelo's "The Creation of Adam." This bacteriograph was created using 160 individual images...dimerize and activates transcription. The team created two constructs, among others: pBLADE-mCherry:...about a decade ago. To create these images, the scientists used a strain transformed with pBLADE expressing...instead of mCherry under the PBAD promoter. They created a bacterial lawn on an agar plate, put a mask over... that Di Ventura’s lab and Khammash's lab have created and shared with Addgene. You can find all the plasmids...