...gRNA are not to scale.   Let’s start with the simplest multiplexing situation: you only need to express... and two U6 promoters. To use this plasmid, you simply order oligonucleotides for your chosen gRNA target...Transcript Multiplexing Plasmids: These plasmids are similar to the Csy4 polycistronic system described above...assembly, and up to 8 gRNAs may be expressed simultaneously. Vectors for both transient expression and ...small insertions. Multiplexing of two targets simultaneously is possible with efficiency >70%. CRMAGE is... delivered on a different plasmid and allows simultaneous multiplex editing of six different genes. This...

... to provide that context yourself. Simple words are not so simple Just as our lab used jargon that required...problematic word. How many different things do you simply refer to as a “tube”? We took steps towards solving... that nearly all Addgene teams used that seemed simple, but they could in fact be interpreted in several...when starting rotations or my postdoc would have simplified the first weeks or months when I was learning...

...these mutants are not toxic to HEK cells and that similar mutations can improve the specificity of Cas9 derived...from S. aureus. This means you can likely apply similar rational mutations to your Cas9 homolog of choice...SpCas9-HF1 Kleinstiver et al and the Joung Lab similarly thought that if they could weaken sequence independent...found in eSpCas9, the effect on specificity was similar; SpCas9-HF1 generated fewer off-target cuts when...variant displays comparable on-target activity and similar or decreased off-target activity across the sites...

...computational methods to identify genes by their similarities to genes in other species, but these studies...gene function wide open. What if two seemingly similar proteins have acquired divergent functions due ...strains are available. Then, staff or students clone similar sequences from a less-well studied organism into...confident that the gene from the test species has a similar function to the gene in the reference species. ...Out Our Molecular Biology Reference Pages Learn Simple Lab Techniques in Our Protocol Videos ...

...through homologous recombination. The concept is simple: deliver a template with a modified version of ...nucleotide sequences are exchanged between two similar or identical molecules of DNA. Gene targeting takes...insert GFP into the genome, we’d create a plasmid similar to the one shown below, where the sequence of GFP...plasmids described in the preceding sections are simple ways of knocking out a non-essential gene of interest...functions normally because one of its exons has simply been replaced with the same sequence flanked by...

...and a low similarity index. In figure 3, we can see that PerCP and PE/Cy5 have a similarity index of 0.87...Cytek’s website has a convenient tool, called the similarity index, which allows the user to compare the emission...when designing a panel.     Figure 3: Similarity index across 12 different colors on a three laser.... PE/Cy7 and BB515, on the other hand, have a similarity index of 0, which would be a better choice for...

...Charles Gersbach’s lab at Duke University developed similar systems based on the light-inducible heterodimerizing...effector (LACE) system, the Gersbach lab utilized a similar strategy to develop an optimized photoactivatable...system was able to recognize the same PAM and had similar targeting specificity as full-length Cas9 (flCas9...Systems.  Listen to the anti-CRISPR podcast segment Simply expressing anti-CRISPR proteins does not provide...that post-UV exposure it showed cleavage activity similar to the wild-type Cas9. This photocaging technique...Posts Read Our Primer on Optogenetics by Derek P. Simon Read Our Interview with Ed Boyden Resources on ...

...than following it. The Cpf1 crRNA is also much simpler in structure than Cas9’s; only a short stem-loop...found two that display robust cleavage activity similar to that of Cas9. These two nucleases, AsCpf1 and...respectively), both cleave in a staggered pattern similar to FnCpf1. Potential advantages of Cpf1 over Cas9...CRISPR systems based on Cas9 were thought to be the simplest CRISPR systems and the easiest to adapt to genome...et al. suggest that AsCpf1’s off-target rate is similar to that of high fidelity Cas9s eSpCas9 and SpCas9...

...2012). This concept is a little more complex than simply competing for replication control factors and tends... daughter cell during cell division. They don’t simply rely on chance (or random diffusion) to make this...plasmids, incompatibility due to partitioning is similar to incompatibility due to having the same replication...some types low copy plasmids during cell division simply does not allow for enough time for correct partitioning... of bacterial incompatibility groups based on similarity of replication and partitioning systems where...

..., which is not well protected by Cas9. This is similar to the proposed mechanism for engineered pegRNAs...related voltage indicator Ace2N-mNeon also led to similar improvements in performance.      Figure...10.1016/j.neuron.2023.03.009. PMID: 37015225.  Simple and highly specific targeting of microglia with...combination with miR124T targeting sequences, provides a simple yet valuable tool for microglia research and broader...AAV plasmids here! Serrano, C., et al. (2023). Simple and Highly Specific Targeting of Resident Microglia...

