...protocol was developed for A549 cells but can be adapted to a variety of target cell lines and selection...

...Escherichia coli TadA, a tRNA adenosine deaminase. Like cytosine base editors, the evolved TadA domain is fused...on a second ADAR variant that displays improved specificity, and editors carrying an ADAR truncation that...break, Cas3 initiates single-strand DNA degradation. This degradation can continue for many kilobases in one...Publications CRISPR Overview Bacteria have an interesting adaptive immune response that detects and eliminates foreign...target. Due to its comparative simplicity and adaptability, CRISPR rapidly became the most popular genome...inhibiting Cas9’s endonuclease domain; or inducing degradation of the Cas9 protein. Most Acr proteins are specific... or dCas9 to create the adenine base editor. The TadA domain has also been further evolved to catalyze...

...protocol was established using 293T cells but can be adapted to alternative cell lines. Workflow Timeline Day...

...Dana-Farber Cancer Institute. Targeted protein degradation In the Bradner and Gray labs, we synthesize compounds...small molecule degraders that achieve selective degradation of endogenous proteins (notably, BRD2/3/4, CDK9... Hippel-Lindau (VHL) to bring the endogenous degradation machinery into close proximity with the target...polyubiquitination of the target protein and proteasomal degradation. Remarkably, small molecule degraders provide...prolonged biological effects. While targeted degradation of endogenous proteins is an exciting strategy...tag-based strategy, which we refer to as the degradation tag (dTAG) system, to co-opt the endogenous ubiquitin... evaluate the effects of rapid and selective degradation of protein targets in time scales that are not...

...protocol uses a dry transfer device but can be adapted for wet and semi-dry transfer approaches. Last ...

...editing portion of the protein, they examined ADAR1 and ADAR2, which deaminate adenosine to inosine in RNA... functional A->G change. They fused ADAR deaminase domains (ADARDD) to dPspCas13b, but observed low RNA... editing, they employed hyperactive ADAR constructs, like ADAR2DD(E488Q). They also adjusted the structure...RNA. Using an ADAR2DD catalytic mutant, they showed that editing is mediated by ADAR2DD, not PspCas13b...tested truncated ADARDDs to see if they could shrink the construct. They identified ADAR2DD(delta984-1090...mutating residues in ADAR2 that interact with duplex RNA, they hoped to destabilize ADAR-RNA binding to decrease..., 2016) as a RNA-targeting CRISPR enzyme, they adapted Cas13b for precise RNA editing (Cox et al., 2017...

...leading to ubiquitination and proteasomal degradation. Adapted from Nabet et al., Chemical Biology, 2018...of a protein and can inducibly facilitate the degradation of the tagged protein. Degrons are destabilizing...various enzymes and sent to the proteasome for degradation. The tags we will discuss here are all conditional...conditional, meaning that they only facilitate degradation of their fused protein under certain conditions...are reversible mechanisms of control. Protein degradation can be induced by the addition of a small molecule...to normal once the drug has been removed. The degradation and reversal are rapid compared to other systems... entire tagged protein to the proteasome for degradation. In addition to having the coolest name of all...

...NNNRRT 8 ABE SpCas9 TadA-TadA* NGG 9-12 SpCas9-VQR TadA-TadA* NGA 8 SaCas9 TadA-TadA* NNGRRT 9 SaCas9...SaCas9-KKH TadA-TadA* NNNRRT 8 SpCas9-VRER TadA-TadA* NGCG 8  Tips for designing base editing experiments...evolution and engineering gave rise to a mutant TadA (TadA*) that satisfactorily accepts DNA as its substrate... of different basic BE technologies and their adaptation for plant genome editing. Cytosine base editors...mutant library of E. coli tRNA adenosine deaminases (ecTadA), fused it with dCas9, and transformed it into ... at The Pennsylvania State University (PSU) is adapting and improving base editing platforms for plant...editing using hybrid prokaryotic and vertebrate adaptive immune systems." Science 353.6305 (2016): aaf8729...

...evolved TadA domain. Because ABE7.10 contains a heterodimeric fusion of wild-type TadA and TadA7.10, they...wild-type TadA and the evolved TadA region. The ABE8.x-m version contains only the evolved TadA and is ~...3 components:  a deaminase (TadA, originally from E.coli, named TadA7.10 in ABE7.10)  a catalytically...nickase)  a guide RNA that targets the complex of TadA and dCas to the genomic DNA of interest  ...When ABEs are in action they bind DNA so that the TadA domain can edit the short single-stranded DNA stretch...2017) in combination with a synthetic library of TadA sequences to create the eighth generation of adenine...antibiotic selection. As a result, 8 mutations in TadA could create all three edits and were thus more ...

...testing numerous TadA variants, they identified TadA-8e, which edits 590-fold faster than TadA-7.10. It is ...deoxyadenosine deaminase, TadA-7.10 (Gaudelli et al., 2017). The idea was to evolve a TadA-7.10 version with ...evolution of TadA variants with much higher base editing activity compared to the original TadA-7.10 enzyme...additional mutation (V106W) into TadA-8e and the resulting ABE8e(TadA-8e V106W) was as efficient in on-target...codons within T7 RNAP. A phage encoding the deaminase TadA-7.10 A mutagenesis plasmid controlling the phage...

