... editors here! Testing and improving RNA editing from the Cas13b REPAIR system To test the robustness ...editor, the lab started with the Cas13 scaffold, testing a whopping 21 Cas13a orthologs, 15 Cas13b orthologs...and averages only ~50% RNA knockdown. In initial tests, Cas13b from Prevotella sep. P5-125 (PspCas13b) ...ClinVar database. They successfully edited 33/34 sites with a maximum of 28% editing efficiency in HEK293T...to fit into adeno-associated viral vectors, they tested truncated ADARDDs to see if they could shrink the...ADAR-RNA binding to decrease off-targets. Of the tested constructs, ADAR2DD(E488Q/T375G) (REPAIRv2) had...transcriptome-wide comparison, REPAIRv2 reduced off-target sites from the 18,385 observed with REPAIRv1 to merely...

...resistance gene, NPTII, facilitates phosphorylation of kan, which inactivates it. The NPTII gene confers...recovery period after transformation (the dreaded 60 minutes shaking at 37 degrees) can be shortened since the.... As a result, a shorter recovery period of 30 minutes is possible with AmpR. So why even look at another...to produce satellite colonies when used on agar plates due to the fact that beta-lactamase is excreted...Cons: Prone to satellite colony formation on agar plates, less stable at high temperatures and in acidic... and is less susceptible to degradation in agar plates. This prevents satellite colonies from forming ...transformation recovery period than amp or carb (60 minutes). Pros: Widely available, cost-effective, confers...

...cleavage at 1-12 off-target sites; in contrast, SpCas9 may cleave at ~90 sites, according to Kim et al. ...Zetsche et al., 2015. How was Cpf1 found and tested? Class 2 CRISPR systems, including the type II ...Again, they were in for a surprise! Whereas Cas9 generates blunt ends 3 nt upstream of the PAM site, Cpf1...activity in mammalian cells. From 16 diverse Cpf1 candidates, Zetsche et al. found two that display robust...homology-directed repair (HDR). Cpf1 also expands the number of sites that can be targeted by CRISPR to AT-rich regions...regions or AT-rich genomes that lack the 3’-NGG PAM sites favored by SpCas9. Since Cpf1 doesn’t require a ...confirmed. To prevent new editing post-HDR, repair templates should remove the PAM sequence. Comparing Cpf1...

...scientists using pLKO.1 variants learn about those updates? This is a problem many scientists struggle with...molecular cloning protocols, including making LB agar plates, restriction digest of plasmid DNA, and bacterial...is the creation of ZappyLabs. Currently in beta testing, the software aims to be a life science protocol...research labs to create their own lab or group websites. OWW is managed by BioBricks Foundation and is...detailed tutorials and easy to copy and modify templates (e.g. http://openwetware.org/wiki/Protocols/Template...Publishing Group. Like the previously mentioned sites, NPE allows scientists to join labs or groups and...Protocol Online is one of the early protocol sharing websites. It was created in 1999 and allows scientists ...

...CNS transduction and transduces the majority of astrocytes and neurons across multiple CNS regions. PHP....discovered as an antigen upregulated on activated lymphocytes and is commonly used to enrich adult mouse hematopoietic...splicing, or protein coding regions. In total, they tested 13 strains, with seven strains identified as permissive...in the mouse brain.  Both labs then took similar routes to verify LY6A’s role as a receptor for PHP.B. ...LY6A’s expression affected PHP.B’s transduction rates. Knock out of Ly6a in primary C57BL/6J brain endothelial...endothelial cells decreased transduction rates while overexpression of the C57BL/6J version of Ly6a in ...in 293T cells enhanced PHP.B transduction rates. The group also determined the effect of mice strain specific...

...currently being tested in clinical trials to detect mesothelioma earlier than ever. From breath tests to blood...large in size and may have spread while Stage 4 indicates that the cancer has spread from where it originated...blood tests, researchers are hopeful that they can come up with new ways to discover the cancer earlier, ...most exciting developments recently is a new blood test for HMGB1, the high mobility group box 1 protein...injury, this protein leaks out into the cytoplasm, migrates out of the cell, and binds cells that can begin...yet Those who have not been exposed to asbestos Tests like these will allow doctors not only to detect...the future. It can also potentially be used as a test to see how well treatments are working to fight ...

