... cloning efficiency measurements. We performed additional efficiency measurements using similarly sized...number of transformants); the greater the number of inserts, the lower the number of transformants. To compensate... Table 1). These improvements (5- to 12-fold increases in the number of transformants while maintaining...reaction volumes were 20 μl (1, 12 fragments) or 25 μl (24 fragments). The future of assembly DNA assembly...Assembly is commonly used to assemble 2–10 DNA fragments in a single “one-pot” reaction to form complex... site is no longer present, allowing multiple fragments to be assembled in an ordered, pre-determined ...background by antibiotic selection. Screening of transformants by colony PCR confirmed the insertion of the...

...responses in patients. They were able to tweak this assay to study SARS-CoV-2 spike variants. “We created...circulating variants. This allowed us to create pseudoviruses that represent lineages and variants that are...we’ve seen the emergence of several SARS-CoV-2 variants coincide with increased vaccination efforts. So...effective are the current vaccines against the variants? Alejandro Balazs, a Group Leader at the Ragon... of current vaccines differed depending on the variants (Garcia-Beltran et al., 2021). Antibodies from...Moderna or Pfizer vaccine could neutralize many variants, including the B.1.1.7 (Alpha variant), B1.1.298...well as they could for the wild-type. However, variants such as the P.1 (Gamma variant) and the B.1.351...

...CRISPR applications in plants.     Transgenic yeast as source of dietary supplements Dr. Mrinalini Nair,...well as developing yeast as source for dietary supplements! Development of a bacterial expression system..., bacteria, and fungi that reside within living plants and that produce compounds with various antimicrobial...More Optogenetic Tools Like pDawn  Making cassava plants resistant to cassava mosaic virus Dr. Fiona Robertson...make use of the CRISPR/Cas9 technology to engineer plants that are able to destroy DNA viruses, similar to...interested in applying gene technology to bacteria and plants. Her research involves cloning cry genes from the...against local insect pests. By developing biological agents such as insecticidal proteins, the use of chemical...

...share these improvements? You could publish a paper that happens to include your improvements, or maybe ...the thousands of other scientists using pLKO.1 variants learn about those updates? This is a problem many...information, you’ll have the option to add additional documents. By uploading your protocols onto your plasmid... to your protocols when they begin their own experiments. Need to submit an updated protocol? Just email...them public for others to use, and add your own comments. Currently you can find protocols from NEB, OpenWetWare...storage and inventory, managing orders, tracking students and research projects, electronic lab notebooks...trials and pricing options for academic labs, departments, industry, and more to fit every lab's needs....

...Quantifying the abundance of thousands of protein variants simultaneously  With increasing sequencing power...individual’s genome. But when they find genetic variants, what does that mean? Previous research shows ...approximately 75% of all protein-coding pathogenic human variants are decreased in protein abundance, possibly due... due to misfolding. But measuring variants one at a time is time consuming and unscalable.  Melissa Chiasson...Washington sought to address the effects of genetic variants in a high throughput manner. She helped develop...a method that measures the effects of sequence variants on abundance simultaneously called VAMP-seq, for...protein variant library is monitored by fusing the variants to eGFP followed by an internal ribosomal entry...

...and their research. These researchers span four continents and research diverse topics including microbiology... University of Ibadan. She works with medicinal plants such as Launaea taraxacifolia (Willd), an annual...renewable sensor, a bacterium that can sense contaminants and emit light as a response. To create this...fluorescent protein transcription in response to river contaminants and transform these plasmids into a bacterial...geographical locations. In addition to providing reagents and educational resources for scientists globally... Check out our CRISPR resources and CRISPR for plants Bacterial Expression Systems Synthetic Biology ...

... also shorter than those needed for Cas9 (42 nt vs 100 nt.) However, the newly described non-CRISPR protein...Additionally, whereas Cas9 generates blunt ends 3 nt upstream of the PAM site, Cpf1 cleaves in a staggered...smaller than any of these options (2.7 kb with 24 nt guides), so it has the potential to eclipse any of...into AAV when you add in a gRNA and regulatory elements. To solve this problem, the Zhang lab has previously...Kleinstiver et al., 2016. Until recently, two major elements of Cpf1 characterization were missing: on-target...

...mismatches in target RNA from bases 12-26 of a 30 nt target sequence. For the editing portion of the protein...sequencing, they confirmed A->I editing and found that 50 nt spacers increase editing efficiency but also increase...efficiency. They note that RESCUE is most active in a 30 nt guide when C or U base-flips are present across from...

...for a surprise! Whereas Cas9 generates blunt ends 3 nt upstream of the PAM site, Cpf1 cleaves in a staggered...doesn’t require a tracrRNA, crRNA guides are only ~42 nt long. Direct synthesis of these crRNAs should be ...be significantly cheaper than that of the ~100 nt crRNA/tracrRNA hybrid guides needed for Cas9 function...depletion assay to discover FnCpf1’s PAM sequence requirements. Cpf1’s preferred PAM is 5’-TTN, differing from...tracrRNA. Once they had determined the minimal elements of CRISPR-Cpf1, Zetsche et al. turned to characterizing...

...Sequence NGG CRAA (R = A or G) Spacer Length 20 nt 22 nt Temperature Range for Optimal Activity 35 - ... organisms: mammalian cells, yeast, drosophila, plants, worms, zebrafish, frogs, some bacteria; but not...s love of heat in two ways. The first set of experiments tested the ability of GeoCas9 RNPs to be incubated... following heat treatment. The second set of experiments tested the ability of GeoCas9 RNPs to cleave ... we haven’t thought of yet? Let us know in the comments below. And if you want to give GeoCas9 a try, ...

