...each other very much undergo recombination, two smaller plasmids are formed. Viral vectors are prone to... the 5’ and 3’ LTR regions, it splits into two smaller plasmids. The ~3.7 kb recombined vector backbone...can improve growth and DNA yield. Test multiple, small colonies After streaking LB agar plates, you may...may notice that there are both large and small colonies. Bacteria containing the recombined product tend...tend to grow faster, so we recommend picking the smaller colonies which are more likely to carry the full...whereas the round tan colonies that look similar to normal E. coli cultures contain the recombined vector ...nicked, linear, and supercoiled. Here we find a smaller, recombined plasmid around 1.5 kb. When cut with...

...protein that enhances prime editing in mammalian cells: the small RNA-binding exonuclease protection factor...specialized prime editors (PEs). PE6a and PE6b are small prime editors with RT domains derived from the E...pombe Tf1 retrotransposon RT, respectively. Their small size comes at the cost of improved efficiency, although...and M-MLV RT enzymes, respectively, to obtain PEs small enough to deliver with AAVs but still efficient ...improves editing efficiencies compared to PEmax with normal pegRNAs, but combining PE7 with epegRNAs resulted... ✓ Evolved reverse transcriptase domains Smaller size and/or improved efficiency         ...used to rescue a genetic disease phenotype in an animal model, which was made possible through their use...

...relatively small and easy to work with, making them the go-to choice for studying mammalian biology. Similar...from S. pyogenes and has the advantage of being smaller (Zhang et al., 2016). For editing the genome, the...be electroporated into the oviduct of pregnant females, a technique called Genome editing via Oviductal...either an oocyte or into the oviduct of pregnant females (Yoon et al., 2018, Mizuno et al., 2018). Alternatively...for generating mutations by NHEJ and generating small mutations with HDR, when it comes to larger scale... SL, Kutny PM, Zhang Y, Cheng AW, Jillette NL, Malhotra A, Geurts AM, Chen Y-G, Wang H (2015) Efficient...srep32565 Resources on the Addgene Blog CRISPR 101: Mammalian Expression Systems and Delivery Methods Learn ...

... are much smaller and would be ideal for such AAV packaging, but their activity in mammalian systems has...packaged into AAV particles for delivery into mammalian cells, especially when fused to effector proteins...this problem. A miniature CRISPR-Cas system for mammalian use Stanley Qi’s lab has developed a miniature...activation, base editing, and genome editing in mammalian cells. The “V4” version (named CasMINI) had the...noted that Cas12f1 had an extra-long gRNA for its small size. They were able to increase activity and decrease...10kb but it is also capable of coding concurrent small insertions to allow in-frame deletions and introduce...program large genomic deletions with or without small insertions. But, instead, PEDAR couples this deletion...

...Polyclonal antibodies are generated by injecting an animal with an immunogen, then isolating and purifying....com. To generate polyclonal antibodies, an animal (such as a rabbit or a goat) is injected with an...response starts to wane about a month later, the animal is given additional booster immunizations every...reached - typically within 2-4 months. After the animal has the desired level of antibodies in its bloodstream...retrieved first by drawing blood from the immunized animal. The red blood cells and the serum are separated...different affinities for IgG subtypes from different animals so it’s important to choose the reagent that works...Only up to 5% of the IgG antibodies produced by an animal are specific for the immunogen. A different type...

... with in vivo experiments to determine the optimal/minimal injection titer and volume needed to achieve... now be able to make smaller adjustments to a set of injections into your animal model. For example, in... injection titers and volumes to determine the optimal injection parameters needed to transduce your cells...weeks allows the researcher to determine when the optimal time to conduct an experiment begins. This can ...the DIO/FLEx vector Once you have determined the optimal injection parameters for the serotype that best..., except now you will already have a starting “optimal” injection volume and titer to begin with. Now ...lab (Lavin, TK. 2020) it was determined that the optimal titer to prevent off-target expression of the sensitive...

