...them! PE2: Improving reverse transcriptase efficiency Building upon prior reverse transcriptase research...humans and cataloged in the ClinVar database. Previously developed genome editing methods using nucleases...prime editor consists of a Cas9 nickase fused to a reverse transcriptase. The Cas9 nickase is guided to the...sequence hybridizes to the target site, and the reverse transcriptase domain copies the desired edit, directly...wild-type Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase to the C-terminus of Cas9 H840A nickase...every ~8 base pairs on either DNA strand, many previously developed base editors with a <8 base-pair editing...the edit, the Liu lab turned to a strategy they previously used when they developed base editing. By nicking...

...systems reviewed here. The only required components are 1) a SMASh-tagged protein and 2) asunaprevir (small...and exogenously expressed proteins. Here we will review what advantages degron tags can offer over other...systems – CRISPR, siRNA, etc. Firstly, they are reversible mechanisms of control. Protein degradation can...the drug has been removed. The degradation and reversal are rapid compared to other systems because the...comes to degrons, you have options! Below we will review the 4 most common types of degrons and some of ...the presence of a specific protease inhibitor, asunaprevir, the viral protease can no longer self-cleave...addition to having the coolest name of all the tags reviewed here, the SMASh tag does offer a few other unique...

...formal peer review. Some organizations, including some journals, also support open peer review or curation... peer review itself can be prone to biases or errors, with a lack of transparency in most review processes...authors on a public server independent from peer-reviewed publishing in a journal.  Accelerating Scientific...scientists and advocates have argued that while peer review is valuable, modern scientific publishing has a...or endorsements. In contrast, journal-based peer review is usually a confidential process among a few individuals...productivity that can be shared with hiring and review committees, as opposed to the opaque (and, admittedly...preprints, and a growing number of funding bodies and review committees encourage and consider preprints as ...

...L-hisitidine no S. cerevisiae  no yes   URA3 pyrimidine (uracil) yes - Grow with 5-FOA. S. cerevisiae yes - This...of a nitrogen source. S. cerevisiae  no no   LEU2 L-leucine no S. cerevisiae yes - This can complement...explain how they differ from the bacterial elements previously described. Why do scientists use yeast vectors...pertains to ORIs in budding yeast, Saccharomyces cerevisiae; however, we’ve also noted some features required...Schizosaccharomyces pombe, vectors at the end.) In a previous post, we briefly discussed how the regulation ...if specific regulatable elements are included (reviewed here). Plasmids for use in S. pombe, on the other... TRP1 L-tryptophan yes - Grow with 5-FAA. S. cerevisiae no  no TRP1 alters some yeast phenotypes. This...

...publication of peer reviews so we can see how and why papers change throughout the review process. This will...reproducibility. Cause 1: Inability to repeat previously performed experiments Researchers should be able...incredibly useful technique simply by following the abbreviated methods section found within a published manuscript...additionally averse to producing papers that repeat previously published results. This over-reliance on the ...will hopefully push reviewers towards more productive criticisms that focus less on perceived novelty ...recognizing that we’re biased and taking steps to prevent our biases and goals from effecting our experiments...things I’ve read comes to this: we need others to review our experimental designs and we need to be transparent...

...and it grows fast to boot. The yeast strains S. cerevisiae and S. pombe have dominated the research scene...are there even other yeasts? In this blog we will review an alternative model yeast strain and discuss why...used yeast strains: S. pombe (fission) and S. cerevisiae (budding). Yeast make for an excellent model ...genetic manipulation The first eukaryote (S. cerevisiae) to have its genome fully sequenced There are...wondering if the “biotech yeast” is S. pombe or S. cerevisiae. Actually, it’s neither. The most commonly used...phaffii and other yeast strains.     S. cerevisiae S. pombe K. phaffii Growth properties... is only distantly related to S. pombe and S. cerevisiae; evolutionarily it evolved much slower and bears...

...only active under specific circumstances. In a previous post, we discussed inducible promoters, which ...bind to the operator. The bound repressor then prevents transcription from occurring, which means that...to the tetracycline operator sequences (TetO), preventing transcription. In the presence of tetracycline...binds tTA, resulting in a confirmation change that prevents binding to the promoter and turning transcription...as well as the inducible manner detailed in our previous post. In the past 30 years since the original ...binds cTA, resulting in a confirmation change that prevents binding to the promoter and turning transcription...as well as the inducible manner detailed in our previous post. While originally developed for use in mammalian...

