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We narrowed to 919 results for: IRE;

Showing: 121 - 160 of 919 results

  1. CRISPR References and Information

    Type
    Collection
    ...zebrafish. Developed by the Jason Berman Lab . CRISPRdirect (Link opens in a new window) Identifies candidate...

  2. Immunology Research Plasmids and Resources

    Type
    Collection
    ...reticuloendotheliosis viral oncogene homolog B I-REL, IREL RFX5 regulatory factor X, 5 (influences HLA class...HUMARA, HYSP1, KD, NR3C4, SBMA, SMAX1, TFM AREG amphiregulin AR, CRDGF, MGC13647, SDGF ARMET arginine-rich...MVCD2 EPOR erythropoietin receptor MGC138358 EREG epiregulin ER ESM1 endothelial cell-specific molecule 1 ...

  4. A Practical Guide to Optimizing AAV DIO and FLEx Vector Expression

    Type
    Blog Post
    .../hum.2019.277 Lavin TK, Jin L, Lea NE, Wickersham IR (2020) Monosynaptic Tracing Success Depends Critically...Many neuroscience experiments that require gene expression in a specific cell type rely on transgenic...lox2272, that are in the opposite orientation (direction of arrows) the inverted transgene is reverted ...promoters, such as CAG are very strong and may require less viral particles or relatively short expression...such as hSynapsin, are relatively weak and may require longer waiting periods and/or higher injection ...recombination rate (0.1% and 0.13% respectively) still required a 200-300 fold dilution to minimize off-target...Conclusion Using AAV vectors in your experiments requires a full understanding of vector performance in ...

  5. Antibody Validation Using the Indirect ELISA Method

    Type
    Protocol
    ...Validation Using the Indirect ELISA Method Antibody Validation Using the Indirect ELISA Method You may...This protocol describes how to run an indirect ELISA against a purified antigen to demonstrate the expected...Introduction This protocol describes how to run an indirect Enzyme-Linked ImmunoSorbent Assay (ELISA) against...antibody and an isotype control antibody to the desired concentrations in the antibody dilution buffer....HRP-conjugated isotype-specific secondary antibody to the desired concentrations in the antibody dilution buffer....Check the plate periodically to determine when the desired color change has occurred. The color will change... to optimize experimental conditions. When the desired color change has occurred, gently remove the plate...

  7. Chemogenetics Guide

    Type
    Guide
    ...window) Guettier JM, Gautam D, Scarselli M, de Azua IR, Li JH, et al. (2009). A chemical-genetic approach...through PSAMs and LMOs is direct, while neuronal control of DREADDs is indirect. DREADD ligands affect signaling... DREADDs in experiments in this species may be entirely due to back-metabolism of CNO to clozapine, demonstrating...that all respond to the same ligands was that bidirectional experiments (e.g. using both activating and ...hM3Dq can be used in the same organism to allow bidirectional control of neuronal activity. Table 1. Descriptions...Unlike DREADDs, which manipulate neuronal activity indirectly through GPCR signaling, another class of chemogenetic...chemogenetic receptors that confer more direct control of neurons through manipulation of ion channels arePharmacologically...

  8. Trimmer Lab NeuroMab Collection

    Type
    Collection
    ...window) together with the laboratory of NeuroMab Director, Dr. James Trimmer, have produced numerous monoclonal...

  9. CRISPR Guide

    Type
    Guide
    ...combination of wild type alleles, NHEJ-repaired alleles, and the desired HDR-edited allele. Therefore, it is...presence of the desired edit experimentally and to isolate clones containing the desired edit (see: Validate...) and the desired edits on a RT template. The PBS base pairs to the 3’ flap, and the desired edit is incorporated...transcription-activator-like effector nucleases (TALENs) required scientists to design and generate a new nuclease...the Cas enzyme. Basic Mechanisms of CRISPR The requirements for a CRISPR knockout assay are simple. You’...continue to anneal to the target DNA in a 3′ to 5′ direction. The location of any potential mismatches matters...of the PAM sequence. The resulting DSB is then repaired by one of two general repair pathways: the efficient...

  10. Plan Your Experiment

    Type
    Guide
    ...BioRender.com. Select Your Desired Genetic Manipulation Different genetic manipulations require different CRISPR...percentage of DNA breaks will be repaired by NHEJ instead of incorporating your desired edit through HDR. Base ...methods and often require screening a larger pool of cells to find one with your desired edit. Lastly, you...base edits Can be paired with restriction enzymes for further customization Usually paired with a sequencing...must be experimentally verified Cut Nick Homology Directed Repair (HDR) Generate specific genomic edits, ...or Cas9 fusions with proteins that promote HDR Requires a repair template Lower editing efficiency compared...Activate Design and Clone Your gRNA Achieving your desired edit comes down to location, location, location...

