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...of this pathway and does not consider the missing or damaged nucleotides that are common to biological... in Mammalian Cell Lines [Video] Pooled CRISPR Libraries Offer Genome-Wide Control for Large-Scale Functional...less heterogeneous when constrained by sequence identities in flanking sequence (“microhomologies”). NHEJ...the Cas9 DSB (blunt or near-blunt ends without nucleotide damage) such products are rare, probably accounting...of the canonical Cas9 approach. A pair of CRISPR guides that flank regions of hundreds of base pairs or... by NHEJ-dependent repair (“pop-in” insertion) provided a template containing compatible overhangs is ..., Zhang Y, Zhang F (2013) Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome Editing Specificity...

...keep up on all of the latest protocol videos, lab tips, and more video content from Addgene, subscribe to...viral genome sequencing strategy to confirm the identity and purity of all AAV viral lots. Aligning with...community. Here are a few virus-related protocol videos that may help to save you time and improve your... by iodixanol gradient ultracentrifugation This video goes over AAV vector purification using an iodixanol...critical tool for many scientists. Mammalian cells provide scientists with the means to study biological processes...If you’re new to tissue culture, check out this video for some tips and best practices for getting started...the Addgene blog Learn about Addgene's Lab Tips video series Resources on Addgene.org Find lab protocols...

...imbalance of electrical charge between the inside and the outside of the cell. This imbalance is maintained...Hyperpolarizing chloride channel SwiChr++ - A channelrhodopsin converted into a chloride-conducting anion...Addgene Optogenetics guide Primer on Optogenetics blog series Addgene Biosensors guide Other resources ...alter cell types and patterning There is mounting evidence that signaling via ion channels and even between... also affect patterning and in some cases even override genetically encoded patterning. Based upon trends...Planarian worms cut into three segments “remember” which side should grow a head and a tail, and when gap junction-mediated...encoded fluorescent voltage indicators (GEVI) are ideal tools to track bioelectric states (Matthew et al...

...increase confidence become more important. For many difficult-to-study protein targets, HiBiT provides an excellent...that are low-throughput, semi-quantitative, and provide only snapshots rather than continuous views of ...often used with knock-in models, ectopic models provide researchers with a means to quickly iterate, work...introduce mutations. This vector-based use of HiBiT provides a means to quickly answer research questions before...tools can also be combined with the HiBiT tag to provide complementary validation of protein expression ...research Researchers can incorporate HiBiT with a wide range of workflows, from classic overexpression ...utilized HiBiT to explore TP53 biology. They first identified that in a set of patients with gastric cancer...

...significantly reduce the risk of an accident (the most common reason for lab accidents is not following safety protocols...article we will discuss general viral safety considerations and best practices for handling lab-grown viruses...replicate and continue the infection cycle once inside the host. While the virus can still enter (and ...and safety department if they are not currently provided. Eye protection or face shields are required ...contamination. As most centrifugation has to be done outside of a biosafety cabinet, you’ll need to take extra...equipment are one of the most common forms of lab accidents, so extra precaution should be used to reduce ...than cell cultures do, and tired researchers are accident-prone researchers. Note that safety policies ...

...selection of guideRNAs. Of particular note, ABE8s reported in this work did not cause genome-wide, guide-independent...human cells (Gaudelli et al., 2020). One of the widely used base editing systems, ABE7.10 (and the starting...catalytically impaired Cas protein (dCas or Cas nickase)  a guide RNA that targets the complex of TadA and dCas to... Gaudelli et al., 2020. Directed evolution identifies adenine base editors with improved editing in...the “x” is the placeholder for the number that identifies the mutation in the evolved TadA domain. Because...ABE8.20-m/d) to evaluate further.  The creation of accidental insertions and deletions (indel) by the catalytically...motif (PAM) made up of 5′-NGG-3′ (“N” can be any nucleotide base, followed by 2 guanines). This limits the...

... E. coli genome-wide inhibition library To expand the tools available for genome-wide studies in E. coli...tools for your research! This time: E. coli genome-wide CRISPR inhibition Covalent tethering of DNA template... Nature Communications Find the E. coli genome-wide CRISPR inhibition library Covalent tethering of...enhance HDR repair using a single-stranded oligodeoxynucleotide HDR template covalently attached to the ...base editor (CBE) with rat APOBEC1 causes genome-wide deamination of RNA cytosines in human cells. Thus...Nme2Cas9 with gRNA cassette Erik Sontheimer’s lab identified a Cas9 protein from Neisseria meningitidis, Nme2Cas9... Nme2Cas9, that recognizes a simple dinucleotide PAM (N4CC), lending to high target site density (average...

