...and found that 50 nt spacers increase editing efficiency but also increase off-target editing within the...1090), which decreases the REPAIRv1 construct size from 4,473 bp to 4,152 bp without decreasing editing efficiency..., which deaminate adenosine to inosine in RNA, creating a functional A->G change. They fused ADAR deaminase...to dPspCas13b, but observed low RNA editing. To increase A->G editing, they employed hyperactive ADAR constructs... they hoped to destabilize ADAR-RNA binding to decrease off-targets. Of the tested constructs, ADAR2DD...

...CRISPR screen to disrupt these enhancers. The third and final step is to conduct our CRISPR screen. We start... the marvel of DNA compaction, an enhancer can increase the expression of a gene that is millions of base...stem cells, and conclude with a targeted CRISPR screen that can disrupt these enhancers. A real-world ...that have NANOG binding sites 2. Design a CRISPR screen to target and disrupt the NANOG enhancers 3. CRISPR...you’re interested in designing your own CRISPR screens for enhancers, check out the Appendix. Conclusion...precision medicine. We can apply this knowledge to create targeted disease therapies with minimal side-effects...

...Website: http://protocols.io/ Protocols.io is the creation of ZappyLabs. Currently in beta testing, the software...courses, as well as a space for research labs to create their own lab or group websites. OWW is managed... cells. If you’re familiar with the process of creating and editing Wikipedia pages, OWW will be relatively...The site allows you to comment on protocols and create lists of your favorite protocols for future reference...of the early protocol sharing websites. It was created in 1999 and allows scientists to contribute protocols...web-based lab management tool that allows labs to create accounts in which their members can share protcols...your lab’s needs. At Addgene, we’ll continue to create new molecular biology educational resources, such...

...Fluorescent Protein (GFP) has likely existed in creatures such as jellyfish for millions of years; however...physiological processes. Re-engineering GFP to increase its range of color and application Almost as.... Tsien described an S65T point mutation that increased the fluorescence intensity and photostability ...derivatives Mutation Known Function S65T Increased fluorescence, photostability, and a shift of the... the major excitation peak to 488 nm F64L Increased folding efficiency at 37C Y66W Causes the chromophore...Blue-shifts the wavelength (blue derivatives) Y145F Increases quantum yield for BFP Y145A and H148D Stabilizes...to identify particular cell populations in drug screens, to visualize micrometastases in nude mice in cancer...

...level of antigen in the sample. This figure was created with BioRender.com  Direct ELISAs A direct ... bind non-specifically to assay components and increase background staining. Direct ELISAs also lack the... antibody. Incorporating a secondary antibody increases the sensitivity of the assay, since multiple secondary... both primary and secondary incubation steps, increasing the workload and time commitment for users. This...antibodies for the capture and detection steps, which increases the specificity of the assay. The assay is also...antibody availability Longer protocolLess specificityIncreased chance of cross-reactivity Sandwich ... High specificityHigh sensitivity Longer protocolIncreased chance of cross-reactivityChallenging to develop...

...they function in nature can inform us on how to create better recombinant vectors. In this blog post, ...provide tips on how we can harness those features to create broad host range recombinant vectors.  A plasmid... host range in plasmids? Not all plasmids were created equally. Only a handful of plasmids have been identified..., from broad host range plasmids can be used to create recombinant vectors that can replicate in different...vinelandii, Brevibacterium methylicum, Caulobacter crescentus, Desulfovibrio vulgaris, Erwinia carotovora, ..., Bordetella spp., Brucella spp., Caulobacter crescentus, Escherichia coli, Gluconacetobacter xylinus,...recombinant vectors can be modified further to increase the range of hosts in which proteins can be expressed...

