...transformed E. coli expressing Cas7-11, they found no evidence of growth inhibition when the system was retargeted... more they worked with it, the more they found evidence contradicting their initial assumption. Eugene... Type III-E, and then used this information to identify a large variety of related bacteria with similar...with lower knockdown efficiencies. The ability to identify related Cas systems opens up the potential of ...proteins and a putative Cas11-like protein, and a guide RNA. The enzyme has dual activities: processing ...CRISPR-driven precision into a genome, can be considered the fourth generation of CRISPR, one which drops...drops itself neatly into that gap. Consider, for instance, a disease like cystic fibrosis, which can be...

... past decades, neuroscientists have made great strides to map brain regions and allocate specific functions... modulating. Viral vectors, in contrast, are considerably more versatile and specific in how they manipulate...manipulate neuronal function, with far fewer side effects compared to lesioning methods -  they may be the...ve previously discussed some of the general considerations for performing behavioral tests, but now we...used since the 1970s to test the efficacy of antidepressant treatment in rodents. In this test, an animal...BDNF in the hippocampus causes a shift towards antidepressant behavior in rats in the forced swim test as...neurons (Drd1) in mPFC is responsible for the antidepressant effect. To specifically activate the Drd1 neurons...

..., with residence times on DNA of approximately 6 hours. To our surprise, we measured identical dissociation...With 36bp on the PAM-distal side of the break and 91bp on the PAM-proximal side, consistently supported ... Jacob Corn’s lab. CRISPR-Cas9 (Cas9) is an RNA-guided nuclease that targets and cuts genomic DNA. The...DNA post-cut. Using substrate DNA labeled on both sides of the break, we determined that Cas9 held tightly...introduces functional Cas9 protein and the targeting guide RNA into cells by electroporation. Results from ...PMC4383097. 3. Kim, Sojung, et al. "Highly efficient RNA-guided genome editing in human cells via delivery of purified...HDR Basics Learn How to Design Your gRNA Find the Ideal CRISPR Software for Your Needs Resources on Addgene.org...

...we’ll go through some of the most important considerations to make when planning and performing an IF ...try the Introduction to Fluorescence Microscopy video from iBiology). IHC and ICC are essentially just...antibodies is a time consuming and expensive process. Consider the vast number of possible primary antibodies...experiment The process to perform IF can be broadly divided into the several steps outlined below and in Figure...steps include: counterstaining, mounting sample on slides, and confocal   Figure 2: High-level overview...indirect immunofluorescence procedure with key considerations about each step. Image created with BioRender.com...’ll start there. Some of the most important considerations are to make sure your secondary 1) will recognize...

...in industry. The news is good. In their ideal form, ELNs provide users with a holistic means to manage ...will always be a part of everyday work to capture ideas, write down certain notes or brainstorm, but we ... able to understand the situation in depth and provide the solution that fits the most labs, our team ...of budget and ease of use of the software. We identified that when talking about ELN costs and limited... to require on-going maintenance and support.” Besides that, academic institutions tend to think about...intellectual property across various software providers used to supplement their paper notebooks. Instead...witnessing and audit trails. How does the ELN provider protect and respect your IP rights and data ownership...

...is generally error-free provided the DNA template used for repair is identical to the original DNA sequence...directed repair pathways HDR mechanisms can be divided into two main camps: non-conservative and conservative... many applications. Your favorite Cas enzyme, a guide RNA to your site of interest, and a donor DNA molecule...you need to generate a knock-in.   General considerations for designing a repair template to create mutations... modification should be very close to the DSB, ideally less than 10 bp away if possible. Check out our...ssDNA templates (referred to as ssODNs (oligodeoxynucleotides)) are commonly used for smaller modifications...cells than their plasmid counterparts. Another consideration for dsDNA templates is their ability to randomly...

...Cycle Quick Guide to All Things Lentivirus More resources on addgene.org Lentiviral Guide   Popular lentiviral...staple in molecular biology for over three decades. Widely used across various research applications, they..., for some applications, integration might be considered a double-edged sword with unintended consequences...detailed explanation, check out Addgene's Lentiviral Guide). To produce lentiviral vectors, the lentiviral ...AKA transduction in the viral vector world). Once inside the host cell, the viral payload is integrated ...especially concerning in clinical settings, as evidenced by retroviral gene therapy trials for X-linked...nucleic acid for only a limited time, IDLVs are ideal. As the episomes are lost during cell division, ...

...information, see the manual provided by the Weiderschain lab. Find Tet-pLKO-puro. lentiGuide-Puro - This plasmid...infect many different cell types so it is useful in a wide range of applications. This plasmid was the top ...sgRNA scaffold and cloning sites for inserting the guide sequence (pSpCas9(BB)). The 2A-GFP allows for screening...produces a ~10 fold higher titer virus, making it ideal for CRISPR pooled library screening experiments....Chimeric_BB-CBh-hSpCas9 - The S. pyogenes Cas9 (SpCas9) has been widely popular in genome engineering. For use in human...annealed oligos can be cloned to create a chimeric guide RNA. Find pX330-U6-Chimeric_BB-CBh-hSpCas9. pSpCas9...with cell lines already expressing Cas9. Find lentiGuide-Puro. pSpCas9n(BB)-2A-GFP (PX461) - Similar to...

... encoding multiple guides. While two guides require two arrays for Cas9, two guides can be streamlined...enAsCas12a), minimizing off-target effects by identifying guide selection rules, and reducing recombination...through our hot plasmids articles. These articles provide brief summaries of recent plasmid deposits and ...optimized genetic screening technique, researchers identified a synthetic lethal interaction between MARCH5...Cas12a-based screening method was used to create a genome-wide library capable of performing similarly to Cas9 ...target multiple genes within a single cassette provides increased simplicity for combinatorial genetic...cost-effective alternative to commercial ladders which also provides a useful yet low-pressure teaching opportunity...

...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...

...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...

...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 ...

... 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...

... 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...

... 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...