...adequate quality, but this upfront cost will save headaches and expense later. At a minimum we recommend the...

...assays Long-lived protein degradation: This approach measures the degradation rate of radio-labeled long-lived...this is indicative of protein degradation). To control for protein degradation due to other pathways, it ...cytosol and organelles to the vacuole/lysosome for degradation and recycling. The key to Rosella’s autophagy-sensing...short-lived proteins primarily via proteasome degradation. Autophagy is then induced and quantified by ...it is standard practice to compare degradation rates between samples treated with or without an autophagy... measure of autophagy, but, like the protein degradation assay above, it’s slow and only measures bulk...because their contents can be at various stages of degradation. Additionally, electron microscopy analysis is...

...systems to generate ABE8e(TadA-8e V106W), which edits ~590-fold faster than the TadA from ABE7.10 without ...Engineered TadA domain for cytosine base editing Lam et al., 2023 CBE-T, CABE-T   Engineered TadA domain...evolution to create one from the RNA adenine deaminase TadA.   After seven rounds of molecular evolution, they...approach, engineering and directed evolution of the TadA domain has yielded base editors that are equally... Neugebauer et al., 2023 TadCBE, TadDE   Evolved TadA domain for cytosine base editing (TadCBE) or dual... D. (2023). Re-engineering the adenine deaminase TadA-8e for efficient and specific CRISPR-based cytosine...). Improved cytosine base editors generated from TadA variants. Nature Biotechnology, 41(5), 686–697. ...

...members and get to know them! Khandan Baradaran, PhD Khandan Baradaran’s expertise in viral safety, gene therapy... with health authority regulations,” Baradaran says.  Baradaran’s path to science began in high school...new members to the Board of Directors: Khandan Baradaran, Frank Stegmeier, and Luk Vandenberghe. We’re ...in viral genetics. In regards to open science, Baradaran thinks the best way to solve problems is to work...think will get the research funded,” she says. Baradaran met Addgene Executive Director Joanne Kamens several...

...binding and subsequent DNA degradation appears to be conserved. This DNA degradation portion can be bypassed.... These alterations are achieved through DNA degradation, thanks to the unique capabilities of Cas3 and...of DNA, Cascade caught up to it, and then DNA degradation continued. However, Cas3 cannot bind DNA on its...Hopefully more people will want to utilize Cas3’s degradative power in the future! References and Resources.... (2018). Structure basis for RNA-guided DNA degradation by Cascade and Cas3. Science, 361(6397). https...., Sonpho, E., Xu, H., Sasakawa, N., Naito, Y., Nakada, S., Yamamoto, T., Sano, S., Hotta, A., Takeda,...

...contributed by Adam Chin-Fatt, a Ph.D. student at the University of Western Ontario. Adam summarizes Zalatan...Transcriptional Programs with CRISPR RNA Scaffolds." Adam has also created a video to help scientists visualize...and flexible technique that it should be on the radar of every molecular microbiologist. Summary  The...transcriptional programs. Thank you to our guest blogger! Adam Chin-Fatt is a Ph.D. student at the University of...recombinant pharmaceuticals. Follow him on Twitter @AdamChinFatt     References Qi, L. S. et al. Repurposing...

...more quickly and efficiently, they’ve recently adapted the CRISPR/Cas9 system for protein tagging. We .../crispr/tagging. Addgene: What motivated you to adapt CRISPR for use in protein tagging? Myers: Our labs...transcription factors. Our groups got together to adapt CRISPR for tagging experiments and it’s already ... improvements in efficiency and throughput. In adapting the CRISPR/Cas system, what we are doing is adding... is so flexible. Other investigators can easily adapt these vectors for use with their favorite selection...who’ve developed their own system or who use and adapt our reagents to think about adding them to Addgene...

... Fig. 3 dTag mechanism of protein degradation. Adapted from Nabet et al. Check out Addgene's...with a degron tag can provide inducible protein degradation. These systems work by tagging the endogenously...mark the tag and whatever it is fused to for degradation. Since the tagged protein of interest is normally...off (SMASh), auxin-inducible degron (AID), and degradation tag (dTAG). These control systems function at...system for immediate and target-specific protein degradation. Nature Chemical Biology. May 2018. 14(5): 431...

...post was contributed by guest blogger Chinmaya Sadangi, a postdoc at the University of Toronto. The Addictive...communicating science to non-scientists. Chinmaya Sadangi, a postdoc at the University of Toronto, created...underrepresented minorities to study STEM subjects. Here, Sadangi and his team answer a few questions about The Addictive...guest blogger, Chinmaya Sandangi! Dr. Chinmaya Sadangi is currently a postdoc at the University of Toronto...Germany in 2017. Apart from The Addictive Brain, Dr. Sadangi is an eLife ambassador, SfN community leader, and...

... leading to the degradation of the AID-tagged protein. To quantify protein degradation the Ward lab used... that allows for both spatiotemporal protein degradation and quantification of this depletion using imaging-based... inducible degron sequence) tagged proteins. Degradation of your protein interest would also result in...system that brings Cas9 to ubiquitin ligase for degradation.      New from the viral service By Jason ...