... insertion with PASTE. The PASTEsystem involves insertion of landing sites via Cas9-directed reverse transcriptases...systems and technologies, the AbuGoot lab often tests samples of new bacteria from anywhere they can find..., black pellet was sent to the AbuGoot lab for testing, they weren’t sure what they would find… but it...and inserted it into E. coli to express and then test for any proteins that could cut RNA or DNA via a...new CRISPR effector protein they named Cas7-11. Testing showed that Cas7-11 cut RNA with unprecedented ...for clinical applications. Specifically, efficacy rates of Cas7-11 were reported as up to 85%, broadly similar...measurable cell toxicity – using Cas7-11. This included tests done in cell lines known to be more sensitive to...

...and genetics. She loves to communicate the latest and greatest in scientific content.      References 1....ready for purchase. But what is the easiest and fastest way to use the system in mice? We'll have a post...either scan the genome yourself for potential gRNA sites, or you can utilise several online tools that do...supplied by the Zhang lab. This simple website invites you to input the target region (approximately 500bp...control, 1; very dark). Brighter fluorescence indicates a more successful gRNA design. Adapted from Mashiko...plasmid using standard cloning techniques (The XX indicates where your target is inserted, disrupting the ...details on this plasmid, see Mashiko et al., 2013). Testing the efficacy of your gRNAs is then a simple matter...

...University Cancer is one of the greatest examples of survival of the fittest. Cancer cells find a way to ... oxygen in the tumor, activates hypoxia inducing factor (HIF-1), which promotes the production of VEGF...effector cells of the immune system - cytotoxic T lymphocytes (CTLs), natural killer (NK) cells, and helper...increase angiogenesis around the tumor. This in turn regulates the expression of the tolerance-producing antigens...cells and T cells activated ex vivo are also contestants for cell-based immunotherapy. Using activated...Apoptotic and necroptotic tumor cells have also been tested as vaccines and therapeutics. Their uptake by DCs... patient deaths have occurred during CAR-T cell tests because treatment regimens were not chosen carefully...

...organelles with older (yellow to orange) protein. Image courtesy of Roberta Gottlieb. Laker et al., J Biol ...Your protein of interest does x, but it also participates in y. Depletion of your protein may upregulate...involving recombination elements such as FRT or Lox sites, and provides a modular system for easier exchange...known as SpyLigase and is a protein domain that promotes the formation of an isopeptide bond between 2 ... the event of a DNA break. Next, the gRNA being tested is expressed along with Cas9. If the gRNA effectively... 2014..) Our CRISPR-Cas collection of plasmids updates frequently, so visit our CRISPR pages often to ...Scientists can engineer unique enzyme recognition sites that flank a DNA module in an inverse orientation...

...Subscribe to CRISPR updates on our blog to keep current with the latest and greatest! Addgene resources...editing patterns with ssDNA repair templates. Their work demonstrates that different repair template strategies...out #KSgenome on Twitter if you missed our live updates! My most popular tweet from the conference is...for cellular HDR.   Having had time to digest my notes, since the conference, I think I may have finally...high on-target editing and low off-target editing rates across multiple systems. Read our introductory blog... been challenging, with protocols using dsDNA templates achieving ~1-10% efficiency. Easi-CRISPR uses ...

... plasmid, but are often seen in them.  Lox sites Lox sites, in combination with the Cre recombinase, are...two pairs of lox sites which are part of a dual lox system called FLEX or DIO. Lox sites are recognized ...using enzymes, like SmaI, which have recognition sites in the ITRs. Regular Sanger sequencing typically...and temporally control transgene expression. Lox sites are directional 34 bp sequences that can flank the...flips the sequence between the first pair of lox sites, and then excise the sequence between the second...transgene. By restricting the availability of Cre, lox sites lets researchers control the spatial and temporal...nuclear export of RNA and RNA translation, and promotes RNA transcript longevity. The polyA is located...

... is very suspicious! Refrigerate your plates: Placing plates at 4C for few hours after the initial overnight.... (To be completely accurate, IPTG binds and inactivates the lac operon repressor, thereby allowing lac...from the lacZ gene (a mutation called lacZΔM15) creates a non-functional β-galactosidase enzyme. Providing... rush the process: It is important to give your plates enough time for any intact β-galactosidase to be... and white colonies. Take care in making your plates: X-gal is light and temperature sensitive and needs...after autoclaving. If spread on top of pre-made plates, make sure it is evenly distributed and allow sufficient...of false positives: Blue-white screening only indicates the presence of AN insert, not necessarily YOUR...

