...new Cas enzymes on the block? The Cas family of proteins has many useful genome engineering enzymes, each...other Cas enzymes is its method of DNA cleavage. Instead of cleaving DNA at a defined site to form a clean...Unlike many other Cas enzymes, Cas14 targets ssDNA instead of dsDNA. It cleaves ssDNA with high fidelity -...nuclease, however, unlike Cas9, it targets RNA instead of DNA. This makes it particularly useful in generating...composed of the fusion of several Cas7 and Cas11 domains. Cas7-11 cuts ssRNA and is capable of pre-crRNA...   Table 1: Fast facts on Cas family proteins and genome editing abilities   Choosing a...adaptable Cas currently available, the other Cas proteins should not be overlooked. The unique features ...

...However, instead of weakening interaction sbetween the non-target strand and Cas9, Kleinstiver et al disrupted..., researchers from the Zhang lab at the Broad Institute, the Joung lab at Harvard Medical School, and ...groove formed by the Cas9 HNH and RuvC nuclease domains 2. Interactions between the target strand and ...specificity. Design and testing of SpCas9-HF1 Kleinstiver et al and the Joung Lab similarly thought that...mutations N497A, R661A, Q695A, and Q926A (SpCas9-HF1 contains all four mutations). Although the precise mutations...from Addgene here! Future possibilities for Cas proteins With their enhanced specificity, eSpCas9, SpCas9...25079318. PubMed Central PMCID: PMC4176945. 4. Kleinstiver, Benjamin P., et al. "High-fidelity CRISPR–Cas9...

...precise insertion of a DNA sequence ranging from a single nucleotide change to large insertions like the...MLV)-like particles loaded with Cas9-gRNA ribonucleoproteins (RNPs). According to the authors, they named...polyprotein Nanoblades are formed when viral structural proteins, such as the MLV protein Gag, multimerize and ...cleaves the proteolytic site connecting the two proteins, thus releasing Cas9. Nanoblades can also deliver...loaded with 1-4 different gRNAs. Viral envelope proteins To alter the cell tropism of Nanoblades, different...alternative method for generating transgenic mice strains and for non-invasive in vivo gene editing. The ...viral-derived Nanoblades loaded with Cas9-sgRNA ribonucleoproteins." Nature communications 10.1 (2019): 45. ...

...detect, purify, quantify, deplete, and visualize proteins of interest (Greenfield, 2014). Traditionally,...Therefore, researchers from over 100 scientific institutions have proposed a shift to recombinant DNA-based...expression. If starting from a protein sample (for instance, affinity purified antibodies from a patient’s...the line and subsequent cloning of the antibody chains (Crosnier et al., 2010; Andrews et al., 2019). ...well in animals because of similarities to host proteins. After the antibody of interest has been cloned...non-antibody based affinity reagents (e.g., monobodies, DARPins) (Helma et al., 2015).    The Many Benefits of...and sequences for antibodies are not shared. For instance, through open plasmid sharing, the CRISPR community...

...shipping materials (i.e., a deposit kit) with instructions on how to send liquid DNA or bacterial streaks...lab. Plasmid Type Choose from: Encodes one insert, encodes gRNA/shRNA, or empty backbone. Purpose...plasmid will work for them. Species of Gene or Insert Choose from: H. sapiens (human), M. musculus ...luciferase, cre/lox, yeast expression, worm expression, insect expression, plant expression, mouse targeting, ... cannot be propagated in any of these standard strains, choose other, indicate strain in the “Growth Strain...showing the first eight columns and a pop-up instruction box for the “Purpose” field. If you ever have...should go into a cell, hover over the cell and instructions will pop up. When you’re finished filling out...

...Arg-mutant proteins in living baker’s yeast cells, including Arg-mutant yeast homologs of human proteins that...past, we've been working with the Rare Genomics Institute to provide plasmid grants to researchers working... his lab’s research is the mechanisms by which proteins assemble into functional multi-subunit macromolecular...function of even a small fraction of Arg-mutant proteins could improve health outcomes for millions of ...treatment for rare diseases caused by Arg-mutant proteins, but there is currently no evidence of Arg-mutant...large number of other disease-linked Arg-mutant proteins both in yeast and human cells in order to determine...rescue of a variety of disease-linked Arg-mutant proteins in the lab, clinicians might consider prescribing...

