...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 ...

...samples were harvested after the behavior, or the animals being monitored were physically hooked up to invasive...whistles. The fibers are made from a process called thermal drawing, essentially where blocks of polymers are... – including microscale light emitting diodes, thermal sensors, and microfluidic channels. These features...temperature, study electrophysiology, and deliver small molecules.   Fun fact: A 1 cm block of polymer ...channels and can also record tissue temperature proximal to the implanted fibers. As important as its capabilities...device has a rechargeable battery, is relatively small, and can be detached easily whenever necessary. ... induce stimulation in target tissues of a live animal. They started by delivering an AAV construct expressing...

...molecules have a specific wavelength that they maximally absorb. The spectrophotometer measures these absorbances...contaminants. Nucleic acids (DNA and RNA) absorb maximally at 260 nm. Proteins on the other hand absorb best...nm and organic compounds and chaotropic salts maximally absorb at 230 nm. The A260/A280 ratio is used ...quantifying DNA using gel electrophoresis. Except, it’s smaller. And automated. Unlike gel electrophoresis, you...and the sample is separated by electrophoresis. Smaller fragments migrate quicker than larger fragments...fluorescent dye that intercalates into the DNA such that smaller fragments are quantified first before larger fragments...electrophoresis which shows fluorescence units over time. Smaller fragments migrate more quickly than larger fragments...

...highly specific in mammalian cells without significant off-target effects, yet small enough to be packaged...Ladder Engineered miniature CRISPR-Cas system for mammalian genome regulation and editing Anti-FLAG Frankenbodies...favorite protein! While the target only requires a small epitope tag (FLAG is eight amino acids, HA is nine...x Engineered miniature CRISPR-Cas system for mammalian genome regulation and editing by: Rachel Leeson... the size of either Cas9 or Cas12a. Despite its small size, CasMINI is comparable in efficacy to dCas12a...can be done via a nuclease-active CasMINI. This small yet powerful system opens up new possibilities in...

...AAV) is a small, single-stranded DNA parvovirus. AAV can infect humans and some other mammals but is not... allows it to replicate episomally, essentially remaining an extrachromosomal part of the host genome ...gamma-retroviruses have been associated with cancer in mammals, which is thought to be due to their propensity...cool tool in the laboratory! It is ideal for whole animal administration, such as in mouse experiments. AAV...which is non-enveloped. It can infect humans, many mammals, and even the avian population. Some adenoviruses...

...into our cells of interest. Although they have a small packaging capacity, AAV vectors were suitable for...response. Any investigator who is using an in vivo animal model should consider the possible elicited immune...immune response from viral infection. Animal safety is a top priority in our lab so we opted for AAV. ...serotypes may perform better (3). Brain Region  Optimal Serotype Striatum AAV5>AAV9>AAV8>AAV1>AAV6>AAV2...outlined in the table below. Cell Type Cortex Optimal Serotype Oligodendrocytes AAV8 > AAV2 & AAV6 ...desirable, transport can be reduced by injecting a smaller dose of AAV and using a serotype with weak CNS ...scAAV) (7). As single stranded DNA viruses, AAVs normally require second strand synthesis before gene expression...

...applications, smaller affinity reagents means higher resolution. Many of the below are also small enough to...enough that their bulk can be a barrier at times. Smaller, slimmer affinity reagents can have deeper penetration... a suitable antibody has been identified but a smaller or less immunogenic version is needed.  Single-...likelihood of aggregation (Bates, 2019). They are small enough to be produced in bacteria and are generally... nature. Single-domain antibodies are extremely small at 12-15 kD but can have weak signals due to their...proteins, or DARPins, are genetically engineered small affinity proteins, around 14-15 kDs in size. These...

...gRNA was present or b. when a gRNA binding a non-mammalian sequence (GAL4) was supplied. In addition to ...distances from 2-75 Mbp. Labeling multiple, intrachromosomal loci Building on the work of Chen et al., ...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...performed sophisticated tracking of multiple chromosomal loci in live cells. However, they note that certain...Hanhui, et al. “Multicolor CRISPR labeling of chromosomal loci in human cells.” Proc. Natl. Acad. Sci. ...

...was contributed by Neil J. McKenna and Bert W. O’Malley. Survival of all organisms depends on efficient...information such as nutritional status. Within the animal kingdom, this information is relayed through factors... receptors across multiple different tissues in normal and disease states, their circadian regulation,...Signaling Atlas (NURSA) at www.nursa.org. Bert O’Malley, M.D., is Tom Thompson Professor and Chair of ...Science 294, 1866-1870. (PubMed) Lonard DM and O’Malley BW. (2007). Nuclear receptor coregulators: Judges...Molecular Cell 27, 691-700. (PubMed) McKenna, N. J.and O'Malley, B. W. (2002). Combinatorial control of gene expression...coregulators. Cell 108, 465-474. (PubMed) McKenna NJ and O'Malley BW (2005) An interactive course in nuclear receptor...

...Vector Mammalian Lentivirus pRDA_512 (RSV-driven; lentiGuide-based) Mammalian Lentivirus... over 512) Mammalian Lentivirus pRDA_789 (CMV-driven; CROPseq) Mammalian AAV pRDA_889...AAV2) Mammalian Transposon pRDA_757 (Piggybac; constitutive expression) Mammalian Plasmid...features of your vector, which are: Organism (Mammalian or drosophila) Delivery type (lentivirus, AAV...

...constructs available at Addgene includes bacterial and mammalian mCyRFP1 or CyRFP1 expression plasmids, FRET constructs...method to increase CRISPR editing efficiency in mammalian cells. Their marker-free coselection, analogous...mutations in ATP1A1 can render cells resistant to the small molecule ouabain - and editing at the ATP1A1 locus...create SpCas9 base editors with editing windows as small as 1-2 nucleotides! To reduce off-target effects... HF-Cas9 (NGG)  Very low off-target activity; mammalian expression vector Rees H, et al. Nature Communications...looking for the production of ECT-DNA (extra-chromosomal telomeric DNA), which accumulates when T-loops...inhibit Spy Cas9 function in both bacteria and mammalian cells. These CRISPR inhibitors may provide a layer...