...table below) have different PAM requirements. These different PAM requirements allow researchers to target...encodes a single crRNA. The direct repeat regions contain sequences required for processing pre-crRNA into...variants are being discovered all the time and the requirements of these different systems can vary (for example...processing foreign DNA as well as the RNA guides required for endonuclease function, when used for genome...genome engineering, the only CRISPR protein required is the Cas9 endonuclease or a variant thereof. This ...contains both the 20 nucleotide target sequence to direct Cas9 to a specific genomic locus and the scaffolding...researchers simply need to express a gRNA designed to direct Cas9 to their target sequence of choice (see tips...

...Cas9s with looser PAM requirements compared to SpCas9. xCas9 has a PAM requirement of NG, GAA, or GAT, ...A-to-G.) In short, the impaired Cas9 localizes the deaminase to where the edit is desired, and it performs ...with a variety of impaired Cas9 proteins (such as SpRY) with altered PAM requirements, giving it more flexibility... al., 2019), it does not require an expertly positioned PAM without undesired bases nearby that may undergo...Site-specific deletions and knockouts Some experiments may require the introduction of a mutation at a specific site...alternative PAM Cas9s can be found in “The PAM Requirement and Expanding CRISPR Beyond SpCas9.” Single base...flexibility. A consideration with this tool is that the desired edit will be performed on all relevant bases within...

...: dCas9-targeted point mutations for directed evolution Directed evolution is an important method for ... function, strength, and binding (e.g. DHS-seq, FAIRE-seq, and CHIP-seq), genome-wide identification and...pooled libraries enable researchers to screen the entire genome for genes that regulate a wide variety of...the NCC-1 library of approved drugs against the entire kit; a parallel analysis that successfully identified...Researchers can use plasmids from the PRESTO-TANGO kit directly for drug screening or easily shuttle the optimized...optimized, expression validated GPCR sequences to any desired backbone.  Kroeze, et al. Nat Struct Mol Biol....oligonucleotides (oligos), and thereby rapidly create desired allele combinations and combinatorial genomic libraries...

...familiar with when talking about IF, direct and indirect. For direct IF, researchers use a single antibody...allowing one to “directly” visualize their target. In contrast, indirect IF requires two antibodies: a...post to learn more about these techniques!  Direct vs. indirect immunofluorescence There are a couple different...Figure 1: Comparison between how direct immunofluorescence and indirect immunofluorescence work. Image ...with BioRender.com   Indirect IF is a much more common approach than direct IF, so we will focus on... a primary and secondary. For indirect IF, the primary antibody targets the antigen of interest but is...on this technique here. One of the reasons that indirect IF is more common is that secondary antibodies...

...flow cytometry can use both direct and indirect staining methods. In direct staining, the primary antibody...binds to the primary antibody. Direct staining is quicker than indirect staining and eliminates potential...assessed for the desired parameter. A droplet containing a cell that is positive for a desired parameter is...with protocols that require fixation and permeabilization as these methods require damaging the cell membrane...as it passes through and scatters in different directions. The degree of scatter is captured by detectors...about the cell. Light that continues in the same direction as it was initially traveling is called forward...against a target is conjugated to a fluorochrome. In indirect staining a primary antibody binds to a target ...

... live on set, but peering at a 7-inch wireless monitor. Wireless video technology has gotten very impressive...   Figure 4: The remote directing setup, complete with wireless video receiver, camera control,... we decided to focus on a protocol that didn’t require any lab shooting, just animation. The plan was ...to shoot everything from her home, while I would direct “virtually” via a webcam. In the office, we use...this a lot easier and I didn’t need to be there directing. Everyone’s schedules were busy, so it was important...playing over the voice-over in videos), I sent some directions and (rules) to some volunteers, who filmed with... that I wanted to be able to talk to people and direct them. For interviews, being able to ask follow ...

...terminator sequence), since this would directly compete with the required single-stranded annealing and priming...modular overlap-directed assembly with linkers) which brings modularity to overlap-directed methods (Casini...they can all work at the same temperature, so the entire reaction takes an hour or less to complete at 50... resulting in scar-free assembly that does not require the presence of specific restriction sites (or ...sequentially.   How does Gibson assembly work? The required homology between neighboring fragments can be ...be because the exonuclease could chew through an entire fragment shorter than 200 nucleotides before the...techniques described above, this resulted in the direct and seamless cloning into a vector where no other...