... event’s success is evidence that, to share science, all you need is a great idea and a group of people...one-day conference aimed at the general public that provides an accessible introduction to various scientific... engage with the public. The organization runs a wide variety of programs, including fall and spring lecture...science cafes, and Reddit AMAs. Their website also provides a variety of short- and long-form written content...year graduate student at Harvard, who wanted to provide a conference-style experience that was accessible...SITN’s strength in the biomedical sciences and decided to offer a varied format event to keep the audience...improve our communication and organizational skills provide great training for a number of alternative careers...

...post has provided some insight on these differences in viral production and will help you decide which is.... In this post, we will compare two of the most widely used systems, mammalian and insect cells. Whether...cells are also easy to cultivate, fast-growing, and widely available. The viruses Lentiviruses, AAV, and adenoviruses...lentivirus production). The packaging plasmids are widely available, so you’ll only have to worry about cloning..., they are easy to cultivate, fast-growing, and widely available. Unlike mammalian cells, they do not ... (about three to four days), this second round provides the added benefit of producing a stock P1 virus... your viral supernatant. Baculovirus replicates inside insect cells, and its lifecycle ends in cell lysis...

...important factor to consider is your desired library coverage, since this will help decide what transformation...and how will you ensure this?). You should also consider if there are any features of the library backbone...difficult to transform.    Figure 1: Factors to consider when creating a pooled library amplification protocol...amplification protocol is the Chong Zhang E. coli Genome-wide Inhibition Library. DNA and cell amounts After ... work! Selecting your media Lastly, you need to decide between growing your library in liquid media or...similar in size and composition as examples. Table 1 provides an overview of pooled library protocols you can...DOCX, 15 KB) CRISPR Activation Human Genome-wide CRISPRa-v2 Libraries 104,540–209,080 20 bp ...

... tumors, and moreso than other methods, the identification and isolation of rare cell types. In recent...which spectral flow cytometry requires special considerations. Conventional flow cytometry During conventional...flow cytometry, light emitted by a fluorophore is guided to a single detector using a series of mirrors ...Instead of using a single (primary) detector to identify a fluorophore, spectral flow cytometers use all...thought of as a series of histograms flipped onto its side and compressed into a heat map. BV421 peaks in V1...cytometry for a given experiment, it is important to consider how many parameters you want to use in a given...and spectral flow cytometry, it is important to consider the expression levels of the markers you are including...

... or ddPCR is a method that provides ultrasensitive and absolute nucleotide concentration, unlike qPCR ...withstand changes in temperature. Taq was first identified in the late 1960s during research at hot springs...addition to Taq DNA polymerase, PCR requires free nucleotides (dNTPs), template DNA to amplify from and unique...polymerases require an existing strand of DNA to add nucleotides to.  Using these reagents and a series of heating...temperature is then raised slightly to Taq polymerase’s ideal temperature (70-75oC). At this temperature Taq polymerase...procedure check out our PCR protocol page, protocol video and reference on how to design PCR primers.  Types...PCR is used commonly in disease or pathogen identification. Scientists can simultaneously detect several...

...lab provide expression selectively in Parvalbumin containing cortical interneurons. We now provide ready-to-use...through our hot plasmids articles. These articles provide brief summaries of recent plasmid deposits and ...cell’s surface. The Bloom lab used the library to identify RBD mutations which do not interfere with ACE2....1074/jbc.RA120.01289     Novel protein cage provides simple enzyme protection in yeast bioproduction...enzymes in a reaction pathway, the MPyV system provides a simple, efficient method for protecting a single...- Ampl133 targets S. cerevisiae genes with 6-12 guides per gene where gRNA expression is induced by anhydrous...anhydrous tetracycline. Three new anti-CRISPRs were identified that inhibit the S. aureus Cas9, a Cas protein...

