...complex with guide RNA and target DNA. Nishimasu H, Ran FA, Hsu PD, Konermann S, Shehata SI, Dohmae N, Ishitani... sgRNAs for each transcription start site in 15,977 human genes. The transcriptional repressor (CRISPRi...SAM is an engineered protein complex for the transcriptional activation of endogenous genes and it consists...The next insight was to assemble a synthetic transcription activation complex consisting of multiple distinct...together synergistically, modeled after natural transcription activation processes. Konermann and her colleagues...consequences of repressing or inducing individual transcripts. The activator (CRISPRa) sgRNA library uses the...CRISPRi) library contains 10 sgRNAs for each transcription start site in those 15,977 human genes. Weissman...

...fragments of these transcripts. I remember when small RNA was just something we ran off the bottom of ...’t give tRNAs much thought. High throughput transcriptome sequencing methods including work done by the...hypothesized that life started with RNA and a simple cell membrane. His recent work shows that they are making fairly...knew…I had barely heard of these). These single stranded, covalently closed RNAs seem to be conserved in...

...that produce GMO food plants. When a press piece ran on the detector plants, we expected to see that same...below. We’re happy to answer questions about this transition (so far) and what we’ve learned.  Thank you ...

...doi.org/10.1038/s41587-021-01039-7 Nishimasu, H., Ran, F. A., Hsu, P. D., Konermann, S., Shehata, S. I....nicking of the unedited strand and encourage the cell to use the edited strand as a template during DNA...on a fusion enzyme of Cas9 nickase + reverse transcriptase and a prime editing guide RNA (pegRNA). The ...) complementary to the genome and a reverse transcriptase (RT) template including the desired edit. ...although sequences with G/C content outside this range can still be optimized (Anzalone et al., 2019). .... Test different lengths of reverse transcriptase template, starting with about 10–16 nucleotides. For...pegRNA primer binding site (PBS) and reverse transcriptase template (RTT). Created with BioRender.com. ...

... same output regardless of who or which computer ran the process. Another design choice we appreciated...automating the bioinformatics steps needed to transform the raw sequencing data into a complete plasmid...appropriate place in our workflow without having to translate or convert programs to a uniform language.  The...concepts and vocabulary of Nextflow so that we could translate our pipeline steps into processes and then connect...

...just make their own buffers when the kits buffers ran out. Homemade buffers performed the same as those...rather a glass powder slurry that has a milk-like appearance. To make DNA stick to the glass particles in ...the Patterson Protocol was shown to purify DNA ranging in size from 100 bp to 48 kb with high yields and...could interfere with interpreting the results of transfection assays. Go forth and perform your DNA extractions...

...Genome engineering using the CRISPR-Cas9 system. Ran FA, et al. Nature Protocols Nov;8(11):2281-308; (2013)...dHJs are four-stranded branched structures that form when elongation of the invasive strand “captures” and...lesion results from breakage of both DNA strands - a double-strand break (DSB). DSBs can be caused by intracellular...proteins required for strand invasion and a primer for DNA repair synthesis. The ssDNA strand then displaces ...displaces one strand of the homologous DNA donor and can template repair off the other strand; this results in...pathways: classical double-strand break repair (DSBR), synthesis-dependent strand-annealing (SDSA), and break-induced...on the crossing strand (horizontally at the purple arrows) or on the non-crossing strand (vertically at...

...the most out of their meetings. Amanda Gunn ran randomized coffee trials for us at ASCB|EMBO 2019, matching...within a bin as Horizontal Transfer. This comes from horizontal gene transfer, which occurs when bacteria...in Atlanta, GA, in 2018. A lot of my experiences rang true with theirs.  It was at this dinner and the...Singletons logo. Image: James et al., 2020. Help me stranger - Finding mentors for the meeting Just like analysis...

... 556:57–63 . https://doi.org/10.1038/nature26155 Ran FA, Cong L, Yan WX, Scott DA, Gootenberg JS, Kriz...SpCas9 variants effectively double the targeting range of CRISPR/Cas9 within the human genome. Another ...known to produce off-target editing with SpCas9 (Amrani et al., 2018). When choosing a Cas9, remember to...formerly Cpf1), a nuclease that generates double strand breaks in target genes resulting in the formation...nature24049 Find plasmids from this paper at Addgene. Amrani N, Gao XD, Liu P, Edraki A, Mir A, Ibraheim R, ...018-1591-1 Cox DBT, Gootenberg JS, Abudayyeh OO, Franklin B, Kellner MJ, Joung J, Zhang F (2017) RNA editing...