... can be used to generate multiple mutations simultaneously (Jiang et al. 2013). Genome editing tools for...small insertions. Multiplexing of two targets simultaneously is possible with efficiency >70%. CRMAGE is...et al., 2021). This pEcCas/pEcgRNA system works similar to the original pCas/pTargetF but has many other...pCRISPomyces-2 contains Cas9 and a gRNA cassette. The simpler system of pCRISPomyces-2 displays a higher editing...recombinogenic than E. coli, so this system functions more similarly to CRISPR/Cas9 systems adapted for eukaryotes ...knockdown expression of multiple target transcripts simultaneously. Marraffini lab's CRISPRi/a plasmids In addition...

...that you need. The Golden GATEway cloning kit simplifies the cloning process for complex DNA constructs...Grusch et al. demonstrate that Opto-RTKs have a similar level of background activity and activation as ...integrating DNA sequences either sequentially or simultaneously. This method, called by the authors the “Clonetegration...terminator). Next, up to six Level 1 modules can be similarly assembled into a Level 2 vector, thus forming ...repeat-variable di-residue (RVD) module plasmids and simultaneously increases the success rate of module assembly...in place of Brainbow’s Cre/loxP, allowing for simultaneous use of the two systems in different tissues ...the same transgene as the Brainbow cassette, simplifying experiments when additional cross-breeding steps...

...context, they should be expressed at similar levels and under similar conditions as their wild-type counterparts... the fluorophore is pH-dependent. Signal is simultaneously acquired from the the fluorescent and luminescent...now take any open reading frame (ORF) and with a simple recombination reaction, generate a protein with...(MuLE) system from the laboratory of Ian Frew simplifies the process of creating complex polycistronic...tamoxifen-inducible CreERT2 gene deletion, (3) simultaneous expression of cDNAs and shRNAs with a reporter...imaging of multiple endogenous genomic loci simultaneously in live cells. This system allows the study...mCherry), the authors were able to visualize simultaneously several parts of the genome and to determine...

...250 kDa Similar in size 8% 20–250 kDa Similar in size 10% 10–100 kDa Similar in size ...conjugated to primary antibodies). Direct westerns are simpler and faster than indirect ones and can be used in...visualize proteins on a PVDF membrane without staining; simply wet your membrane with 20% methanol and place it...proprietary systems are fast (often <10 minutes) and simple, utilizing individually packaged membrane ‘sandwiches...

...generation simpler, as described in an earlier post on generating CRISPR mouse models. For simple knockout.... It’s also made targeted insertions relatively simple in C. elegans and bacteria. But CRISPRing typical...microinjection, the efficiency of Easi-CRISPR for simple knock-ins and floxed models varies from 8.5-100%...Easi-CRISPR have also recently developed i-GONAD as a simple method for delivery of genome editing components...

...molecules in molecular biology labs (for a review, see Simpson et al., 2015). Visit Addgene's MIcrobiology Pages...following is a description of a technique published by Simpson, Sacher, and Szymanski in January 2016 in Viruses...to remove unbound cells. To detect bound cells, simply lay each membrane on an agar plate selective for...27536293. PubMed Central PMCID: PMC4971087. 3. Simpson, David J., Jessica C. Sacher, and Christine M. ...Chemical Reviews (2016). PubMed PMID: 27319741. 3. Simpson, David J., Jessica C. Sacher, and Christine M. ...

... With this technique, multiple genes can be simultaneously silenced if a consensus sequence is used (~...micro RNAs (amiRNAs) are frequently used. Although similar, amiRNAs are engineered from endogenous precursors...respectively. Short hairpin RNA (shRNA) is conceptually similar to the microRNA precursor mentioned above and is... tissue for downstream characterization. To simultaneously address both limitations of the traditional...Artificial MicroRNAs for Multiplex Gene Silencing and Simplified Transgenic Screen. Plant Physiol 178:989–1001...

...SciFi saw the rise of literary giants such as Isaac Asimov, Arthur C. Clarke, James Blish, and John W. Campbell...strung together, it can read many base pairs simultaneously to increase specificity. This modular approach...follow a DNA-binding and DNA-cleaving strategy similar to the ZFNs, but instead of recognizing nucleotide...base editor (ABE) enzyme in 2017. ABEs rely on a similar strategy as CBE but use a ssDNA adenosine deaminase...research focuses on the use of molecular dynamics simulations and machine learning to develop a predictive ...

...your antibody can distinguish between two very similar proteins. Additionally, they require variable expression...reduced or eliminated antibody signal. If you see a similar antibody signal between your control and your knock-out...protein expression experimentally increased. The simplest version would be to use cells that do not express...you more confidence when both antibodies exhibit similar results. Unfortunately, it can be difficult to ...antibodies to your protein of interest give you very similar results, while the third looks entirely different...

... being inserted into new target sites. This is similar to how retroviruses, like HIV, replicate. Class...can be inserted between the transposon’s LTRs, similar to when packaging viral vectors.   Let’s take a...vertebrates and integrates in human cells at rates similar to retroviral vectors. The hyperactive version ...piggyBac has an integration efficiency in human cells similar to retroviral vectors. The hyperactive PB transposase...with a maximum cargo capacity of ~200 kB of DNA. Similar to piggyBac, Tol2 also prefers to integrate at ...