...nuclease domain. Feng Zhang's group utilized the bi-lobed architecture of Cas9 to engineer a series of "split...memory One of the goals of synthetic biology is to engineer cells capable of recording permanent "memories...display and high throughput binding assays, to engineer photoswitchable dimers which show over a 50 fold...activating endogenous genes Moritoshi Sato's Lab has engineered CRISPR-Cas9 to create a photoactivatable transcription... or (4) multiple sgRNAs with Cas9 for genome engineering. The versatility and ease of generating complex...potential applications of CRISPR/Cas9 in genome-engineering. One particularly attractive application involves...genes involved in neurogenesis, which had proven challenging using the existing dCas9-VP64 activator. The ...

...Addgene to advance their research. Louis Bengyella Dr. Bengyella’s work has focused on elucidating the molecular...our first two plasmid grant awardees: Drs Louis Bengyella and Kwabena O. Duedu, both from the University...in Ghana. Read on to learn more about doctors Bengyella and Duedu and how they plan to use plasmids from... microbial pathogens. This work has given Dr. Bengwella expertise in using bioinformatic and proteomic...associated with pathogenesis. In his future work. Dr. Bengyella would like to use the expertise he’s developed...development of uterine tumors. According to Dr. Bengyella, this class of tumors is “a major public health... Ghana. We’re excited to see what impact Dr. Bengyella’s work will have on women’s health in Ghana and...

...funding, bioethics in the age of CRISPR, and the challenges of science becoming increasingly politicized...., “The Professional Scientist,” addresses the challenges of succeeding in science if you’re anything other... course the rapidly changing world of genome engineering via CRISPR.  Though the individual topics are...ramifications. Readers unfamiliar with academia’s challenges with publication and funding may be dismayed ...The three chapters in this section describe the challenges of trusting science in a “fake news and post-...has played in these fields, and seeing names like Feng Zhang, Jennifer Doudna, Karl Deisseroth, and Ed ...describes the author’s thoughts on science’s biggest challenges over the coming years. It is arguably the most...

... in a variety of cells and organisms. However, lengthy genetic payloads and insufficient transcription...assembled tRNA-gRNAs is placed downstream of a lengthy promoter, which is used to drive the expression...efficient MBE with such a strategy, the need for lengthy promoters, such as U6 or EF1α promoters, has limited...derived from human or plant source), by tuning the length of 5’ leader sequences of a given tRNA to achieve...either from human or plant sources) in both the strength as a promoter and the activity to release gRNAs...results of their MBE efforts, the Gao Lab also engineered a DAP array for prime editing, achieving up to...also enable more convenient multiplex genome engineering of crops or allow for sophisticated CRISPR screenings...

...photobiology: why use light? In synthetic biology and bioengineering, scientists seek to directly probe and build...problems; inexpensive tools such as LEDs can be engineered to deliver precise, consistent, and controllable.... Complicated patterns of light intensity and wavelength can be used to study dynamic processes. Conversely...Conversely, varied input patterns may also be used to engineer new pathways with sophisticated gene expression... of edge detection. Tabor et al. subsequently engineered a second photosensitive TCS activated by green...-friendly. They experimented with the promoter strength of various components, removing inducible promoters...could be made suitable for various scientific or engineering applications.   References Refactoring and optimization...

...Cas9 and Cas12a, creates challenges for in vivo delivery and cell engineering applications. CasMINI, a...gene perturbation The Penn State Protein Ladder Engineered miniature CRISPR-Cas system for mammalian genome...flexible, modular tool for labeling. Here, Liu et al. engineer a chimeric single chain variable fragment (scFv...gels and can be detected on Western blots via engineered immunoglobulin binding domains.  Santilli, R.T.... https://doi.org/10.1038/s41598-021-96051-x Engineered miniature CRISPR-Cas system for mammalian genome... Figure 4: Overview of CasMINI for genome engineering applications. Image from Xiaoshu, et. al. 2021...system opens up new possibilities in both cell engineering and gene therapy.  Xiaoshu X, et al. Molecular...

...technology since its initial applications for genome engineering in 2012.  Many of these advances have stemmed... of dCas9; and 2) CRY2FL-VP64, a fusion of full-length CRY2 and the transcriptional activator domain VP64...PAM and had similar targeting specificity as full-length Cas9 (flCas9). When triggered by blue light (470nm...light-dependent (Yu et al., 2020). To express the N-Cas9, Haifeng Ye’s lab used the bacterial phytochrome BphS which...employed a photoactive strategy which had been engineered from naturally occurring photoactive proteins... specific site on the Cas9, the group used an engineered pyrrolysyl tRNA/tRNA synthetase pair which would... Grimm D, Correia BE, Eils R, Niopek D (2018) Engineered anti-CRISPR proteins for optogenetic control ...

