...the field of gene therapy, more specifically AAV-based retinal gene therapy. The year was 2001 and I started...with the death of Jesse Gelsinger in an adenovirus-based gene therapy clinical trial gone horribly wrong....cell) therapies. One may wonder why the first AAV-based gene therapy treatment for a genetic disease approved...forward with ocular gene therapy The pipeline of AAV-based ocular gene therapies is impressive: clinical trials...for prevalent diseases - to name a few. CRISPR-based tools are also particularly well suited to be combined...and Wu Z., 2018. In vivo Applications of CRISPR-Based Genome Editing in the Retina. Front Cell Dev Biol...

...any plasmid-based experiment, untreated cells should be included as these provide the baseline/standard ...one thing should be present in every experiment-based investigation of a hypothesis: proper controls! ...the various controls that can be used for plasmid-based experiments. What are control plasmids? Generally...the abstract and follow an example of a plasmid-based experiment, delineate its requisite control plasmids...interpretation of the results. Typically, plasmid-based experiments employ transfection, negative, positive...completely. These controls may or may not be plasmid-based depending on the experimental needs. Using our experiment...

...homodimerization of FPs targeted to the endoplasmid reticulum based on the appearance of OSER whorls. This assay is ...random insertions can be generated with a transposon-based strategy (Sheridan, 2002). Avoiding protein over-expression...first express the fusion protein from a plasmid-based system to assess whether the chimera is still functional...verified and it can be compared to what is expected based on immunofluorescence or properties of the protein...independent of GDI-mediated extraction and actin-based trafficking." PLoS biology 13.4 (2015): e1002097...Klarenbeek, Jeffrey, et al. "Fourth-generation epac-based FRET sensors for cAMP feature exceptional brightness...PMID: 30886412. Mastop, Marieke, et al. "A FRET-based biosensor for measuring Gα13 activation in single...

...controls for antibody-based experiments Primary antibody specificity Antibody-based assays have their own...remains the same.    Figure 2: Microscopy-based antibody experiments should include a primary antibody....   Application-specific controls Some antibody-based applications have very specific controls, which ...you to detect dead or dying cells so that you can base your analysis on living cells only (for example,...some of the more critical controls for antibody-based experiments, it is by no means an exhaustive list... more information about antibodies and antibody-based experiments, check out these blog posts.       ...protocols elicited the expected response and have a baseline with which to compare the experimental group. ...

...other forms of antibody-based (e.g., nanobodies, ScFVs) and non-antibody based affinity reagents (e.g....institutions have proposed a shift to recombinant DNA-based antibody technologies (Bradbury & Plückthun, 2015a...

...PBMCs with episomal vectors Episomal vectors are based on the Epstein-Barr Nuclear Antigen-1 (oriP-EBNA...

...guides employed by Cas9 or Cpf1. These short, 24 base DNA guides may be more user-friendly, since they...working with small capacity viral vectors such as AAV. Base removal RNA-guided CRISPR enzymes cause double-stranded...unfavorable. In a natural context, it’s hypothesized that base removal helps prevent the invading genome from “...property could be an advantage. However, repair after base excision would alter the target sequence and preclude...

...one-size-fits-all approach. You will have to select a tool based on your project and not general opinion. I could... features and enables you to select the tool(s) based on your project needs. There are 8 major categories... is important for users wishing to select tools based on their comfort level with operating a computer...sequence”, “identifier”, and “load”. Trends: Sequence based input is by far the most common with most tools ...for the first time, it is best to choose a tool based on immediate needs and re-evaluate after several...have come across to offer classes of parameters based on the type of CRISPR experiment being pursued. ...implementation to be too slow for practical use. The R based package CRISPRseek offers great utility and coupled...

...biarsenical dyes such as FlAsH (green, based on fluorescein) or ReAsH (red, based on resorufin). SNAP-tag: In 2003...Johnsson’s lab developed a self-labeling fusion tag based on the human DNA repair protein AGT that gets covalently...Promega developed HaloTag in 2008 (Los et al., 2008), based on a bacterial haloalkane dehalogenase that binds...in 2008, Kai Johnsson’s lab reported another AGT-based tag named CLIP-tag, which reacts with O2-benzylcytosine...benzylcytosine(BC) ligands (Gautier et al., 2008). Although based on the same original enzyme, SNAP-tag and CLIP-tag...the other. Finding the right tag for you Protein-based tags typically provide better labeling specificity...Cornish, V. W. (2009). An In Vivo Covalent TMP-Tag Based on Proximity-Induced Reactivity. ACS Chemical Biology...

...CD20 and CD73 on specific solid as well as blood-based tumors. Monoclonal antibodies have been shown to...activated ex vivo are also contestants for cell-based immunotherapy. Using activated DCs ex vivo to generate...Recently, China spearheaded the first ever CRISPR-based clinical trial in 18 lung cancer patients. This ...differ in their genotypic and phenotypic identities based on their level of progression, anatomical location...Participants in clinical trials should be picked based on the immunotherapy target using diagnostic tests...During clinical trials, expansion of participants based on early results can enhance the positive effects...most important: How will cancers evolve and mutate based on the immunotherapeutics we use on them? Answering...