.../10.1101/2023.12.12.571321.  Revolutionizing Gene Therapy: The REVeRT Dual AAV Vector System By Vaibhav...sensors AAV vectors for targeting microglia The REVeRT Dual AAV Vector System Neurodegeneration Collection..., 2019, including modifications to the Cas9 or reverse transcriptase enzymes, inhibition of host cell ...traditional AAVs, the Becirovic Lab has developed REVeRT, for gene delivery using reconstitution via mRNA...2023 under a CC-BY license.  The authors used REVeRT to functionally reconstituting coding sequences...disease-relevant large proteins. They demonstrated REVeRT in various contexts in vitro and in vivo, including...human retinal organoids and live mice. Importantly, REVeRT avoids the introduction of extraneous genetic material...

...-depth review of Cpf1, check out this blog post or see Addgene's CRISPR guide page for a review of Cas9... cleave the lentiviral RNA, preventing packaging of the virus.  Reversing the orientation of the direct...RNA from Cpf1-mediated cleavage. This reversing would also prevent processing of the crRNA array when expressed...generally more efficient. Pro-Tip Make sure to reverse the orientation of the crRNA’s direct repeats when...expressed in cells, but notice how Zetsche et al reverses the orientation of the promoter driving expression...(bolded red arrow).  This undoes the original reversing of the direct repeats, leading to expression of...

...Performing peer review at the request of your supervisor is not considered peer review. The peer review request...achievements. Participating in the scientific review process as a reviewer as proof of your judging the work of...already reviewing papers that you should start contacting journal editors to request to be a reviewer. Proactivity... Participating as a judge (including as a peer reviewer for journals, acting as a journal editor) of the...you specifically. You must also complete the peer review for it to be counted as proof of judging. Merely...case.  You can still assist your PI in the peer review process, but remember you cannot use it as documentary...evidence for your case – the invitation to be a peer reviewer needs to be addressed to your own personal email...

... useful. Step 4: Read and learn from the reviewers Reviewers may take 6-12 months to give feedback on...of the art technologies. Therefore, take time to review the literature and offer educated insight into ... the agency and benefits the field reminds the reviewer why your project is the project to fund. Using...limits) provided in this section will convince the reviewers that the proposal is strong, well-thought out,...your advisor to supply a budget form used in a previously funded grant application to see how funds were...application. Make sure to invest time and consider the reviewer's comments. Whether the proposal is funded or not...for improvement, deliver outsider opinions, and reveal items to include for the next application. Although...

...was to be revisited at meetings when sufficient time, additional public fora for data review, and more... Bioethics. On November 25, the MIT Technology Review dropped a bombshell report. A scientist working... were born in a twin live birth several weeks previously. While this claim has yet to be independently...report on the ethics of “Novel techniques for the prevention of mitochondrial DNA disorders.” This document...public views on emerging IVF techniques designed to prevent the transmission of mitochondrial disease;” The...Council began their research into both novel and previously established ethical issues raised around mitochondrial...stop shuffling our feet on necessary actions to review and decide on the future of genome editing technology...

...guide always gets an expert review, separate from the author, and a second review from someone less familiar...channel just for asking people if they can write or review things and another for asking specific science ...answers from our incredible scientists. Expert reviews on blog posts are done on a case-by-case basis,...case of the antibody guide, primary research and previously written Antibody 101 blog posts were used, as...panel recommendations. A few discussions in the review process led to quick dives back into the literature...sharing repositories. Then we format, do a final review to catch those pesky typos, and hit publish. Making...content is an iterative process. We are constantly reviewing the resources we currently offer to identify gaps...

...off-target activity, and prevent cytotoxic effects. For a more detailed review, check out our blog post...externally control the location, timing, and reversibility of the genome editing process. Read on to learn...expression while imparting spatiotemporal control, reversibility, and repeatability. The system developed by ...ASCL1 gene could be spatially controlled in a reversible and repeatable manner. With their light-activated... after 30 hr. The system was also shown to be reversible and repeatable when blue light (450nm) was cycled...paCas9-2 system could be spatially controlled and reversibly activated by turning blue light on and off. As...repressor (dCas9). The activity of all variants was reversible and repeatable. Since these initial studies ...