...that better human health.     References Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu...3.4% of founders establishing germline transmission of the transgene, this technique is still in need of ...and chimeric adults mated to produce completely transgenic offspring. Although powerful, this technology... a mouse model, and its success is not always guaranteed. To learn more about this process check out our...the Cas9 endonuclease that creates the DNA double-strand break (DSB) at the target site, and a donor oligonucleotide...

... . https://doi.org/10.1016/s0764-4469(00)01250-6 Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang...of the DNA. Finally, in 1953, Franklin along with James Watson and Francis Crick resolved the DNA double...writers have been curious about the capability of transforming the genetic code of living organisms and its...that Streptococcus pneumoniae bacteria could be transformed from one organism to the other. This is now famed...Hershey-Chase experiment. In the same year, Rosalind Franklin and her student Raymond Gosling took the first...double helix. In the 1960s, Har Gobind Khorana, Marshall W. Nirenberg, and Robert W. Holley cracked the genetic...explained how the four letters of the DNA get translated into the twenty amino acids. Once scientists ...

...- CHGA chromogranin A (parathyroid secretory protein 1) CGA CHGB chromogranin B (secretogranin 1) SCG1...superfamily, member 11 CD254, ODF, OPGL, OPTB2, RANKL, TRANCE, hRANKL2, sOdf TNFSF12 tumor necrosis factor (ligand... perforin and granzyme B, two enzymes that create pores within an infected cell membrane, triggering cell...nuclear transcription factor Y, alpha CBF-A, CBF-B, FLJ11236, HAP2, NF-YA NFYB nuclear transcription factor...RAET1G retinoic acid early transcript 1G ULBP5 RAET1L retinoic acid early transcript 1L - RELB v-rel reticuloendotheliosis...(sodium/bile acid cotransporter family), member 2 ASBT, ISBT, NTCP2 TAP1 transporter 1, ATP-binding cassette...

...for glutamatergic neurotransmission Targeted sensors for glutamatergic neurotransmission. eLife. 2023 Jan...trafficking to the plasma membrane Acute disruption of the synaptic vesicle membrane protein synaptotagmin...measurements of membrane charges A FRET sensor enables quantitative measurements of membrane charges in live...847-56. David Raible pH Red or Orange pH biosensors (pHuji and pHoran4) pHuji, a pH-sensitive red fluorescent...Proteins: Biosensors Metal Ions Small Molecules Neurotransmitters Cell Physiology Browse All You may also like...fluorescent protein (cpFP) or Förster resonance energy transfer (FRET) efficiency of a pair of fluorescent proteins...Calcium Calcium sensor in astrocytes (GCaMP6f), membrane-tethered Khakh Lab pZac2.1 gfaABC1D-lck-GCaMP6f...

... Monomer pET mOrange LIC cloning vector (u-mOrange) - Bacterial Expression pCS2+8CmOrange - Zebrafish/...pCS2+8NmOrange - Zebrafish/Xenopus/C.elegans/Sea urchin mOrange-N1 - Mammalian Expression mOrange-C1 -...Expression mOrange-pBAD - Bacterial Expression mOrange2 549 565 35 6.5 4.5 hr Monomer mOrange2-N1 - Mammalian...Expression LSSmOrange 437 572 23 5.7 2.3 hr Monomer pLSSmOrange-C1 - Mammalian Expression pLSSmOrange-N1 - ...Structure Plasmids mKusabira-Orange (mKO) 548 559 31 5 4.5 hr Monomer pQC mKorange IX - Mammalian Expression...Expression mKusabira Orange-C1 (mKO) - Mammalian Expression mKO-pBAD - Bacterial Expression mOrange 548 562 49 ...Mammalian Expression mOrange2-C1 - Mammalian Expression mOrange2-pBAD - Bacterial Expression mKO2 551 565...