  11. Antibody Guide

    Type
    Guide
    ...antibodies? Will you be using a direct or indirect detection method? If indirect, do you already have secondary...signal amplification through the indirect detection method. In the direct detection method, the signaling...most common types of ELISAs are direct or sandwich ELISAs. For direct ELISAs, an antigen or protein of...antibody methods, it can be done using either an indirect or direct method. Since IF uses fluorophore color to...cytometry requires a negative control, a small subset of the sample with no antibodies. It also requires single-antibody...complex. Due to their increased manufacturing requirements, monoclonal antibodies are typically more expensive... Polyclonal antibodies are typically collected directly from animals and contain antibodies targeting ...

  12. Molecular Biology Reference

    Type
    Guide
    ...., a strong promoter directs high expression, whereas weaker promoters can direct low/endogenous expression... few hundred or few thousand basepairs from the entire human genome. Digesting the human genome with restriction...functionality. In their simplest form, plasmids require a bacterial origin of replication ( ori ), an antibiotic-resistance...Origin of Replication ( ori ) DNA sequence which directs initiation of plasmid replication (by bacteria)...mammalian promoter sequence. The promoter can also direct cell-specific expression, which can be achieved... and the inserted gene. The promoter region is required for the generation of RNA from the insert DNA ... regions of interest. In vitro DNA replication requires the 4 nucleotides, a DNA polymerase enzyme, the...

  13. Antibodies 101: The Four ELISAs and When to Use Them

    Type
    Blog Post
    ...four types of ELISA are direct, indirect, sandwich and competitive. In a direct ELISA a reporter enzyme...competing antigen. In direct, indirect and sandwich ELISA the reporter signal is directly proportional to the... the bench.   Indirect ELISAs The indirect ELISA process is similar to that of a direct ELISA, but the...figure was created with BioRender.com  Direct ELISAs A direct ELISA is the quickest and simplest of all...source appropriate antibodies for an indirect ELISA than for a direct. In addition, a lab can use the same...available to bind to the reporter. Unlike for direct, indirect, and sandwich ELISAs, in a competitive ELISA...are similar to those of the direct method. Namely, since the assay requires a single conjugated primary...

  14. Cloning

    Type
    Guide
    ...four nicks that are repaired by the bacteria after transformation. LIC does not require site-specific recombination...product may be transformed directly into E. coli , where the nicks will be repaired by the normal replication...for it to then be digested and ligated into your desired plasmid. It is important to note that restriction..., that cut your insert or backbone at only the desired location for your cloning project. Restriction ...simplifying the process and reducing the amount of time required for cloning. To utilize this approach, the fragment... of interest one time into a donor plasmid (or acquire one that already has your gene into it) and then...advantage of type IIS systems are two-fold. First, the entire cloning step (digestion and ligation) can be carried...

  15. Antibodies 101: Secondary Antibodies

    Type
    Blog Post
    ...all use either the direct approach or indirect approach, depending on the requirements of the specific assay... also be. This is the direct approach to immunoimaging assays. But if the direct approach isn’t giving... associated, indirectly, with each protein of interest.    Figure 1: The direct approach (left...always use the indirect approach, while flow cytometry’s standard approach is the direct method. ELISAs...secondary antibodies require their own incubation and wash steps, which means that indirect methods are often...sometimes assumed that the direct method is more quantitative than the indirect method. But in reality, ...quantification can be done using either the direct or the indirect method.  New Methods Time is a precious...

  16. Lentiviral Vector Guide

    Type
    Guide
    ...the laboratory, the non-required components have been removed, and the required components have been separated...genes. However, they are different viruses and require different isoforms of these packaging components...particles. Wild-type lentiviruses additionally require the regulatory genes tat and rev , along with virus-specific...and wild-type LTRs Packaging plasmid — contains entire viral genome (packaging, regulatory, and accessory...as a promoter on the transfer plasmid, and this requires Tat for activation. Second-generation plasmids... to the additional plasmid. Second, tat is not required in the third-generation system. Instead, the transfer...plasmids instead of three and eliminates the requirement for Tat. Always includes deletion in the 3' LTR...

