...systems Gamma-retroviral vectors have a relatively high transduction efficiency, but in contrast to lentiviral...small-molecule libraries and can be combined with high-throughput sequencing to perform multiplexed analysis... cassette. It can be used to produce a ~10-fold higher titer of lentiviral particles than the original...lentivirus provides significant genetic power and allows high throughput genetic modulation studies in mammalian...

...NGG) in both distribution and GC-content, which might improve editing in AT-rich genomes. Additionally...editing efficiency for the Cpf1 orthologs was only slightly lower than that of the widely used SpCas9 and ...that AsCpf1’s off-target rate is similar to that of high fidelity Cas9s eSpCas9 and SpCas9-HF1. Both AsCpf1... cleaves relatively far away from the PAM, NHEJ might retain the PAM site. Therefore, if HDR does not ...

...elements. A high affinity terminator may be desired for multi-cistronic constructs where high termination...terminators, such as T7 and rrnB, as well as engineered high-efficiency terminators such as T0. Rho-independent...poly-uracil tract as shown in the figure to the right. The tertiary structure of the hairpin-DNA complex...

...RNA. Measuring p24 levels in a viral prep is straightforward and rapid as there are several commercially...Studies have found that values can be 10-1000 fold higher than for functional assays depending on the vector... collected lentiviral prep may be significantly higher than the same prep that has been stored in the ...routinely produce virus may want to choose more straightforward approaches such as the FACS-based or colony-forming...

...Maya Kostman for the IGI.     Here are some highlights of the plasmids shared over the last year and... more tools, head over to this blog post that highlights five different methods to use CRISPR to detect...

...location bind to Cas9 with canonical sgRNA structure (right). Created with BioRender.com. What to edit The...3 or more mismatched bases can often evade MMR (right). Created with BioRender.com. For specific prime...prime editors bound to sgRNAs only drive nicking (right). Start with nicking sgRNAs ~50 bp from the prime...2024). Figure 9: epegRNAs (left) and PE7 (right) offer two different strategies for protecting pegRNAs...

...genome is made of protein-coding genes, and you might be wondering what the rest of our genome could possibly...non-coding RNAs! You probably didn’t see that coming, right? Non-coding RNAs (ncRNAs) are transcribed but do...versatile option. It can be used to target lncRNAs with high specificity, particularly in applications requiring...Hazan J, Bester AC. CRISPR-Based Approaches for the High-Throughput Characterization of Long Non-Coding RNAs...

...just for lab specimens — there is evidence that lightning can also promote gene transfer in soil bacteria...microbial offense One way to triumph over your neighbors is to eliminate the competition, and Penicillium...specialized degradative capabilities are often tightly regulated to be active only in the presence of ...Simonet, P. (2001). Laboratory-scale evidence for lightning-mediated gene transfer in soil. Applied and Environmental...

...If you’ve ever been looking for just the right CRISPR vectors on Addgene and found instead ones that ...Doench reports even the greenest members of his lab, high school interns, were able to use Fragmid to design...making it easier to get the vectors that are just right for your experiment. As always, we recommend checking...components by clicking the blue text in the upper right-hand corner.      Step 3: Reviewing the ...

...the Mobile CRISPRi cassette flanked by left and right Tn7 end sequences. Donor bacteria with a chromosomal...scheme. Mobile-CRISPRi integrates downstream of the highly conserved glutamine-fructose-6-phosphate aminotransferase...cell, the CRISPRi DNA (purple) flanked by left and right Tn7 end sequences (green) integrates downstream ...mirabilis, had only ~1 transfer per 1,000,000 colonies. Higher transfer rates are better suited for genome-wide...

...of them: Characteristic ESC ASC Self-renewal High Low once isolated from the human body Usage Universal...specific cell types can be produced Ethical concern High. Embryos are destroyed during ESC isolation. Low...post-transplantation rejection when used therapeutically High Low (if ASCs are isolated from the patient) For...each other in many aspects. Like ESCs, iPSCs have a high self-renewal rate and can be used universally to...

...organelle, and protein localization. For instance, you might dye your cells and look at them under a microscope...population of timer indicates the age of the pool: the higher the ratio, the older the pool. FP timers have ... al. Protein Excitation (nm) Emission (nm) Brightness pKa Maturation Plasmids Fast-FT 403 (blue),...sections are formed. In synthetic biology, FP timers might be able to tell researchers whether old or new cells...

...but, to start, let's review why NgAgo is in the spotlight and take a moment to remember that NgAgo has only...a single experiment. Also, the NGG PAM sequence might not always be available at an optimal distance from...mean the technology is dead in the water. On the bright side, successful indel generation and epitope knockin...supposedly alien 5’-phosphorylated ssDNA guides, which might be rapidly degraded by the cellular machinery, leaving...

...At Addgene, we've set out to fill this gap by highlighting that researchers learn many transferable skills...the lab and by offering advice on areas where you might need some help. Today in our transferable skills...Although this approach to negotiation is the most straightforward, it can mean that everyone leaves disappointed...

...Cas effectors to light. One lesser-known Cas is waiting for its chance in the spotlight — Cas3. Cas3 can...Cas effectors can’t, while maintaining relatively high efficiency. These alterations are achieved through.... E., Bravo, J. P., Ramos, D., Hibshman, G. N., Wright, J. T., & Taylor, D. W. (2023). Structural snapshots...

...added to media, buffer, etc., which will bind and light up a tagged protein for imaging. Given its popularity... like the SMASh tag does. However, it is a straightforward system – HaloTag and its PROTAC are all that...tag Are all tags created equal? No! Choosing the ‘right’ tag does depend a lot on your unique experimental...function is easily perturbed by tags, then a small tag might be the way to go (dTAG/SMASh). If you don’t want...

...being used to perform genome-wide screens and highlight some of the reagents that have been made publicly... library. Choosing the CRISPR library that is right for you There are several factors to consider when... Findings from this type of experiment can shed light on the mechanism by which cells gain resistance ...

...are studying a novel gene, is not always a straightforward process. Rather than try to directly detect...cells that are expressing your gene of interest at high levels. Unlike vectors that express screenable or.... (J Virol. 1988 Aug;62(8):2636-43.) PubMed. 3. Highly Efficient Multicistronic Lentiviral Vectorswith...):845-60. doi: 10.1089/hum.2008.188.) PubMed. 4. High cleavage efficiency of a 2A peptide derived from...

..., so it is pretty important to get this process right the first time. I’ll lay out some guidelines here...transformation process The process itself is straightforward. When your agrobacterial cultures are ready...s flowers inside. 50 mL falcon tubes or plastic weigh boats are both great options for this, depending... et al. (2007) ‘Improved Gateway Binary Vectors: High-Performance Vectors for Creation of Fusion Constructs...

...contributed by guest blogger Tessa Cressey. The highly pathogenic Ebola virus belongs to the group of ...necessity for working with this virus under the highest biosafety level conditions, BSL-4. In this regard..., it is important to choose cell lines that are highly transfectable.  The amounts of each plasmid transfected...vary between labs, initial titration experiments might be helpful to achieve optimal results. It is important...