... you are trying to do: gene knockout, a specific base edit, or modulation of gene expression. Location...with BioRender.com. The jigsaw: Editing by HDR, base editing, and prime editing For a specific edit, ...constraints are even more exquisite for the so-called base editor Cas9, which makes DNA changes in the absence...dsDNA breaks (Rees et al., 2018). For C>T and A>G base editors, the intended edit must be in a 5 - 10 nt...others have examined the ability to use sequence-based and other features to nominate gRNAs that are likely...(2020) Genome editing with CRISPR–Cas nucleases, base editors, transposases and prime editors. Nat Biotechnol...doi.org/10.1093/nar/gkw583 Rees HA, Liu DR (2018) Base editing: precision chemistry on the genome and transcriptome...

...studies of E3 ligase cereblon Optimized mScarlet-based tags for live imaging in C. elegans CRISPR/Cas9 ...proteins) and performs well in many different antibody-based applications.  IPI’s recombinant version is a fusion... O. (2024) Comparative analysis of new mScarlet-based red fluorescent tags in Caenorhabditis elegans. ...high resistance to antibiotics. Several CRISPR/Cas-based systems have been developed for other mycobacteria...and Hung, D. (2024). A dual-plasmid CRISPR/Cas9-based method for rapid and efficient genetic disruption...STReTCh (11 kDa) are significantly smaller than FRET-based mechanosensors (~60 kDa) and are compatible with...

...substantial immune response or toxicity. More AAV-based CRISPR systems Smaller Cas proteins SaCas9 isn't...both is within AAV’s packaging limit. For CRISPR-based RNA editing, the REPAIR (RNA Editing for Programmable...eliminate the need to deliver larger cargo such as base editors and prime editors. While using AAV to deliver...activation. Split inteins have also been used to express base editors. References Chew WL, Tabebordbar M, Cheng...Comander J, Liu Q, Liu DR (2020) Cytosine and adenine base editing of the brain, liver, retina, heart and skeletal.../nbt.3055  Wang D, Zhang F, Gao G (2020) CRISPR-Based Therapeutic Genome Editing: Strategies and In Vivo...

...steel wire into a backbone structure of a protein, based on its atomic coordinates. These wire models were...momentarily forgetting that the physical model is based on their own modeling of the protein’s structure...in 2014 to ask if we could make a model of Cas9 based on the structure published by Martin Jinek’s group...recognize a PAM sequence, allow the complementary base pairing between the guide RNA and the target DNA...conformational shifts. This backbone model of Cas9 is based on Fuguo Jiang’s structure of Cas9 – primed for ...

...Kanemaki lab previously developed a CRISPR/Cas9 based method to tag endogenous proteins in HCT116 cells...sites for editing. PAM sequences can be from 2-6 base pairs in length and follow the DNA sequence targeted...SpCas9-NG) with relaxed preferences for the third base of the PAM so that it now recognizes NG rather than...of seven ChRs derived from marine microorganisms based on metagenomic data. MerMAIDs (Metagenomically discovered...

... expected sequence. Here at Addgene, we use NGS-based quality control to confirm the sequence of all the...used to confirm the rough structure of the plasmid based on the predicted sizes and organization of different...estimate how far down the gel your bands have traveled based on the dye! Always run a ladder. Ladders allow you...you get in your sample lanes. Choose your ladder based on the expected band sizes. Always run control uncut...

...FlipGFP-based activity reporter of SARS-CoV-2 main protease Mpro. (Unpublished) A FlipGFP-based activity... A Scalable, Easy-to-Deploy, Protocol for Cas13-Based Detection of SARS-CoV-2 Genetic Material. Rauch ...

...spaced peaks …like these…. 3. Expect to get 500-700 bases of clean reliable DNA sequence Anything less and...uncertain terrain. 4. Never trust the first 20-30 bases of a DNA sequencing read The peaks here are usually...unfortunate side effect of messing up the reaction around base 70-75 of the read (see image below), so I would ...

...modify, whether it be editing CRISPRi, CRISPRa, or base editing. Multiplexing gRNAs: the basics One common...Gibson Assembly or SLIC cloning methods. A BsaI-based E. coli multiplexing plasmid is available from the... efficiently cleaves gRNAs sandwiched between 28 base Csy4 recognition sites. If Csy4 is not expressed...inserted into a Cas9-containing destination vector based on pCAMBIA. Cas9 is plant-optimized, and plasmids...you to assemble type II-A CRISPR arrays for dCas9-based transcriptional repression. The CRISPathBrick plasmid... method that combines CRISPR and recombineering-based MAGE (Multiplex Automated Genome Engineering) technology...only 2-3 hours incubation. Kondo Lab multiplexed base editing in E. coli The Kondo lab expressed a cytidine...

... Figure 2 ). The Bogdanove group also hosts web-based software to help scientists find potential TAL effector...assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Cermak T, Doyle EL...