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Showing: 261 - 280 of 902 results

  1. A New Generation of Adenine Base Editors Improves Editing in Primary Human Cells

    Type
    Blog Post
    Published
    May 7, 2020, 1:15 p.m.
    ...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...

  2. Viral Vectors 101: Virus Safety

    Type
    Blog Post
    Published
    June 22, 2023, 1:15 p.m.
    ...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 ...

  3. What's New in CRISPR - May 2019

    Type
    Blog Post
    Published
    May 28, 2019, 12:49 p.m.
    ... 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...

  4. Science Communication Snapshot: DayCon 2015

    Type
    Blog Post
    Published
    June 17, 2015, 9:04 p.m.
    ... 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...

  5. Plasmids 101: 5 factors to help you choose the right cloning method

    Type
    Blog Post
    Published
    Aug. 21, 2018, 12:31 p.m.
    ...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...

  6. Adenoviral Delivery of CRISPR/Cas9 Aims to Expand Genome Editing to Primary Cells

    Type
    Blog Post
    Published
    Dec. 10, 2020, 1 p.m.
    ...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...

  7. Polymerase Chain Reaction Overview and Applications

    Type
    Blog Post
    Published
    Aug. 10, 2021, 1:15 p.m.
    ... 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...

  8. Hot Plasmids and Viral Preps - May 2021

    Type
    Blog Post
    Published
    May 18, 2021, 1:15 p.m.
    ...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...

  9. Production of Virus in Insect Versus Mammalian Cells

    Type
    Blog Post
    Published
    Nov. 5, 2024, 8:32 p.m.
    ...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...

  10. Antibodies 101: Conventional vs Spectral Flow Cytometry

    Type
    Blog Post
    Published
    Aug. 3, 2023, 1:15 p.m.
    ... 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...

  11. Pooled Library Amplifications

    Type
    Blog Post
    Published
    May 30, 2024, 1:30 p.m.
    ...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 ...

  12. Plasmids 101: Choosing an Antibiotic Resistance Gene

    Type
    Blog Post
    Published
    Oct. 10, 2023, 1 p.m.
    .... 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...

  13. Viral Vectors 101: The Retroviral Lifecycle

    Type
    Blog Post
    Published
    July 27, 2023, 1:15 p.m.
    ... 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...

  14. Interview: Hodaka Fujii on enChIP, New CRISPR Tools, and More

    Type
    Blog Post
    Published
    Dec. 2, 2014, 7:23 p.m.
    ... 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 ...

  15. Your Lentiviral Plasmid FAQs Answered

    Type
    Blog Post
    Published
    April 23, 2014, 1:08 p.m.
    ... 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...

  16. Typing CRISPR Systems

    Type
    Blog Post
    Published
    March 18, 2025, 1:15 p.m.
    ... 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, ...

  17. Finding nucleic acids with SHERLOCK and DETECTR

    Type
    Blog Post
    Published
    April 16, 2020, 1 p.m.
    ...UnLOCKING Cas13 (C2c2) was first identified in 2016 by the Zhang lab as an RNA guided RNase (Abudayyeh et al 2016...-related single nucleotide polymorphisms (SNPs) from human saliva. SHERLOCKv2 provides a method to detect... expensive, time-consuming, and complex to use outside of standard laboratories. In the case of the COVID... 2016). Cas13 can be guided by a single CRISPR RNA (crRNA) to cleave ssRNA or mRNA. It also exhibits a...based on DETECTR technology with the mission to “provide a CRISPR-based platform on which an infinite number...2016) C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector. Science 353:aaf5573... Koonin EV, Zhang F (2015) Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. Cell...

  18. Beyond the Blood Brain Barrier: PHP.B Enters the Central Nervous System Via the LY6A Receptor

    Type
    Blog Post
    Published
    July 23, 2019, 12:46 p.m.
    ...searched for this unique sequence of DNA and both identified lymphocyte antigen 6 complex (LY6A) as a receptor...library of AAV9 mutants that had a small randomized peptide loop inserted in the AAV9 capsid. This mutant library...then screened in vivo in C57BL/6J mice strains to identify variants that reached the CNS following IV injection...hematopoietic stem cells. Its ligand(s) has not been identified. While LY6A or other Ly6 gene family members ...susceptibility. The Deverman and Wilson Labs independently identified LY6A as a receptor for PHP.B by looking for a...total, they tested 13 strains, with seven strains identified as permissive and six as non-permissive to PHP.B...the PHP.B permissive phenotype of mouse strains beside C57BL/6J and BALB/cJ, and the effects of overexpressed...

  19. CRISPR 101: Which Cas9 Do I Choose for My CRISPR Experiment?

    Type
    Blog Post
    Published
    Oct. 13, 2022, 1:45 p.m.
    ...Any ∼20 nucleotides of DNA can serve as the target for Cas9’s single guide RNA (sgRNA), provided it meets...unique experiment. Here, we will introduce you to the wide array of Cas9s (many available through Addgene’s...Specificity concerns? If off-target effects are a worry, consider a dual-sgRNA system. This system uses a “nicking...little homology to other regions of the genome and consider using a Cas9 variant with higher specificity (...residue of a protein or knocking out a very small peptide. In these cases, you have little flexibility as...specific? Perhaps you need to introduce a single nucleotide modification at a defined amino acid to mimic...requirements, giving it more flexibility. A consideration with this tool is that the desired edit will...

  20. Plasmids 101: Broad Host Range Plasmids

    Type
    Blog Post
    Published
    April 11, 2024, 1:15 p.m.
    ...range, identify a few of the features that confer broad host range in plasmids in nature, and provide tips... meliloti, Rhodobacter sphaeroides, R. capsulatus, Rhodopseudomonas spheroides, Salmonella spp., Serratia...equally. Only a handful of plasmids have been identified as broad host range plasmids. The properties ...are not fully understood, but scientists have identified some of the features that confer broad host range...metabolic load (Meyer 2009). Figure 1: Idealized examples of a narrow host range plasmid with only...Porphyromonas gingivalis, Proteus mirabilis, Providencia spp., Pseudomonas spp., Rhizobium leguminosarum... M. xanthus, Proteus rettgeri, P. mirabilis, Providencia stuartii, Pseudomonas spp., Rhizobium leguminosarum...
Showing: 261 - 280 of 902 results