...Villarreal. By the late 1980s, scientists even created a codon adaptation index (CAI) that is based on... (Sharp and Li, 1987). In 2000 the Ikemura lab created the Codon Usage Tabulated from GenBank database...interest based on an organisms codon usage bias to increase translational efficiency and thus protein expression...across an entire polypeptide. Many researchers have created codon-optimization algorithms/resources (Athey ...study, codon optimization resulted in a 6-9 fold increase in expression of these channels providing scientists...to codon optimize? Although codon optimization increases protein production in certain systems, synonymous...optimization may not always be the optimal strategy for increasing protein production. If you are trying to express...

...significant knowledge gap in your field or even creating a new field. A knowledge gap is an impediment ...of experiments or a laundry list of molecules to screen has unclear utility. You cannot predict what will... have no hook. A battery of new assays for your screen result may or may not actually help. Ultimately...distinguish mechanistic questions from open ended screens and data collection. Organize your research vision...want to know the answer to your research problem. Creating a coherent unified story and model can help make...Consider going beyond the whiteboard. You may wish to create a sculpture or print a 3D model to better illustrate... His interests include the quality control of secretory proteins in the Endoplasmic Reticulum, optimization...

...USA have used AAV1 and AAV2 serotypes in order to create an atlas of the mouse brain. This atlas enables...antibody recognition of epitope tags. Thus they have created antigenic protein tags called “spaghetti monster... FPs fused to multiple epitope tags. Indeed, to create the smFPs Looger’s team strategically inserted ...antibody-based amplification of their signals is weak and increases background fluorescence. Furthermore, many of ...instance, smFPs have been used to visualize “thorny excrescence” (TE) spines in the Hippocampal CA3 pyramidal...sites and primary antibody-based detection, smFPs create much brighter signals for weakly expressed proteins...: Making the journey from axon to nucleus AAVs CREATEd for gene delivery to the CNS and PNS Resources...

...s lab performed a series of positive selection screens in bacteria to identify mutants of S. pyogenes ...sequences (Kleinstiver et al. 2015). From these screens, they identified four novel SpCas9 variants with...some cleavage with an NGA PAM. This variant may increase the specificity of genome modifications at DNA...allows for expanded PAM recognition as well as increased specificity and lower off-target activity (Hu ...proteins has and will continue to dramatically increase the number of CRISPR applications. The first non-Cas9...formation of “sticky ends” rather than the blunt ends created by Cas9. Cas12a  displays lower off-target editing...) system fuses Cas13b to RNA deaminase ADAR2 to create a specific RNA editor. Cas13 enzymes are advantageous...

...was Addgene's first distributed plasmid and was created in the lab of my thesis advisor, Dr. Bruce Spiegelman...requested item of 2005. shRNA - In 2006, we saw an increase in popularity of empty backbones for shRNA expression...requested. Ubiquitin - One particular gene we saw increased interest in from 2008 to 2009 was ubiquitin. The...popular with scientists, but 2012 was marked by increased deposits (and requests) of biosensors, optogenetics...and for so many different purposes that we had to create a CRISPR page to keep up with all the new plasmids...of plasmid sharing? Check out this infographic created by our own Addgenie, Laura Veckerelli. Click on...

...Harvard. There’s an astounding number of ways to create chromosomal mutations in bacteria, so many that...of surrounding genes. However, there are ways to create scarless mutations, ones that don’t leave any undesired..., CRISPR-Cas9 technology has also aided in the creation of scarless mutations, both in stand-alone systems...systems (Jiang et al., 2013), and those which increase the efficiency of Lambda red (Pyne et al., 2015;... recombineering (Khetrapal et al., 2015). They created vectors for Lambda Red that contained one of eight...aeruginosa. These toxins were a mix of Type VI secreted effector toxins, toxin-antitoxin type toxins, ...Enterobacter cloacae, and Serratia marcescens and have created dozens of deletions as well as insertions with ...