...icosahedral capsid made of 3 types of proteins: fiber, penton and hexon proteins. The viral genome DNA is further... which contains the adenoviral genes necessary for virus production. For detailed instructions on how ...consisting of a capsid surrounding an inner core which contains the adenoviral genome. The genome itself is a ...further protected by histone-like proteins forming the nucleocapsid. B) Three generations of adenoviral ...ceases relatively quickly due to immune response against AdV. Importantly, recombination-competent adenovirus...helper adenovirus is required to provide the viral proteins in trans. The Gutless AdV have generated high ...

...at the Korea Advanced Institute of Science and Technology (KAIST) and Institute for Basic Science (IBS...protein. After fusing the CRY2 and CIB1 domains to separate proteins of interest, a researcher can cause ...robust and efficient oligomerization of target proteins in response to blue light. This tool, CRY2clust...2017. PubMed PMID: 28288122. New fluorescent proteins for use as FLIM-FRET acceptors  Listen to the...(YFP) with an excitation peak of 519 nm. These proteins have improved photophysical properties and can...maturation kinetics than other dark fluorescent proteins, it has good signal stability allowing for minimized...

...student and then instructor at Singularity University at NASA Ames, and an instructor at Biocurious.org...makerspaces around the world. Hackerspaces.org maintains a list of active spaces near you.   Learning ...complexity using existing C. elegans mutants. For instance, we use a mutant known as CX51 that exhibits motor...Minnesota has declared our makerspace an educational institution meaning that we can obtain known mutants at $7.00...motor behaviour, and ultimately submit our new strains back to the CGC for other researchers to use. Although...equipment that would otherwise be discarded. For instance, the centrifuge in the picture had a broken controller... the fantastic optics and fluidics platform it contains. Biotech equipment becomes obsolete amazingly ...

...add a bit of fun. He mixed together 10 E. coli strains producing different pigments, and tasked the students...perfect fit for the streak plate challenge. Chromoproteins are a subset of the fluorescent protein family...off color in ambient light. Expressing these chromoproteins in E. coli would make it easy to distinguish...single colonies, these chromoprotein-expressing strains are easily amenable to other experiments to teach...have the students transform them into different strains of E. coli,” says Westenberg. During the transformation... how different genetic backgrounds of the host strains affect transformation efficiency.  Aside from the...blog Read our other blog posts on fluorescent proteins Check out educational resources on the blog Resources...

...coding genes in the human genome with fluorescent proteins. For each gene, we needed to design guide RNAs...ranked by specificity of targeting or by cut-to-insert distance for HDR. (The notable exception is TagIn...compressed CFD score) * (gaussian normalized cut-to-insert distance) This gives each guide a score ranging... can be used in the rank equation above. Cut-to-insert distance was fitted to experimental data of its...the greater this distance between the cut and the insert, the more poorly the guide performs HDR. Optimal...sequences to prevent re-cutting of the sequence inserted by HDR. Although this practice is generally advised...enough to prevent re-binding. Lastly, the sequence inserted by HDR may inadvertently re-create a match for...

...modulation of endogenously and exogenously expressed proteins. Here we will review what advantages degron tags... the tagged protein. Degrons are destabilizing domains which will be identified by various enzymes and...manipulate endogenously and exogenously expressed proteins. Thirdly, degrons and their drugs are titratable...not amenable to being tagged, e.g. tagging some proteins can affect their function, the SMASh tag self-...developed as an affinity tag for visualization of proteins of interest. A fluorescent Halo ligand can be ... molecules which target E3 ligases to specific proteins by simultaneously binding both their target protein...their effectiveness with over a dozen different proteins. In the end, you can’t predict with absolute certainty...

... pC5Kan-P2A P2A Insect pUltra P2A and T2A Lentiviral Ac5-STABLE2-neo T2A Insect   For the co-expression...vectors simultaneously express two or more separate proteins from the same mRNA. We've discussed promoters ... detect your gene of interest, scientists have instead developed novel methods to co-express your gene...IRES Elements Translation in eukaryotes usually begins at the 5’ cap so that only a single translation...site, thereby resulting in co-expression of two proteins from a single mRNA. IRES was originally discovered...between your genes of interest and you can then insert the whole multicistronic cassette into a backbone...Although it is recommended to use the 2A peptides instead of an IRES when stoichiometrically equivalent levels...