... by the Rinehart lab: pCRT7 NT TOPO E17TAG GFP and pGEX-6P-1. The pCRT7 NT TOPO vector contains a mutant...68292 C321.ΔA bacterial strain – Addgene #68306 pCRT7 NT TOPO E17TAG GFP plasmid - Addgene #68295 SupD plasmid...identifies a staggering ~250,000 phosphorylation events in the proteomes of higher mammals (2). How can... the importance of any of these phosphorylation events on the activity of a given protein? Classical methods...5). Optimization of this system defined three components required for translation (Figure 1): A tRNA ...the pSer-incorporated polypeptide. These three components were shown to effectively incorporate phosphoserine...inactive without requisite dual phosphorylation events on its activation loop, but can be made constitutively...

...efficiencies of multiplex editing at each site. A 75-nt human cysteine tRNA stood out from their initial ...array, DAP arrays use tRNA itself, as short as 75-nt, to both drive and process the tRNA-gRNA arrays, ...both efficiency and multiplexity, with these constraints? The Gao lab first thought to enable MBE by ...the DAP array, the Gao Lab packaged MBE and MPE elements for deliveries via adeno-associated virus (AAV...

... unprocessed or processed gRNA array (22 nt spacer with 30 nt direct repeat) mediating potent knockdown...

...CRISPR/Cas system makes use of 3 components, a guide RNA (gRNA) of about 125 nt that specifies the target, ...the mouse genome is made up of 3 x 109 nucleotides (nt), and encodes 23,000 or so genes. It would be great...the gRNA will seek out its target among the 3 X 109 nt of genetic content in the mouse genome and the Cas9...gRNA, Cas9, and donor oligonucleotide or plasmid components are brought together and microinjected into either...eggs. Or, to avoid handling embryos ex vivo, the components can be electroporated into the oviduct of pregnant...CRISPR is so efficient that you can inject the reagents directly into fertilized mouse eggs, circumventing...friends and colleagues while working with these reagents. Other than that, be nice to your microinjectionist...

... kb ~3.9kb crRNA/gRNA length gRNA: ~100 nt crNA: ~42 nt Requires tracrRNA? Yes No dsDNA cleavage...Cas9 and Cpf1 CRISPR Nucleases   Cas9 Cpf1 Components crRNAtracrRNACas9 crRNACpf1 Endonuclease...AAV transduction.  In most of Zetsche et al’s experiments, a single vector co-expressing Cpf1 and a crRNA...culture of mouse neurons and in vivo. For these experiments, cells were infected with a 1:1 ratio of two ...editing with CRISPR-Cpf1 is one of the latest developments in CRISPR technologies.  It’s a simple and effective...thoughts about Cpf1 multiplexing, leave them in the comments below.        References 1. Zetsche et al. "Multiplex...

...)  Editing window ~2 nt  85175  pBK-EE-BE3  SpCas9 (NGG)  Editing window ~2 nt  85176  pBK-YE2-BE3...BE3  SpCas9 (NGG)  Editing window ~2 nt  85177  pBK-YEE-BE3  SpCas9 (NGG)  Lower on-target activity than... than BE3; editing window ~1-2 nt  87438  pET42b-HF-BE3  HF-Cas9 (NGG)  Very low off-target activity...Campbell’s lab added two new mCherry variants to the repository. These variants can be used as tools to learn... Podcast Segment The impact of KLF4 N-terminal variants on iPSC generation Deriving iPSCs (induced pluripotent...transposons, the Woltjen Lab compared different variants of the polycistronic cassettes and discovered ... developed a new AAV approach using regulatory elements that restrict expression to this cell type. To...

...prepared in large amounts and frozen for future use or directly ligated with the annealed 24 nt oligos to generate...the spacer(20nt) on pEcgRNA plasmid as only two 24 nt oligos are required and the BsaI linearized pEcgRNA...not function properly in E. coli BL21(DE3) as transformants failed to grow after the transformation of pTargetF... This would result in the lack of recovered transformants. Our speculation motivated us to update the ...system While the above systems are great for experiments that require only single chromosomal integration...

...Kleinstiver BP, Prew MS, Tsai SQ, Topkar VV, Nguyen NT, Zheng Z, Gonzales APW, Li Z, Peterson RT, Yeh J-...Addgene. Kleinstiver BP, Tsai SQ, Prew MS, Nguyen NT, Welch MM, Lopez JM, McCaw ZR, Aryee MJ, Joung JK...The number of off-target cleavage events for the VQR and VRER variants is similar to wild-type SpCas9, ...reported the development of near-PAMless Cas9 variants. These variants, named SpG and SpRY, can target NGN PAMs...the number of CRISPR reagents continues to grow, so too will the number of reagents available through Addgene...limitation: 1) the use of novel S. pyogenes Cas9 variants with varying PAM sequences, 2) the use of Cas9...positive selection screens in bacteria to identify mutants of S. pyogenes Cas9 that were able to cleave target...

...more important because you have to target within ~30 nt of your proposed edit, which means there are so few...base editors, the intended edit must be in a 5 - 10 nt window relative to the PAM, and bystander edits are...SaCas9, NmeCas9, Cas12a enzymes, and engineered variants thereof offers additional PAM options that can...HDR for introducing edits. The same locational constraints are even more exquisite for the so-called base... JG (2020) Design and analysis of CRISPR–Cas experiments. Nat Biotechnol 38:813–823 . https://doi.org/...

...acids long, has optimal sgRNA spacer length of 22-25 nts, and is thermostable between 31-100°C. They published...novel organisms and enzymes from these extreme environments. Gearing up for sample collection at the hot...temperature readings using a heat gun, and pH measurements. We collected small water and sediment samples...Cas9 be useful? It can enable applications where reagents must be stored at elevated temperatures for long...