...therapeutic interventions for cancer. This screening platform is an incredible boost to the cancer research community...Barcoding (HiTMMoB) technique (Tsang et al., 2016), creating a collection of plasmids each carrying a single...mutations The researchers used the collection to screen individual perturbations in vitro using Ba/F3 and...~22% of the mutations, however, significantly increased cell viability, and these “activators” may be ...annotation of rare gene aberration drivers of pancreatic cancer." Nature communications 7 (2016): 10500...

... in yeast Base editing CRISPRi in Caulobacter crescentus Multiplexed gRNA for human pluripotent stem cells...editing. Using several rapid and cost-effective screening methods, David Liu’s lab identified and validated... base editor plasmids CRISPRi in Caulobacter crescentus Figure 1: Serial dilutions of CRISPRi in...adapted from Guzzo et al., 2020. Caulobacter crescentus is a popular model organism for studying the ...ctrA sgRNA and saw that the levels of ctrA mRNA decreased after 20 minutes in xylose (which controls expression...methylcytosine oxidation to DNA repair, which increases activation of methylation-silenced genes. Read...

...experiments but we can definitely give you credit for it and increase your impact! Download ReFigure extension...along with the article metadata, legend and user created notes on a “Pinterest for scientists”? ReFigure...Linkedin and ResearchGate profile. To see how to create ReFigures in a few minutes, visit our YouTube [email protected] Please support ReFigure by creating ReFigures and answering this 2 minute survey! ...impact of teaching and doing science is rapid, concrete and open to innovation by all.  Additional Resources...

... biosensor creation, and custom FRET biosensors can be created using the cpFRET kit created by Olivier...Robert Campbell’s lab display calcium-dependent increases in fluorescence of up to 11,000-fold. FRET biosensors...biosensors. Blue flame “cameleon” calcium sensors created by Roger Tsien’s lab contain two fluorescent proteins...calcium binds, it triggers CaM/M13 interaction that increases FRET transfer between the fluorescent proteins...

...positive selection, negative selection, and/or screening. We’re focusing on positive and negative selection...in this blog post, but don’t worry, we’ll cover screens in a future post. Positive selection: Cells that...with or without your inserted DNA) and further screening will help you determine if the plasmid backbone...arises in yeast. For example, Markus Ralser’s lab created a yeast prototrophy kit containing 23 plasmids ...bacteria to grow on sucrose. Plasmid cloning: Creativity with naturally occurring genes  At this point... scientists are finding more and more ways to creatively perform selections in plasmid cloning. Additional...

...are under a Creative Commons 3.0 license, which requires users to give appropriate credit, provide a link...about new scientific techniques and cool plasmids. Creating graphics were usually the last thing I thought..., I found some free design software that let me create graphics that better communicated the science in...reasons why this was the first program I used to create graphics for the Addgene blog. However, it has ...requested, although there’s no guarantee Biorender will create it. Additionally, you can upload your own images...

...membrane depolarization increases the BRET signal whereas hyperpolarization decreases the signal. The authors...technologies that can accelerate your research. The creation of NanoLuc® Luciferase from a rather dull 19kDa... by NanoLuc® luciferase (NLuc) and set about to create an in vivo imaging reporter by linking two CyOFP1...-Lanterns with NLuc and called their improved creations Enhanced Nano-Lanterns. These great imaging tools...the two fluorescent proteins together thereby increasing their FRET signal. FRET is a non-radiative transfer...overlap with this optogenetic tool, they needed to create a calcium sensor with the following characteristics...

...International Mentorship Program (IMP) USA-Europe was created. IMP as an idea was conceived and developed by ...the specific technical and logistic support to increase the chances of success, but also nurture a supportive...country of origin with an international mentor creates a closer and more approachable relationship for...at IMP In these sessions, mentors helped mentees create “Career Development Plans” which provided paths...developmental needs and resources needed to pursue them, increased their network of contacts important for their ...Conference highlights included remarks by the Spanish Secretary of Research, Development, and Innovation, D. Carmen... as they witness their student’s progress and increases in self confidence. These improvements, combined...