...™ transposase facilitates the integration of the transposon specifically at ‘TTAA’ sites randomly dispersed...achieved using the piggyBac™ transposase, which facilitates direct integration of the donor-transposon (carrying...carrying your cargo of interest) into random ‘TTAA’ sites throughout the genome. When using piggyBac™, the...likely to be cell line-dependent, and should be tested in the cell line of interest. Importantly, by varying...significant rearrangement (such as when a stem cell initiates differentiation), transgene silencing may occur...the excision-only piggyBac™ transposase. Image courtesy of Transposagen.   How does piggyBac™ enhance...selection cassette by excision-only piggyBac™. Image courtesy of Transposagen.   How does the piggyBac™...

...400 vials in 35 minutes compared to a lab assisstant filling about 200 vials in 45 minutes by hand. How ...96 tips for easy sample transfer between 96 well plates. The second pod can pick up 8 tips and position...useful if you’re transferring samples from 96 wells plates onto agar q-trays where the samples must be spread...BIOROBOT Universal Platform is ideal because it eliminates the potential of human error or cross contamination...this automated protocol, we can prep two 96 well plates in just under 3 hours or one 96 well plate in about...uses it to fill as many vials as it can in the shortest amount of time. Racks of 48 vials are set up on...using electronic multichannel and single channel pipettes. These can speed up any project in which you need...

...template during DNA repair. Test multiple nick sites, starting with sites about 50 bp upstream and downstream...10–16 nucleotides. For longer templates, it is even more important to test different lengths and sequences...particular edit. These tips are a great starting point: Test different lengths of the primer binding site, starting...about 13 nt (Anzalone et al., 2019). Primer binding sites with 40–60% G/C content are most likely to be successful... can still be optimized (Anzalone et al., 2019). Test different lengths of reverse transcriptase template...This reduces concurrent nicks, which lowers indel rates (Anzalone et al., 2019). When possible, the PE3b...

...lysine 27 methylation. When tested in living cells where methylation of these sites was controlled, the reporter...methylated states. Thus, this system is a reliable tool in reporting methylation status of these sites. This...a variety of diseases, ranging from cancer, to diabetes, to congenital disorders. A series of studies ...disease but increased their risk of death from diabetes. Conversely, paternal grandmothers who experienced...experienced in a period of life when the grandparents’ gametes were developing, an epigenetic change is thought... in the FRET signal. Subsequent demethylation separates the fluorescent units and so lowers the FRET signal...Bygren, and Soren Edvinsson. "Cardiovascular and diabetes mortality determined by nutrition during parents...

...Thursday, or, at the latest on Friday. It’s best to split cells before the plates are more than 80-85%...air and beyond Turn on the hood for at least 15 minutes before you start any tissue culture work and make...Also, avoid unnecessary talking because talking generates microbe-laden aerosols that may enter the hood...70% ethanol immediately. Finally, avoid sticking pipettes into solution bottles directly, rather decant ...they are passaged. This could change their growth rates and morphology, rendering their continued use inadvisable...Anything between orange and light orange usually indicates that the cells have grown enough, are making their...Benjamin Franklin)! It’s good practice to label your plates and/or flasks with your name, date, the cell line...

...so far! Why do we need PAM flexibility? IDT estimates that there is an NGG PAM every 42 bases in the...might be undesirable due to potential off-target sites or low predicted on-target efficiency. In cases ...aureus and N. meningitidis require different PAM sites than SpCas9, in this case, NNGRRT and NNNNGATT, ...activity on non-NGG PAMs, generating xCas9 3.0-3.13. To test xCas9 3.0-3.13, Hu et al. first restored catalytic... that the clones were capable of cleavage, they tested the clones against an NNN PAM library in bacteria...editing rate as SpCas9 at an NGG PAM. When they tested a variety of NG, GAT, GAA, and CAA PAMs, they saw...outperformed SpCas9-VPR in activating transcription. Sites containing NG, GAA, or GAT PAMs averaged 56-91% ...

...be activated from low fluorescent states to high fluorescence states, they can change from one fluorescent... light. The fact that blue light both activates and inactivates Dronpa is not ideal, since this means ...488nm blue light. The T203H mutation in PA-GFP generates a chromophore population that is more neutral,...but then further exposure to blue light also inactivates the chromophore, leading to a non-fluorescent...photoactivation. A high contrast ratio usually indicates that a PA-FP has a low level of spontaneous fluorescence...and display bright and photostable green and red states. Beyond these imaging applications PA-FPs have ... the National Academy of Sciences of the United States of America 101.45 (2004): 15905-15910. PubMed PMID...