... neurons projects axons directly to the prefrontal cortex, but also indirectly via synapses to neurons...dyes usually spread unidirectionally, there are some reports of bi-directionality. For predominately anterograde...specifically AAV6 in the retrograde direction and AAV2 in the anterograde direction (Salegio et al., 2013). Zingg...E.M., Mu, D., Hudry, E., Hyman, B.T., Maguire, C.A. and Ramirez S.H. (2017). Trafficking of adenoassociated...autobiographical events (see Jin & Maren, 2015). Directional flow of information between different parts of...for tracing compounds to outline neurons in the direction of information flow in order to understand which...retrograde tracing outlines neurons in the opposite direction, from the terminals of their axons to their cell...

...experiments can require steps such as protein expression and purification, which will require additional laboratory...being developed. Performing a CRISPR experiment requires delivery of, at minimum, two components: the Cas9...occur due to prolonged Cas9 expression     The requirement for Cas9 transcription and translation delays...which avoids many of these complications, is to directly deliver a ribonucleoprotein (RNP), consisting ...variety of cell types, gene editing using homology directed repair (HDR), and generating large genomic deletions...are not expressed. Because this method does not require the delivery of foreign DNA, and the Cas9-gRNA ...applications where limited expression of Cas9 is required and specificity is a concern, such as knockout...

... endeavor, NEBuilder requires upfront planning. This cloning methodology requires that the fragments to...multi-position site-directed mutagenesis by assembling multiple fragments containing the desired mutations. This...homology and any desired edits to the target region. Assembled plasmids can be directly transformed into...from New England Biolabs® has minimal upfront requirements and allows you to expediently join multiple ...DNA assembly, and allowing fragments to be taken directly from restriction enzyme digests to DNA assembly...plasmids via restriction enzyme digestion, and used directly in DNA assembly will still yield scarless assemblies...plasmids by gene here), designing primers with your desired mutation, performing PCR to generate the mutation...

...components are required? CasPEDIA provides information on crRNA and tracrRNA requirements, along with any... any other required non-CRISPR-associated host factors required for CRISPR processing. For each enzyme...classification system, CasID, to help facilitate direct comparison of different enzymes. CasPEDIA is meant... properties (CasID): Cis/trans activity, PAM requirement, gRNA, and multiplex ability Experimental guidelines...a candidate Cas for your experiment. Target requirements As we all know, a Cas guided by a gRNA is useless...) in some cases. The resource displays these requirements in regard to their sequence (NGG, etc.) as well... gRNA (5’ or 3’). In some cases, there is no requirement, which is also documented. Experimental design...

...crRNA/gRNA length gRNA: ~100 nt crNA: ~42 nt Requires tracrRNA? Yes No dsDNA cleavage Blunt end 5'... work with a transfectable cell line. 2) They require larger expression vectors which are often more ...Cas9 multiplexing vectors are larger because they require regulatory sequences to allow for expression of...vector expresses Cas9 and up to 7 gRNAs Each gRNA requires its own promoter Sakuma et al   Multiplexed...expresses Cas9, eGFP, and up to 4 gRNAs Each gRNA requires its own promoter   Kabadi et al Csy4-Cleavable...gRNAs expressed from a polycistronic transcript  Requires co-transfection of Cas9 Nissim et alTsai et al...gRNAs expressed from a polycistronic transcript Requires co-transfection of Cas9   Xie et al  Multiplexing...

...biallelic modifications. Most commonly, DSBs are repaired by the nonhomologous end-joining (NHEJ) pathway...sequence change the cell must undergo homology-directed repair (HDR), a distinct cellular DNA repair pathway... is relatively rare and DSBs are predominantly repaired by NHEJ. While other groups have focused on strategies...overall HDR rates, we have shown recently that desired genome-editing events by HDR can be generated more...introduced. Although we were able to introduce the desired mutations in genes associated with dementia, such...CRISPR from re-cutting the target sequence once the desired edit has been introduced. We found that blocking... or guide RNA blocking mutations? This depends entirely on the locus you are editing, and if the blocking...

... in the direct repeat region is necessary for cleavage of a target. Cpf1 also does not require an additional... Since Cpf1 doesn’t require a tracrRNA, crRNA guides are only ~42 nt long. Direct synthesis of these crRNAs...domain. Cpf1 cleaves DNA in a staggered pattern and requires only one RNA rather than the two (tracrRNA and...systems, including the type II Cas9-based system, require a single-component nuclease to mediate cleavage...blunt double stranded cleavage and G-rich PAM requirement. Zetsche et al. started from square one to characterize...depletion assay to discover FnCpf1’s PAM sequence requirements. Cpf1’s preferred PAM is 5’-TTN, differing from...length, about the same size as Cas9’s, but with the direct repeat preceding the spacer rather than following...

