... to switch out reporters, Cas proteins, guide cassettes, promoters, N’ and/or C’ terminus, and selection... systems. What’s the error rate? The Doench lab tested the Fragmid system by sequencing 60 individual ... assemblies created over five months. When they tested those clones (in duplicate), 93% had the correct...your backbone and genes contain only the Bbsl cut sites needed for the Fragmid cuts, clone in the appropriate...References McGee, A. V., Liu, Y. V., Griffith, A. L., Szegletes, Z. M., Wen, B., Kraus, C., Miller, N. W., Steger...

...A4 and A8-A10. The researchers selected 8 of the tested ABE8 constructs (ABE8.8-m/d, ABE8.13-m/d, ABE8.17...2 guanines). This limits the use of ABEs as DNA sites that do not contain a suitable PAM cannot be edited...for primary human cell editing, the researchers tested ABE8 in CD34+ hematopoietic stem cells. ABE8 efficiently... gRNA used (Gaudelli et al., 2017). The authors tested ABE8.17-m and found that one base change mutation...overall improved base editing capacity, even at sites that used to be difficult to target. In particular...

...They will come in handy here! Crossing your F1 heterozygotes to each other will generate approximately 1 ...wild type at one or both of the alleles, or heterozygotes, are not a waste. Best practice is to use littermate...transgenic strain, the promoter being used should be tested in cell lines to determine how it impacts the expression...chromosomes recombine during meiosis, creating gametes that contain a mix of genetic material from each...recombination at the floxed site, your control should be littermates that express Cre but not loxP. With this two-...

...characterized by misfolding of the TTR protein, which contributes to the disease phenotype. Intellia initiated ...Biosciences is currently running this trial, with more updates expected in the coming months/years. The CRISPR-HIV... (Hussein et al). It employs Cas9 to cut at two sites within the viral genome to excise a large portion...disease, inflammatory diseases, blindness, and diabetes. Directions and obstacles for future CRISPR therapies... "Functional Repair of CFTR by CRISPR/Cas9 in Intestinal Stem Cell Organoids of Cystic Fibrosis Patients...

...functions due to mutations accumulated over time? Undergraduates can help to answer this question! The Pathways...can be relatively confident that the gene from the test species has a similar function to the gene in the...enzymatic steps (5). In their experiments, students test whether or not proteins from the fission yeast, ...Excel: Producing One Million Additional College Graduates with Degrees in Science, Technology, Engineering...PMCID: PMC4012490. 4. Hoffman, C.S., Wood, V., & Fantes, P.A. An ancient yeast for young geneticists: A...

...review analyzing and explaining the antibody crisis states that up to 50% of all commercially available antibodies...to more complex brain systems such as non-human primates and humans. Since we need larger-volume antibodies...extensive library of monoclonal antibodies; Addgene collaborates with these companies to create and provide general...includes accuracy, strength, and reproducibility. We test the antibodies on human and mouse tissue and compare... distinct imaged pattern, and exposure times. To test the antibody’s reproducibility, each antibody is...

...surprise and delight,” the very first protein they tested — E. coli expressing good ol’ EGFP — showed a slight...and Maria were skeptical. The team was careful to test the possibility that this might just be an artifact...area measures approximately 82 mm across. Video courtesy of Andrew York and Maria Ingaramo.   MagLOV shows...tools.  Embracing science in the open The York Lab operates with a strong commitment to exploration and collaboration...self-publishing directly on his website (and often posting updates on social media) and freely sharing code, raw data...

...mechanism of their voltage sensing component, which separates them into two distinct categories detailed below...phosphatases from sea squirts. This design structurally separates the two components of the indicator, which can...was dipicrylamine, a lipophilic compound that distributes itself within the plasma membrane as well. Another...particularly physiologically relevant in neurons, myocytes, and endocrine cells. It follows that the specific...component (i.e., when the dye is lipophilic and integrates into the membrane), the voltage sensing component...

...it ranks guides by CFD score, and it mutates HDR donor templates to avoid re-cutting when needed. It gives...each gene, we needed to design guide RNAs, donor templates and sequencing primers for quality control. At...sophisticated features of CrispyCrunch is how it mutates guide sequences to prevent re-cutting of the sequence... the above are options, the algorithm silently mutates codons in the guide sequence, one by one, from ...

...quality control, but does not perform functional tests. The newest genome engineer sharing the stage with...be less desirable if one has to target multiple sites in a single experiment. Also, the NGG PAM sequence...nearly 400 times. Researchers have been excitedly testing NgAgo's genome editing applications. So, how does...conducted a survey and asked researchers if they had tested and compared NgAgo head-to-head with CRISPR-Cas9...could not achieve indels with the NgAgo system indicates that it is very tricky to use and fussy to optimize...