...to insert at these sites.  Transgenic animals Transgenic animals are often generated by directly injecting... has a close-to-random integration profile in mammalian genomes. SB is active in vertebrates and integrates... well as in vivo in yeast, plant, insect, and mammalian cells, including human cells. piggyBac is biased...transposase (hyPB) is ~10-fold more active in mammalian cells compared to the codon-optimized wild-type...)TTATAA(G/C)TNA. Tol2 can deliver 10-11 kB to mammalian cells without decreased efficiency, with a maximum...human cells than piggyBac and Sleeping Beauty. Minimal Tol2 or miniTol2 is a truncated version of the ...Tol2 have all been used to generate transgenic animals, including zebrafish, mice, rats, and rabbits (...

...a blastocyst which is then implanted into a female animal for gestation. Nucleases that induce double-...are a common model organism used to understand mammalian traits and genetically engineered mouse models... during the DNA repair process. The addition of small molecules that promote homologous recombination ...microinjected into a zygote, which is then implanted into a female mouse for gestation. All three can introduce multiple...homology to the site of interest or create mosaic animals where not all cells in the blastocyst were successfully...identical mice. Thus, scientists must genotype each animal to identify the ones with the desired genotype....gene expression to be controlled through a drug or small molecule that regulates Cre expression. The most...

...and the best practice to determine optimal DOL is through several small-batch experimental labeling. Size... century, it can be overwhelming to select the optimal dye or fluorescent ligand when designing an experiment...enzyme variant tag that covalently reacts with a small substrate ligand motif attached to a fluorophore...response to a specific stimuli, which leaves only a small volume of fluorophores in an excited state, effectively...to protein labeling and analysis, dyes should be small enough not to hinder protein function and should...affinity and specificity to nucleic acids, with minimal background staining. Additionally, cell permeability...2022). Caveat fluorophore: an insiders’ guide to small-molecule fluorescent labels. In Nature Methods (...

... Protein tags are usually smallish peptides incorporated into a translated protein. As depicted in the...point in crystallizing it or testing its function. Small ubiquitin-related modifier (SUMO) can help with ...with folding and stabilization, as can maltose-binding protein (MBP). Overexpression can also lead to insolubility... issues, but scientists may also choose to add smaller proteins, such as Thioredoxin A (TrxA) that improve...purification An affinity tag, generally a relatively small sequence of amino acids, is basically a molecular...simplest affinity tag is the polyhistidine (His) tag. Small and unlikely to affect function, His-tagged proteins... His HHHHHH  0.8 Affinity and Purification Very small size, rarely affects function S-tag KETAAAKFERQHMDS...

...understand how to use it. TLDR: This is a versatile mammalian expression vector that you can use to express ...express both SpCas9 and your gRNA in a variety of mammalian cell types. Purpose  SpCas9 This plasmid expresses...that the Zhang lab has tested in a variety of mammalian cell lines. Cas9, of course, is the key component...backbone can be used to transfect a variety of mammalian cells and can be modified for viral trasduction...by western blot. Overall, this is a versatile mammalian expression vector that you can use to express ...SpCas9 and your gRNA of interest in a variety of mammalian cell lines. Alternative forms of this vector contain...Some Common Ways to Deliver CRISPR Plasmids to Mammalian Cells Resources on the Addgene Website Visit...

...different animals. Check out the Addgene website for plasmids that contain tinman or the mammalian homolog...character but rather to an extremely important gene in animal development. To understand where sonic hedgehog...that flies with non-functional hedgehog exhibit abnormal segmentation during embryogenesis. Typically, ...hedgehog (Nusslein-Volhard and Wiechaus, 1980). The mammalian hedgehog protein, sonic hedgehog (SHH) was named...pathway, which is a key regulator of all bilateral animal development. SHH plays a key role in proper limb...non-viral system to integrate particular genes into mammalian genomes and easily disrupt gene expression to ...arrest gene required for spermatogenesis One of our smaller conference rooms is named Cookie Monster. cookie...

...candidate in the Yang Lab, Qi Li, a lecturer at Sichuan Normal University, Jieze Zhang, an intern in the Yang ...recently, we’ve developed a new system “Multicopy chromosomal integration using CRISPR-associated transposases...great for experiments that require only single chromosomal integration (ex: genome editing), our MUCICAT...transposase system, INTEGRATE, is best for multicopy chromosomal integration (ex: for protein expression) (Zhang...Zhang et al., 2020). Multicopy chromosomal integration for gene expression is an alternative versus plasmid-based...advantages: Fast and site-specific multicopy chromosomal integration of cargo without using screening ...Molecular Plant Sciences in 2019. She joined Sichuan Normal University and became a lecturer in the College...