...reverse transcriptase domain copies the desired edit, directly polymerizing new DNA onto the nicked target... and more efficient than homology-directed repair Homology-directed repair (HDR) stimulated by double ...both specifies the target site and contains the desired edit (or edits) engages the prime editor protein...the pegRNA — a guide RNA that also encodes the desired edit and homology to the genomic DNA locus. After...base-to-base conversion in principle, but some changes require multiple steps and enzymes. Prime editing can encode...relatively low, meaning that most Cas9-induced DSBs are repaired by non-homologous end joining. As a result, Cas9...still almost always results in higher ratios of desired edits to indel byproducts than Cas9-initiated HDR...

...topics we covered in this post. Besides direct labeling, indirect labeling can be used when suitable fluorophore-conjugated...label, where fluorophore-conjugated antibodies directly bind to epitopes of interest that are found in...through the cell membrane freely, while others require a permeabilized membrane to enter cells. Depending...and 'staining' to refer to small molecule dyes. Indirect labeling While antibodies used for target marker...western blotting or ELISAs.  A popular method for indirect labeling employs a primary epitope-targeting antibody...biotin plasmids at Addgene!     Figure 1: Indirect detection of target markers can be achieved through...Created using biorender.com. Another way of indirect labeling is through antibody host species reactivity...

...which leads to the desired mutation and excision of the vector backbone. This step requires “counter-selection...amplicons upstream and downstream of the desired deletion directly into cut vector with excellent efficiency...scarless mutations, ones that don’t leave any undesired scars in the chromosome. These methods roughly... powerful, but they rely on exogenous enzymes, require multiple transformation and plasmid curing steps... recombination enzymes can also accomplish the desired genomic modification. This technique is frequently...mutations. Ultimately, successful allelic exchange requires two sequential homologous recombination events...events, but because these events are rare, each requires selection. These two steps are (Figure 1): The ...

...usage. Fast facts – PAM requirement: TTN or TTTN. Best for: homology-directed repair or therapeutic delivery...on choosing a Cas9 enzyme. Fast facts – PAM requirement: NGG and an expanding number of others. Best...proceed up to multiple kilobases, but only in one direction. Cas3 is thus not helpful for designer genome ...application potential as a result. Fast facts – PAM requirement: AAG or TTC  Best for: very large deletions. ...single strand DNA generation, such as homology directed-repair. Cas12a is also conducive for targeting...targeting AT-rich genomic loci as the PAM does not require any G’s or C’s. One curious feature of this Cas is...enzyme (about a third of the size) and does not require a tracrRNA (it can self-process pre-crRNA into ...

...lets you fire neurons with the flick of a light switch. Neurons are not typically persuaded to fire when ...and neurons don't fire. With blue light, ions flow into the neuron and causes it to fire. Created with BioRender.com...need high-intensity light for activation which requires implantation of fiber-optic cables into the brain...-throughput proteins engineering methods, like directed evolution, because channelrhodopsins are typically...them construct “designer” channelrhodopsins with desired characteristics. The model was trained to predict...and/or ChR2(H134R). Designer channelrhodopsins paired with a systemic AAV allow for minimally-invasive...express as many copies of the ChRs as compared to direct injection. This lower multiplicity of infection...

...are always required and lab coats are either recommended or required in BSL-1 and required in BSL-2 labs...Lentivirus (a type of retrovirus) and gamma retrovirus require more caution as they are integrating viruses – ...need to be handled in a BSL-2 (or BSL-2+) lab and require additional safety measures.   General viral handling...When working with lentivirus, many organizations require or recommend disposable gowns over your lab coats... provided. Eye protection or face shields are required whenever there’s a splash or spray risk in BSL...BSL-1. In BSL-2 labs, eye protection is required any time you have to work with BSL-2 materials, such as...working in a biosafety cabinet, even if it is not required by their organization. It’s always okay to add...

... a lot of the “soft skills” required for teamwork may not be a requirement. Your manager: Unlike academia...Many companies will hire a postdoc to perform a specific project, while others will hire a group of postdocs...working as a team to develop therapies that could directly impact patients. And, to be honest, I’ve never...be motivated to get your project in a workable direction quickly. Of course, you might have less time to...industry experience” that I’ve seen listed as a requirement on some industry job postings. Also, if you work...academic setting—especially if job applications require teaching experience or high numbers of publications...publications, which may take a longer time to acquire. Learning about drug discovery: Unless your academic lab...

...producing firefly luciferase. Firefly luciferase can readily be measured to quantify (indirectly) transcription...minigenome systems, such as the one developed for Zaire ebola virus (EBOV) are extremely useful, allowing...contains the 3’ and 5’ ends of the genome which are required for replication and transcription (Figure 1). ...replaced by a single (non-viral) reporter gene (e.g. firefly luciferase or eGFP) flanked by the EBOV-specific...minigenome contains all cis-acting signals necessary to direct minigenome replication and reporter gene transcription...reviewed in 2, 3). Other important components of the Zaire ebola virus minigenome system Similar to other RNA...transcribes viral genes. Four viral proteins are required for viral genome replication and transcription...