...pOpen-EcoRIM M.EcoRI DNA methyltransferase 165517 pOpen-Eco31IA Eco31IA DNA methyltransferase 165518 pOpen-NotIM...pOpen-NotIM M.NotI DNA methyltransferase 165527 pOpen-Eco31IB Eco31IB DNA methyltransferase 165536 pOpen-PstIM...pOpen-PstIM M.PstI DNA methyltransferase 165538 pOpen-SfiIM M.SfiI 165538 DNA methyltransferase 165547 pOpen-SapIM1...pOpen-SapIM1 M1.SapI DNA methyltransferase 165557 pOpen-SapIM2 M2.SapI DNA methyltransferase 165558 pOpen-EcoRVM...pOpen-EcoRVM M.EcoRV DNA methyltransferase 165577 pOpen-XbaIM M.XbaI DNA methyltransferase 165578 pOpen-NcoIM ...32 single-stranded DNA-binding protein 165549 pOpen-ETSSB Extreme Thermostable Single-Stranded DNA-Binding...Polymerases Restriction Endonucleases Reverse Transcriptases RNA Polymerases Other Enzymes (Link opens in...

...37351 pQC membrane TdTomato IX Membrane Palmitoylation TdTomato Connie Cepko 22479 FUmGW Membrane Palmitoylation...Mitochondria-Outer Membrane Mff acGFP1 Gia Voeltz 158007 pCMV-mGold-Mito-N-7 Mitochondria Cox8A mGold Francois St-Pierre...pCAG-mGFP Membrane Palmitoylation sequence from GAP43 EGFP Connie Cepko 61099 Lck-GFP Membrane Palmitoylation...Mitochondria-Outer Membrane TOMM20 mCherry* Michael Davidson 118736 pBOB-CARMIL2 BH domain-GFP Plasma Membrane CARMIL2...alpha-tubulin mGold Francois St-Pierre 158009 pCMV-mGold-Actin-C-18 Actin Filaments Actin mGold Francois St-Pierre...EGFP Carol Otey 31923 pPaxillin-PSmOrange Focal Adhesions paxillin PSmOrange Vladislav Verkhusha 79999 pZyxin-miRFP703... Proteins are first translated at the endoplasmic reticulum, then are transported to the Golgi apparatus...

...by plasmids called "transfer plasmids," which can be modified to encode a wide range of gene products. ...Addgene lentiviral packaging, envelope, and transfer plasmids Viral...Libraries All Viral Plasmids Packaging Plasmids Transfer Plasmids Lentiviruses, a subset of retroviruses...types of viruses used in research. Lentivirus can transduce both dividing and non-dividing cells without a...stably into the host genome, enabling long term transgene expression. There are some safety considerations...procedures. Additionally, because this virus integrates randomly into the host genome, it has the potential for...encodes the viral genome (typically called the transfer plasmid ). This table lists common lentiviral ...

...dynamic range, and resistance to photobleaching has made luciferase a common choice in assays ranging from... luciferase. Transcriptional Element Screening Optimized STARR-seq ( S elf- T ranscribing A ctive R egulatory...including transcriptional reporters for NF-kb, TGF-b, c-Myc, p53 and MAPK/JNK transcriptional reporters... of luciferase plasmids used for assays of transcriptional regulation and biosensors. Plasmid...bioluminescence found in a diverse number of organisms, ranging from bacteria to aquatic animals to insects. Luciferase... protein half-lives, making them useful as transcriptional reporters. However, they are not secreted, ...regulatory elements, such as promoters, enhancers and untranslated regions, or the effect of mutations of these...

...enhanced transduction of the retina following subretinal administration and enhanced transduction and spread...cite Boye, et al. 2016. Impact of Heparan Sulfate Binding on Transduction of Retina by Recombinant Adeno-... from the University of Florida for enhanced transduction of the eye and other tissues. As part of our...Y444F) serotype is demonstrated to have enhanced transduction of the retina following intravitreal injection... AAV2(Y444F) serotype also displays enhanced transduction of the trabecular meshwork following intracameral...trpYF) The AAV2(trpYF) serotype shows enhanced transduction of the retina following intravitreal injection...macaque. This serotype also demonstrates enhanced transduction of cells in culture for ex-vivo conditioning...

...6105. https://doi.org/10.1093/nar/gku241 Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N..... Type I systems are also employed as CRISPR transposases, by leaving out Cas3. Type I is further subdivided...complex (blue subunits) with a guide RNA (green and orange) bound to the target DNA with Cas3 (pink). Complex...promoter, allowing for easy multiplexing. The double-stranded breaks caused by Cas12 create short 3’ overhangs...paradoxically called V-K, is a CRISPR-associated transposase (CAST) that can insert large fragments of DNA...use a Cas14 enzyme, and uniquely target single-stranded DNA non-specifically. Type VI Type VI is defined...interference, as it can target and degrade select RNA transcripts, or be engineered to conduct direct RNA editing...