  17. Adeno-associated virus (AAV) Guide

    Type
    Guide
    ...got their name, but they are not related. AAVs require the presence of adenoviral genes E1, E4, E2a and...space available inside of the small AAV capsid and directly correlates to the size of the DNA removed from...adenoviral genes not present in the AAV genome required for viral replication (E4, E2a and VA). These ...AAV Plasmid Elements table . Figure 2: Plasmids required for rAAV vector production. Created with BioRender.com...central (CNS) nervous systems. Targeting the CNS requires crossing the blood-brain barrier (BBB), and a ... in-stock viral preps . AAV retrograde (AAVrg) Directed evolution has also been employed to engineer an...transport, AAVrg. Retrograde transport refers to the direction of movement within a neuron from the terminal ...

  18. Antibodies 101: ELISA (Enzyme-linked Immunosorbent Assay)

    Type
    Blog Post
    ...sample, whereas antigen is bound directly to the wells in a direct and indirect ELISA.   Sandwich ELISA Most...et al., 2019.   Indirect ELISA The indirect ELISA is very similar to a direct ELISA, aside from the...detected. These are sandwich, competitive, direct, and indirect. Sandwich and competitive ELISA use antibodies...the sandwich ELISA as described here, the direct and indirect ELISA (more details below) can also be adapted...from Boguszewska et al., 2019.       Direct ELISA In a direct ELISA, purified proteins or the experimental...for visualization. The indirect ELISA is used for similar experiments as the direct ELISA, the only difference...Antibodies are used in many different experiments that require scientists to detect proteins in their samples....

  19. Common Injection Routes in Mice

    Type
    Blog Post
    ...deliver substances directly to the lungs. It is an advanced technique, as it requires intubation of the...< 0.05 ml Skill required: beginner Oral gavage Oral gavage delivers drugs that require ingestion, such...after initial mouse training. Other techniques require more skill and additional training, going from ...seem like the easier of the two, but it actually requires more technical skill to place the needle in the...is slower, and more institutional oversight is required. Restraint: mechanical restraint or anesthesia...-orbitally Gauge: 27–30 Volume: < 0.2 ml Skill required: beginner (retro-orbital) to intermediate (tail...Like IV injections, intraperitoneal injections require a little bit of practice to ensure the proper injection...

  20. Easi-CRISPR: Generating Knock-In and Conditional Mouse Models

    Type
    Blog Post
    ...variety of other undesired outcomes, including one loxP site insertion and one NHEJ-repaired indel, often ...around the desired insertion site to search for potential gRNAs. As with other homology-directed repair experiments...with your desired insertion feature (e.g., a fluorescent protein), you can amplify the desired region using...the power of ssDNA donor molecules for homology directed repair. Using long ssDNA donors, the Gurumurthy...arduous. First, you’d construct a plasmid with your desired changes, long homology arms and a selection marker...pseudopregnant female mice. If you obtained the desired chimeric mice, you would breed them many times,...times, hoping to produce a mouse with the desired edit. You’d also need to remove the selection marker, likely...

  21. Technical Design of a Western Blot

    Type
    Blog Post
    ...assay run time to two days.  Indirect or direct detection method Indirect detection is the most common... to primary antibodies). Direct westerns are simpler and faster than indirect ones and can be used in ...well, and multiplexing is challenging and limited. Direct westerns typically rely on primary antibodies conjugated...sensitive to lower amounts of protein, and can require more antibody or loading sample, which may not ...conjugate, and changing your reporter molecule requires either re-conjugating or re-purchasing your primary... like the mitochondria or nucleus, will likely require a harsher lysis buffer than proteins easily available... also add time to an already lengthy protocol, require the use of hazardous chemicals, and are not always...

  22. Lambda Red: A Homologous Recombination-based Technique for Genetic Engineering

    Type
    Blog Post
    ...three are required for recombineering with a dsDNA substrate; however, only Beta is required when generating...and no longer requires drug selection.  One drawback to this approach is that it requires the generation...based on homologous recombination. It allows for direct modification of DNA within E. coli and is independent...occur in a test tube. The donor DNA substrate only requires ~50 nucleotides of homology to the target site...2) if the dsDNA was short enough, a ssDNA whose entire complementary strand was degraded. Beta: Beta protects...ssDNA target in the cell.  Only Beta expression is required for recombineering with an ssDNA oligo substrate...substrate should be ~70-100 nucleotides long with the desired alteration(s) located in the center of the sequence...

  23. RNA Interference in Plant Biology: New Tools for an Old Favorite

    Type
    Blog Post
    ...experiments that require exogenous protein, successful induction of RNAi only requires the presence of ...as the entire culture can be harvested instead of microscopic isolation. The plasmids required to conduct...tools exist to perturb biological function in a direct or unbiased way at the DNA, RNA, or protein level...level. The “correct” choice of tool requires careful balancing of the inherent advantages and limitations...Figure 1). The plant kingdom has expanded its repertoire of Dicer enzymes and these different Dicer-like... of an RNAi-inducing transgene. Typically, the desired target of RNAi is expressed as inverted repeats...and Stainier, 2020). Additionally, a study that directly compared CRISPR to RNAi in the context of a high...