... sites in the same orientation preventing the transgene from reverting. Figure created with BioRender.... (FSF) sequence prior to an ATG start codon to prevent transcription in cells where the recombinase was...direction of arrows) the inverted transgene is reverted into the sense orientation and one of the unused... diagram showing the mechanism of spontaneous reversion during AAV plasmid amplification. On the left ...cre-recombinase sites lead to greater frequency of reversion and leaky expression. On the right non-homologous...scrambled recombination sights lead to reduced reversion and less off-target expression. Figure created...2020) it was determined that the optimal titer to prevent off-target expression of the sensitive FLEx-split...

... and identifying >88% of previously identified essential genes while previous Cas9 and shRNA library screens...and non-essential genes for both pathways had previously been identified (Hart et al, Bassik et al). The...10% false discovery rate and identified 67% of previously identified ricin toxicity genes. The screen also... also discovered several genes previously not associated with ricin regulation, including almost all genes...Results from the screen also reproduced several previously observed characteristics that influence gRNA ...A systematic mammalian genetic interaction map reveals pathways underlying ricin susceptibility." Cell...to conduct genome-wide screens with CRISPR/Cas9 Review the basics of CRISPR with this cheat sheet Read...

...structures, and genetic mutations are not easily reversible. Late last year, the Akhmanova Lab published ...This design allows highly localized, rapid and reversible microtubule severing and has very low dark-state...Mahlandt et al., 2023). These Opto-RhoGEFs provide a reversible and non-invasive way to activate a RhoGTPase ...light-induced binding of the plasma membrane. In previous publications, they described tools to control ... fusion protein away from its usual location, preventing it from carrying out its proper function (Figure...with these CIB1-Rabs in other contexts are sure to reveal even more insights. Super-duper lipid editor And...composition of the membrane itself? The Baskin Lab previously published OptoPLD, an optogenetically controlled...

...Cells. Annual Review of Neuroscience. 2022 Jul 8;45:447-469. https://doi.org/10.1146/annurev-neuro-111020... Cells. Annual Review of Neuroscience, 45(1), 447–469. https://doi.org/10.1146/annurev-neuro-111020-100834...The blood-brain-barrier (BBB) exists to prevent substances in the blood, like proteins and viruses, from...interest, while miRNA target sites can be used to prevent expression in cell types not of interest. Systemic...both mouse strains. It is therefore important to review the literature for use of a capsid in your strain...al. 2022. Image created with BioRender.com. Abbreviations: AAV, adeno-associated virus; GRE, gene regulatory.../ENEURO.0373-21.2022 Brown, D., Altermatt, M., Dobreva, T., Chen, S., Wang, A., Thomson, M., & Gradinaru...

...TAAGCAGAATTCATGTGGCATATCTCGAAGTAC-3'. For the Reverse Primer, the design is similar, but we need to use the reverse complement to get...primer and the downstream restriction site to the reverse primer. Next, we need to examine the DNA sequence...homology to the ORF). We now need to generate the reverse-complement of this sequence so that we can successfully...successfully amplify the ORF. You can generate the reverse-complement using existing software (a quick internet...others). If we put the sequence we chose for our reverse primer (5’-TGGCATATCTCGAAGTACTGAGCGGCCGCTAAGCA-...TGGCATATCTCGAAGTACTGAGCGGCCGCTAAGCA-3’) into this calculator we get a final Reverse Primer sequence of 5’-TGCTTAGCGGCCGCTCAGTACTTCGAGATATGCCA...

...is difficult to replicate.  Why is replicating previous research findings so difficult to do?  There are... so it is unlikely to be caught during the peer review process, which often focuses more attention on ...between the original setting and the new one, previously unaccounted for, may lead to different results...finding we can be more certain that some important, previously unknown variable actually makes a big difference...of Open Science. With a Registered Report, peer review occurs before results are known. The author submits...journals before a study is conducted. Editors and reviewers recommend changes, and if everyone agrees that...proposed experiment. Furthermore, the journal and reviewers are incentivized to make sure that any possible...