...translation of RNA to make proteins. That's central to everything that goes on inside a cell. Until we get ...of enough sensor domains. Ultimately, we'll need a couple thousand or so sensor domains for everything ...DNA systems capable of storing data and computing inside living cells. He's also a co-founder of the BioBricks...BioBricks The BioBrick Foundation's Linda Kahl explains the Addgene collaboration this way: "Addgene is...strategy that organizes how we start the work, and it begins with the central dogma - replication of DNA, transcription...and expensive. There's the promise that we could instead have things like peptide or RNA aptamers that act...

...for Science.  Cold and overcast with intermittent rains, it was weather befitting of the ominous political...Science and Engineering at Rensselaer Polytechnic Institute. He also studies energy markets in his spare time...cause breast cancer and infertility or to argue against unnecessary clinic regulations, evidence-based ...issues hit hardest. According to the Guttmacher Institute, most women of reproductive age live in a state... how planets form around young stars and the instruments we use to study them. You can follow her at alexandrazgreenbaum.wordpress.com.... I worried that, because we were speaking out against legislators and because the march had been so well-publicized...scientists – including global warming skeptics – insisted that more data and research funding are critical...

...Harrach, 2014). Given that the early genes encode proteins essential to initiating the whole process – such...used to house a recombinant sequence (transgene) instead.    Although the late genes are usually present...absent, and the late genes (and their structural proteins, which would encapsidate new virions) are not ... is transient due to the fact that AdV surface proteins (especially fiber’s knob domain, and hexon), as... as well as other intracellularly-exposed proteins and downstream cellular events, all recruit strong ...sense of what AdVs are about – and we hope that it inspires further use of existing vectors, as well as research...resources References Bauzon, M., Castro, D., Karr, M., Hawkins, L. K., & Hermiston, T. W. (2003). Multigene expression...

...now available. In this post, we’ll review the inspiration behind ExM, how it works, and some of the limitations...most SRM techniques also require specialized instruments and technical expertise, limiting their accessibility...a straightforward answer to the space problem. Instead of imaging smaller, what if we just made the sample...chemical dyes, and genetically encoded fluorescent proteins (Gambarotto, et al. 2019; Tillberg, et al. 2016...adapted for detecting nucleic acids as well as proteins (Klimas, et al. 2023; Wassie, et al. 2019). Perhaps...et al (2021) Expansion microscopy provides new insights into the cytoskeleton of malaria parasites including...Saad O, Bewersdorf J (2020) Light microscopy of proteins in their ultrastructural context. Nat Commun 11...

...classified as a type V CRISPR system. Like Cas9, Cpf1 contains a RuvC-like endonuclease domain, but it lacks ...off-target editing efficiency. Kim et al. and Kleinstiver et al. characterized genome-wide editing efficiency... to single mismatches, but variably so, with Kleinstiver et al. reporting that Cpf1 can tolerate mismatches...essential function in Cpf1-mediated editing, as Kleinstiver et al. saw no decrease in Cpf1 activity with ... are indicated below each graph. Figure from Kleinstiver et al., 2016. Cpf1’s strength may lie in ...activity than that previously observed with SpCas9. Kleinstiver et al. suggest that AsCpf1’s off-target rate ...eSpCas9 and SpCas9-HF1. Both AsCpf1 and LbCpf1 ribonucleoproteins (RNPs) failed to induce off-target editing...

... In this iteration, the size of the lentiviral insert was minimized to reduce recombination and avoid...transcriptase enzymes, inhibition of host cell proteins, and modifications to the pegRNA. The Britt Adamson...nucelocapsid protein, one of the virus’s most abundant proteins and a critical component for viral genome packaging...-LV [+M], negative control) were immunoblotted against Anti-SARS-CoV-2 Nucleocapsid Protein antibodies...middle panel). Samples were also immunoblotted against vinculin as a loading control (right panel). Image... of ssAAV, a targeting sequence of miR124T was inserted into the vector to silence transgene expression...editing, and delivery of disease-relevant large proteins. They demonstrated REVeRT in various contexts ...

...of the viral capsid proteins are analyzed. The abundance of viral capsid proteins as a fraction of total...relative stoichiometric ratios of the viral capsid proteins VP1, VP2, and VP3. Samples of viral preparations...subjected to gel electrophoresis. Viral capsid proteins VP1, VP2, and VP3 are shown relative to the total.... Three to five days later, cell cultures are inspected for sterility. Transducibility Some viral vectors...virus from Addgene, read our detailed recipient instructions for next steps. Plan an experiment using our...plasmid in the viral preparation by performing PCR against unique regions of the transfer plasmid.   Lentivirus...