...example, the visualization of chromosomal translocations or chromosomal shattering (chromothripsis). As...could then be used to examine both inter- and intrachromosomal dynamics, and how they change in response ...turned their attention to mapping pairs of intrachromosomal loci. The technique successfully resolved ...the degree of chromatin compaction even for this small distance! To the authors’ knowledge, this work represents... represents the first mapping of intrachromosomal loci, a major benchmark in characterizing the 4D nucleome...next! References Multicolor CRISPR labeling of chromosomal loci in human cells. Ma H, Naseri A, Reyes-Gutierrez...

... the identification of unknown GEFs for small GTPases." Small GTPases 7.2 (2016): 93-106. FREQ-Seq To...facilitate gene expression in bacteria, yeast, mammalian cells, insects, and even Xenopus oocytes. The ...promoters followed by leader sequences (encoding small leader peptides) downstream of which you can clone...Synthetic Inverters from Zinc Finger Proteins and Small RNAs." PloS one 11.2 (2016): e0149483. Rogers,...can be generated in a cost-effective manner with minimal bias. The FREQ Seq kit has been used in microbiology...2013): 5373-5383. Walkiewicz, Katarzyna, et al. "Small changes in enzyme function can lead to surprisingly...segments of choice into either an E. coli or a mammalian expression vector in a one-pot reaction. Using...

...in a clinical setting. CRISPR is often used for animal germline modification, to repair or add in disease-causing...recessive disorder affecting approximately 1 in 5000 males. DMD is caused primarily by frameshift mutations... reading frame, leading to the production of a smaller, but at least partially functional protein. Individuals... of the dystrophin gene would take place, with minimal risk of off-target editing. In order for the therapy...improvement, with only 30% correction needed for normal function. Even low-frequency editing could make...inflammation and necrosis compared to unedited animals. In grip strength, specific force, and other muscle...require HDR-mediated precision editing tailored to smaller patient populations. To bring DMD CRISPR therapy...

...Temperature Range for Optimal Activity 35 - 45 °C 50 - 70 °C Genome Editing in Mammalian Cells? Yes Yes ...tool of choice for a number of model organisms: mammalian cells, yeast, drosophila, plants, worms, zebrafish...at temperatures ranging from 30-74 °C, with an optimal growth temperature of 55 °C. For a brief comparison... experimental results. GeoCas9 RNP editing in mammalian cells Besides being able to take some heat, GeoCas9...GeoCas9 can introduce genomic edits in mammalian cells. Harrington et al tested GeoCas9’s editing skills...without the need to grow the organisms at lower than optimal temperatures. GeoCas9’s thermostability could also...

...AMA   Guidelines for giving feedback Use formal and informal mechanisms for feedback (more on this below...her management style. Formalizing the feedback session Giving regular, informal feedback is a very effective...to an event as possible. If you wait too long, a small correction can become an unnecessarily big issue...scientists seem to find it easier to give feedback in formal sessions using a specific list of questions after... it helps to plan effective next steps. For a formalized feedback session, answer the questions below ...

...tag or a fusion protein. Mammalian vectors One of our most popular mammalian expression vectors, c-Flag...the pcDNA series. It has the CMV promoter for mammalian expression, a multiple cloning site to insert ...cassette for growth and cloning in bacteria and a mammalian selection cassette to screen for positive transfected...transiently transfected cells, you will not need a mammalian selection cassette (e.g. 1435 pSG5L Flag HA). .... You can also upgrade your basic mammalian expression system and go for a lentiviral or a retroviral ...to study gene function. From making transgenic animals, gene targeting and genome modifications to reporter...

...lanes or a small portion of the gel.  What a waste?  I started looking for smaller and smaller gel apparatus...electrocution.  These days you can buy small gel boxes, but none as small as the baby gel box and this one ... I was wasting buffer running my tiny gels in a normal gel box. It took a bit more ingenuity to solve ...this problem.  None of the commercial boxes were small enough to minimize the required area and buffer ...