...here to help with that! Four important factors to consider for any experiment are: Speed Cost Difficulty...For cloning in particular, another important consideration is: The number of inserts ...as some methods...versatile Type IIS restriction enzymes. These cut outside their restriction sites allowing you to easily ...little as 30 minutes! Alternatively, you might consider Gibson cloning. In this technique, you first perform...order determined by their homology. You might also consider FastCloning, first described by Chaokun Li and...Finally, TOPO cloning is another good option to consider when time is of the essence. This method requires...-generated insert. When mixed, topoisomerase I provides both the endonuclease and ligase function while...

...homologous recombination (HR). Adapted from an image provided by Manuel Gonçalves; made with BioRender. ...vectors As compared to other viral vectors, AdVs can provide a large transgene packaging capacity. For instance...chromosomal region. The CRISPR/Cas9-derived RNA-guided nuclease-induced gene disruption frequencies in...in corrective genes. For this you would need to provide a DNA template to repair the double strand break...Nature Methods the same year that delivering RNA-guided nucleases or TALENs together with AdV donor DNA...demonstrated by Manuel Gonçalves and his colleagues at Leiden University Medical Center are now available at ... Dystrophy, Alpha1-antitrypsin deficiency (most widely used so far) or for cancer gene therapy such as...

.... Amp is a semisynthetic derivative of the more widely known antibiotic, penicillin, and is a beta-lactam...buys you a little more time, since bacteria won’t divide immediately after it has been heat-shocked or electrically... it by producing a lot of beta-lactamase. Pros: Widely available, cost-effective, timesaving for transformations...Expensive.   Kanamycin Kanamycin, or kan, is an aminoglycoside which inhibits protein synthesis. The kan resistance...The NPTII gene confers resistance to several aminoglycosides, including G418 which is commonly used for ...recovery period than amp or carb (60 minutes). Pros: Widely available, cost-effective, confers mammalian resistance... with all bacteria used in research, it isn’t a widely popular selection agent. Pros: Stable (spectinomycin...

... Addgene's Lentiviral Guide Most popular lentiviral plasmids γ-Retrovirus Guide References Cullen, B....retroviruses have been developed into some of the most widely used tools in molecular biology. “Retroviruses”...immune system directly). It may seem odder when you consider that out of the 6 x 106 virus-like particles that...DNA called a “provirus.” This DNA is synthesized inside of what is called the “target cell” (or host cell...reverse transcriptase are packaged (Wu et al., 1997) inside of the capsid . This is useful for viral reproduction... particle, some of the RT protein gets packaged inside of the vector particle, along with its RT-deficient...clock to the 6 o’clock positions at the left-hand side. These steps are common between natural infection...

... of regulation of genome functions, we need to identify molecules associated with specific genomic regions... genomic regions can be easily isolated for identification of associated proteins, RNAs, and other genomic... retroviral vectors expressing tagged dCas9 and guide RNA. The retroviral enChIP system enables us to ...system will allow researchers to use enChIP in a wide variety of biological systems. We are currently ...addition, we have recently developed the oligoribonucleotide (ORN) interference-PCR (ORNi-PCR) method ...shipping. Addgene realizes the One-Stop shopping for a wide range of plasmids. The merit of depositing plasmids...community is a kind of duty for researchers when considering the peer-review system of journals and grant ...

... While not ideal, transient expression of viral constructs is a useful tool as it provides a means to ...the specific target cell or tissue needs to be considered. Q2: Can I use lentiviral transfer vectors for... for the HIV Tat protein and consequently are considered safer. Second-generation transfer plasmids MUST...of lentiviral vectors in producer cell lines. Identifying less toxic envelope proteins or envelope proteins...to produce viral particles. The components are divided as follows: Transfer vectors contain minimal cis-acting...those three genes plus tat. The envelope plasmid provides a heterologous envelope for pseudotyping and is...to lower the risk. Biosafety should always be considered with respect to the specific experiments being...

... understanding of CRISPR, and perhaps provide insights and ideas for your own research! Classification... abundant of the two, comprising about 90% of identified CRISPR-Cas in bacteria and nearly 100% in archaea...transposases, by leaving out Cas3. Type I is further subdivided into subtypes A–G. These subtypes have similar...Schematic of a Cascade complex (blue subunits) with a guide RNA (green and orange) bound to the target DNA with...with BioRender.com.   Type III Type III is considered the most complex of the CRISPR types and is thought...et al., 2021). IV-A and IV-B lack what you may consider to be an integral component of CRISPR — a nuclease...engineered Cas enzyme, with adaptations for increased fidelity, activation/repression, high precision edits, ...