...has developed a miniature Cas system (CasMINI) engineered from the uncultivated archaea Cas12f (Un1Cas12f1... system. The CasMINI system was developed by engineering the Cas12f scaffold gRNA and making strategic...CRISPR editing with a hypercompact Cas12f1 and engineered guide RNAs Yong-Sam Kim’s lab also created a ...the gRNA scaffold through five rounds of gRNA engineering.  They did not modify the Cas12f1 enzyme. The...facilitate its packaging into virus-like particles. Feng Zhang’s lab adapted the native PEG10 RNA sequences...plasmid tools to optimize prime editing systems. Engineered pegRNAs improve prime editing efficiency In the...the 3’ end with a structured RNA motif. These engineered pegRNAs (epegRNAs) protect the RNA from degradation...

...while writing each section. Research challenge The Research Challenge motivates the request for application...creates strong, well-directed meaning and delivers strength to the sentence. Below are the typical sections...how you, the expert, will address the research challenge. Now is the time to make bold statements. While...solution is the best approach to the research challenge. Demonstrating expertise in the field and past...how your innovation will address a variety of challenges and impact the field - its encouraged to dream...time budget writer, you may find this section challenging because it’s hard to determine the exact value... differs from publication writing and may be challenging to master at first. Additionally, reach out to...

...structure-guided engineering of the PAM-interacting domain of Cas9. After multiple rounds of engineering, they developed...the "perfect" guide sequence for your genome engineering experiment. With CRISPR entering the clinic for... Nureki lab generated SpCas9-NG, a rationally engineered Cas9 with NG PAM compatibility, as the name implies...R.A. et al. PAM-flexible genome editing with an engineered chimeric Cas9. Nat Commun 14, 6175 (2023). DOI...Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants. Science 368,290-296(2020...Plasmids available here)   Nishimasu, H., et al.,Engineered CRISPR-Cas9 nuclease with expanded targeting ...Requirement and Expanding Beyond Cas9 xCas9: Engineering a CRISPR Variant with PAM Flexibility History...

...proteins or TALE proteins, proteins had to be re-engineered for each gene, making wide-scale gene activation...gene activation: rather than requiring protein engineering for each loci, CRISPR/Cas systems only require...methods rely on fusion proteins while others re-engineer components of Cas systems themselves. SunTag, ...Mediator (SAM) Description SAM uses specially engineered sgRNAs to increase transcription. This is done... through creating a dCas9/VP64 fusion protein engineered with aptamers that bind to MS2 proteins. These...activation of bacterial gene expression using an engineered CRISPR-Cas system. Nucleic Acids Research 41:...Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature 517:583–588 . https...

... Kucera, M.Sc. and Eric Cantor, Ph.D. from New England Biolabs. Golden gate assembly limitations Embraced... assemblies that enable biosynthetic pathway engineering and optimization. However, current best practices... reduced these limitations through the use of engineered Type IIS restriction enzymes and careful choice...redesign was guided by both advances in the re-engineering of the original BsaI-HF® Type IIS restriction...In conjunction with BsaI-HFv2 (NEB #R3733), re-engineered to provide improved Golden Gate performance, ... Kucera, M.Sc. and Eric Cantor, Ph.D. from New England Biolabs. Becky Kucera is a Principal Development...Development focusing on DNA cloning and assembly, New England Biolabs, Inc.         Eric Cantor is a molecular...

...recombinant protein purification and metabolic engineering. If instead you want to build genetic circuits...biology MoClo applications, but there are more! Engineering disease resistance through gene stacking, increasing...localization signals, plant markers, and reporters (Engler et al., 2014). This kit is a great starter for ...vectors, several delivery systems which can be engineered into the TUs, and even viral gene silencing vectors...kit has a focus on transformation and genome engineering. This is a specialized system with parts including...confined to that arena! The Mammalian Toolkit was engineered specifically for the types of expression and ...Multiplex MoClo Toolkit for Extensive and Flexible Engineering of Saccharomyces Cerevisiae.” ACS Synthetic Biology...

...excitation wavelength (Salomonnson et al. 2012). They can also be excited efficiently at wavelengths between...development of cell sorters together with the engineering of fluorescently labelled antibodies made it ... lymph nodes at different times after antigen challenge, scientists were able to track where GFP labelled...et al. 2016, Cavanagh et al. 2008)? Technical challenges of two photon microscopy Intravital imaging is... at steady state or under physio-pathological challenge. However technical specificities have to be taken... also important to finely tune the excitation wavelength as it has been shown that even a small incremental...incremental change of the excitation wavelength can significantly affect emission intensities from fluorescent...