  24. Advice on Career Paths and the Green Card Process for International Researchers and Entrepreneurs

    Type
    Blog Post
    ...Buy American, Hire American" Executive Order which sets broad policy intentions directing federal agencies.... or postdoc. Foreign national researchers who require visas, permanent residence or citizenship to continue... likely that an international candidate will be hired for a role that is closely aligned with their education...degree or its equivalent and state licensure, if required to practice in that field. H-1B work-authorization...research organizations, and organizations that require the H-1B employee to work at one of the first three...application meaning that the category does not require a foreign national to have an employer or a job...the individual’s past work on the field. NIW requirements stress “educational background, expertise, publications...

  25. REPLACR Mutagenesis: Replacing In Vitro Recombination Methods

    Type
    Blog Post
    ...No Purified Enzymes Required No No Yes Yes Lab Made Competent Cells Required Yes No No No Comparison...Site-directed mutagenesis (SDM) is one of the key tools researchers use to prove causation in molecular...small point mutations, large deletions/insertions require complicated, often costly in vitro assembly methods... Linearized Plasmids After PCR). Current site directed mutagenesis methods Current SDM methods rely on...you design two oligonucleotide primers with the desired mutation, then use those to PCR amplify the template...but these methods can be expensive because they require particular enzymes, some of which are proprietary...above, you design two primers, each containing the desired mutations, to amplify both strands of a plasmid...

  26. Which Fluorescence Microscopy Technique is Best for Me?

    Type
    Blog Post
    ...the entire axial volume of a sample. Therefore, when a large number of optical slices are acquired all...This post was contributed by Doug Richardson, Director of the Harvard Center for Biological Imaging and...the microscope objective evenly illuminating the entire (wide) field of view. Fluorescent light traveling...visualization. Illuminating a sample in the opposite direction to fluorescence collection is referred to as “...increases the speed at which a confocal image can be acquired. Assembling an image pixel by pixel is slow; therefore...hole is illuminated at a time, the image can be acquired more quickly. 2-Photon Microscopy 2-Photon Microscopy...limiting photobleaching. Similar to widefield, the entire field of view is excited at one time and captured...

  27. Components of CRISPR/Cas9

    Type
    Blog Post
    ...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...

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

    Type
    Blog Post
    ...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...

  29. 27 Hot Plasmids from 2016

    Type
    Blog Post
    ...: 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...

  30. Antibodies 101: Introduction to Immunofluorescence

    Type
    Blog Post
    ...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...

  31. Antibodies 101: Flow Cytometry

    Type
    Blog Post
    ...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 ...

  32. Fluorescent Proteins 101: Luciferases

    Type
    Blog Post
    .... Figure 1: Firefly, from Wikimedia Commons. The North American firefly, Photinus pyralis, ...generation Slower Faster Cofactors/substrates Required Not required Signal strength Lower Higher Sensitivity...most familiar source of bioluminescence is the firefly. Along with charming nighttime displays, these ...with BioRender.com. Bioluminescent reporters require a chemical substrate (and sometimes cofactors) ...fluorescent proteins. On the other hand, they don’t require an excitation light source (usually a laser in ... Natural and engineered luciferases Although firefly luciferase was the first bioluminescent reporter... deep-sea copepod (Gaussia princeps), Japanese firefly (Luciola cruciata), and more. These variant proteins...

  33. Filming Science Videos in the Age of Social Distancing

    Type
    Blog Post
    ... 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 ...

  34. Plasmids 101: Gibson Assembly and Other Long-Homology Based Cloning Methods

    Type
    Blog Post
    ...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...

  35. Using AAV for Neuronal Tracing

    Type
    Blog Post
    ... 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...

  36. CRISPR 101: Ribonucleoprotein (RNP) Delivery

    Type
    Blog Post
    ...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...

  37. Pushing the Limits of DNA Assembly

    Type
    Blog Post
    ... 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...

  38. Multiplex Genome Editing with CRISPR-Cpf1

    Type
    Blog Post
    ...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...

  39. CasPEDIA: A Functional Classification of Cas Enzymes

    Type
    Blog Post
    ...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...

  40. 3 Tips to Improve HDR Efficiency for CRISPR Editing in Human Cells

    Type
    Blog Post
    ...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...
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