...start by looking at the requirements in the job description. For each requirement, ask yourself if you have...the job requirements, strengthened an application. Even so, there were examples of great hires who hadn...Similarly, entry-level jobs are less likely to require cover letters, while higher-level jobs are more...submitted for their entry-level jobs and does not require or request them for these positions. (They are ...how your skills and experience match the job requirements, by giving specific examples of your work and.... Let’s imagine you’re applying to a job that requires ddPCR skills. Your resume lists ddPCR, so the ... explain how your experience matches the job requirements, and address any unusual circumstances or proactively...

...spreading through the neuroscience community like wildfire and for good reason. For the first time in the...spent two months in the lab of Antonello Bonci, Director of the National Institutes of Drug Abuse Intramural...m not an electrophysiologist so I cannot speak directly on this topic but, in numerous studies, optogenetics...optogenetics is used in slice physiology in order to directly show that, for example, the firing of a specific...appropriate test.  Of course, proper controls are required and include, for example, confirmation of opsin...optogenetics experiment and what are the tools required for each one? Behavioral Optogenetics can be broken... the optogenetic constructs Expression of your desired optogenetic construct in the brain region and cell...

... Set up a table of contents in the top directory: A directory is a file system in the computer and is...including background on other analyses that have inspired you to perform this analysis. Having a clearly...name for a folder. For instance, your Desktop directory contains the folders and files that you see on...on your desktop. The location of your master directory or folder for all of your analyses is up to you... (see above). Give every experiment its own directory: In addition to output files, this is also where...README.txt document is invaluable. In the top directory of every experiment, immediately write a text ...text document with a brief description of the directory contents, at a minimum. This may or may not be where...

...appropriately edited for your desired mutation - insertion, deletion, or site-directed knock-in. We’ll also discuss...know a few things about your target cells and the desired edits. First, you will need to know what the ploidy...knockout mutations When a knockout cell line is desired, any mutation that introduces a frameshift in the...gRNA and Cas9 combo and are hoping for homology-directed repair events, or maybe you’re performing a single...the best screening method depends on what your desired edit is. Large knock-ins Are you introducing a...may be the way to go. Design a PCR to flank your desired edit site and run the PCR product on a gel to look...mutation in a neighboring nucleotide along with your desired edit. This silent mutation should be designed solely...

...demands of using these wireless devices are significantly higher than standard “wired” optogenetics but may...difficult portion of optogenetics (though it does require a significant amount of equipment, most of which...written outlining the different steps and materials required for each [1, 2]. This protocol [3] provides, in... in exquisite detail, the various steps required to produce your fiber optic implant. Companies such as...the ferrule implant must be maintained during the entire protocol, otherwise optical stimulation will be...primary one is that some behavioral experiments require stimulation only under certain contexts/locations...without a loss of laser power. More recently, wirelessly-controlled optogenetic implants have been reported...

...scientists should acquire during their PhD and postdoctoral training. Addgene executive director Joanne Kamens...were many changes to the  Board of Directors and the Executive Director changed twice. I had to learn how... to report to the Board of Directors and liaise with the Executive Director. As members changed, I had...official assessment and discussion with the Board of Directors, the Board officially voted me in as Chair of ...Different leadership styles Being an effective leader requires you to understand how to work with many different...format depending on the particular style and requirements of the new Board Members. In some instances .... Some members would need much more guidance, direction, and a clearly laid out plan of action, others...

... techniques have in common? They all typically require secondary antibodies, frequently of the mouse or...certainly aren’t “broken,” their production does require continued animal sacrifice. Could there be an alternative...Antibodies One of the main reasons antibodies are great indirect immunodetection tools is their strong signal strength...expressed in bacteria, which means their production requires animal sacrifice and precludes genetic engineering...one primary antibody. This process can be avoided direct antibody fluorophore conjugates are used, but these...dyes Commercially available Not recombinant; Require animal sacrifice Poor tissue penetration Less ...Less resolution due to larger label displacement Require separate incubation of 1° and 2° Must use different...

...Limited genomic integration Multiple transfections required Need to sequence to verify there’s no genomic ...Transgene-free and vector-free Multiple transfections required Protein Delivery DNA-Free Low High Transgene-free...they have several safety concerns. First, they require the use of potentially harmful viral particles ...integrating viral systems currently lack the safety required for translational use. Viral Vectors Retroviral...transposon insertion site, so sequence-verification is required. Non-integrating iPSC delivery methods Viral...promoters for transcriptional regulation, so it requires a different approach for regulating transgene ...Generating iPSCs with plasmid-based expression requires serial transfection of 1 or 2 plasmids that express...