...Ellis does research in synthetic biology and bioengineering including a recent effort to construct a synthetic... SynBio. Chapter 3: Synthetic biology as the engineering of biology at the level of DNA using modular,...in the Ellis lab including yeast and E. coli engineering, microbial cooperation, and antibiotic production...lectures synthetic biology for the Department of Bioengineering. He graduated from Oxford University and then...supervision of Prof Jim Collins and specialising in engineering gene regulation in yeast. Dr. Ellis is now one...undergraduate projects. Special thanks to our sound engineer/producer, Eddy Page! Edward B. Page is a Digital...Production Specialist at Addgene. He is the audio engineer and producer for the Addgene Podcast.      Additional...

...base editor when they realized that MPG could be engineered to act directly on unmodified G bases, resulting...Tong, Liu, et al., 2023). gGBE consists only of engineered MPG fused to nCas9. First, MPG removes the G ... target guanosine instead, perhaps analogous engineering of UNG, which excises uracil, could allow it ...considerably. And there’s so much more exciting protein engineering going on in the base editing field! From CRISPR-free...., Qu, C., Zhu, Y., Guan, Y., Wang, L., Liu, M., Feng, B., Song, G., … Li, D. (2024). Adenine transversion...deaminase-free base editors for G-to-Y conversion by engineered glycosylase. National Science Review, 10(8), ... T-to-S base editor and C-to-G base editor by engineered human uracil DNA glycosylase. Nature Communications...

...University, Jieze Zhang, an intern in the Yang Lab, and Sheng Yang, a professor in synthetic biology from the ...pEcCas/pEcgRNA system. This system works well for engineering single genome edits but the double strand breaks...interrupt genes (or gene clusters) in metabolic engineering. So far, we’ve used the MUCICAT system in two...compared to the industrial strain. In our metabolic engineering case study of N-acetylglucosamine biosynthesis... systems to choose from. Ready for metabolic engineering with improved cargo dedicated to gene (cluster... optimizing strains with low efficiency.       Sheng Yang obtained his Ph.D. from Shanghai Institute ... phenotypes and to design a new generation of engineered cells for green chemistry and biomedical applications...

...BioRender.com. How does it work? First, an engineered prime editing guide RNA (pegRNA) that both specifies... site and contains the desired edit (or edits) engages the prime editor protein. The prime editor consists...introduced, relies on the PE2 enzyme. PE4 and PE5: Engineering mismatch repair Even in PE3 and PE3b, prime editing...editing outcomes, including indels. Therefore, they engineered a dominant-negative mutant of the protein MLH1... RNA pseudoknots, calling this longer RNA an “engineered pegRNA” or epegRNA. Although the addition of ... which was made possible through their use of engineered virus-like particles. However, there’s still ...(2023). Phage-assisted evolution and protein engineering yield compact, efficient prime editors. Cell,...

... protect samples during cryo-EM grid freezing Engineered and evolved optimizations for prime editing CHARM...https://doi.org/10.1101/2024.02.06.579238.    Engineered and evolved optimizations for prime editing By...applications! Delivering prime editors (PEs) via engineered virus-like particles (eVLPs) offers an option...the donor sequence. The latest work evolves and engineers Bxb1 recombinase variants to optimize PASSIGE ..., P. Z., Palczewski, K., & Liu, D. R. (2024). Engineered virus-like particles for transient delivery of...recombinases and prime editing. Nature Biomedical Engineering, 10.1038/s41551-024-01227-1. doi:  https://doi.org... prion protein by AAV-mediated delivery of an engineered compact epigenetic editor. Science, 384(6703)...

...repair and genome engineering CRISPR-Cas9 has revolutionized the genome engineering world and made targeted...immediately applicable in DSB-induced genome engineering, it has biological importance for the repair ...CRISPR forum moderated by the Zhang lab CRISPR Challenges: Standardization and HDR CrispyCrunch Optimizing... (2018). Pubmed. CRISPR References: Genome engineering using the CRISPR-Cas9 system. Ran FA, et al. ...546; (2015). PubMed. Optimization of Genome Engineering Approaches with the CRISPR/Cas9 System. Li, K...PubMed. Making designer mutants in model organisms. Peng, Y. et al. Development 141; (2014). PubMed. Enhancing..., et al. Poster at Keystone Precision Genome Engineering. 2017.  Easi-CRISPR: a robust method for one-...