... plasmid with multiple, individual expression cassettes, having the genes expressed from the same cassette...described below. IRES Elements Translation in eukaryotes usually begins at the 5’ cap so that only a single...originally discovered in poliovirus RNA, where it promotes translation of the viral genome in eukaryotic ... start with a plasmid that has multiple cloning sites flanking the multicistronic element(s), or you could...efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice. Kim...

...biological questions. The two most common uses are: 1) to test the expression level in a specific system (by measuring...occurs when energy transfer from one FP (e.g. CFP) excites the fluorescence of another FP (e.g. YFP). FRET...create the chromophore. This can be from a few minutes after it is translated to a few hours. For example...However, for most FPs it is a few seconds to a few minutes. Photostability can be affected by experimental...greenfluorescentblog.wordpress.com.   More Helpful Websites & Resources: FP guide at Addgene Interactive ...

...cell types. Variance can be caused by cell cycle rates; rapidly diving cells will have higher HR frequencies...viability of your cells. Enrich S-phase cells HR operates predominantly in S and G2 phase of the cell cycle...carefully (Lin et al., eLife)! Use a Cas that facilitates HR What if promoting HR was as simple as swapping...It can be! Cas12a is a Cas family member which generates sticky ends upon cutting. Cas12a may promote HDR..., Rouet, R., Vejnar, C. E., et al. CRISPR-Cpf1 mediates efficient homology-directed repair and termperature-controlled...

...insert-specific sequence. This is a “yes or no” kind of test, with a positive clone amplifying a product and ...primers. These primers are designed to anneal to sites that flank the insert site. A positive clone will...depends on your preferences. Either way, make sure to test your colony PCR primers before using them to screen...Pros Cons Insert-Specific Simple "yes or no" test results Doesn't tell you the orientation of ...of sterile water.  Pick 3-10 colonies in total to test, depending on the number of background colonies ...

...chemiluminescent substrates are available. Labs with limited equipment often opt for the chromogenic substrates since...antibody buffer components can interfere with conjugates. Make sure to do your research before conjugating...sandwich ELISA. In this method, the target antigen competes with a reference antigen for binding to a primary...is non-specific binding or false positives.  If testing a recombinant protein, especially one with tags...

...back to) is the one-eyed pinhead mutant, which regulates a developmental pathway implicated in congenital...Zebrafish Model Organism Database ZFIN actively curates information on mutants and other resources. Targeted...oligonucleotides called morpholinos. When injected into zygotes, morpholinos complementary to a target mRNA inhibit...help elucidate the gene networks that transform zygotes into adults and explain how signaling pathways ...study human illnesses such as blood disorders, diabetes, cancer, and Alzheimer’s disease. Naturally, the...

...quickly screen and investigate transcription factor candidates and the regulation of genes in the biosynthetic...save you a lot of time. Don't re-invent the wheel. Test different leaf ages Usually younger leaves work ...interest. Find all plasmids from the Lee-Parsons lab. Test different leaf infiltration methods Co-cultivation...transcripts in the plant but not from Agrobacterium. Test different Agrobacterium strains We used the disarmed... This allows you to quickly assemble vectors and test different ideas. We are a huge fan of the Golden...

...visual appeal, instant sharing across multiple other sites), I created a new platform for sharing video abstracts...communicating research. Short videos of 3 to 5 minutes provide more than enough time for researchers to...website that helps researchers go beyond citation rates to measure the impact of their contribution. It ...Vimeo, and other platforms (such as university websites). WeShareScience is a social platform that provides...about her research on plummeting embryo survival rates. Video on We Share Science: http://wesharescience.com...

...factor) promotes editing (Yan, J et al., 2024). They developed a new prime editor that incorporates La’s ...AAV Vector System Neurodegeneration Collection updates   Multiplexed perturbation and decoding in pooled...Figure 6: Illustration of split fluorophore assay to test reconstitution via mRNA trans-splicing. A cis-splicing... New and notable Neurodegeneration Collection updates Last but not least, check out the new and noteworthy...

...typically contains iterons that serve as recognition sites for replication initiation proteins. These iterons...The plasmid does not have many restriction enzyme sites. Hosts often have a restriction barrier as a defense... al. 1977). So if a plasmid has few restriction sites, it may be able to sneak past this checkpoint.  ...incorporate interchangeable antibiotic resistance cassettes (Prior et al. 2010), inducible promoters with ...inducibility (Toh et al. 2023), and additional cloning sites (Keen 1988).  Find broad host range recombinant ...