--- Log opened Sat Oct 07 00:00:39 2017
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06:41 < kanzure> "RNA targeting with CRISPR–Cas13" http://www.nature.com/nature/journal/vaop/ncurrent/full/nature24049.html https://twitter.com/EricTopol/status/915632664027176960
06:42 < kanzure> "Here we demonstrate that the class 2 type VI[6][7] RNA-guided RNA-targeting CRISPR–Cas effector Cas13a[8] (previously known as C2c2) can be engineered for mammalian cell RNA knockdown and binding. After initial screening of 15 orthologues, we identified Cas13a from Leptotrichia wadei (LwaCas13a) as the most effective in an interference assay in Escherichia coli. LwaCas13a can be ...
06:42 < kanzure> ...heterologously expressed in mammalian and plant cells for targeted knockdown of either reporter or endogenous transcripts with comparable levels of knockdown as RNA interference and improved specificity. Catalytically inactive LwaCas13a maintains targeted RNA binding activity, which we leveraged for programmable tracking of transcripts in live cells. Our results establish CRISPR–Cas13a as a ...
06:42 < kanzure> ...flexible platform for studying RNA in mammalian cells and therapeutic development."
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06:45 < kanzure> .tw https://twitter.com/eboyden3/status/915892505224388609
06:45 < yoleaux> Our two-photon image-guided patch clamp (aka imagepatching) paper in Neuron, now with updated/clearer Figure 1. http://www.sciencedirect.com/science/article/pii/S089662731730702X (@eboyden3)
06:46 < kanzure> "Closed-loop real-time imaging enables fully automated cell-targeted patch clamp neural recording in vivo" http://www.sciencedirect.com/science/article/pii/S089662731730702X
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06:49 < kanzure> "Sub-millisecond optogenetic control of neuronal firing with two-photon holographic photoactivation of Chronos" http://www.jneurosci.org/content/early/2017/10/02/JNEUROSCI.1246-17.2017
06:49 < kanzure> "Here, we show that efficient current integration enabled by two-photon holographic amplified laser illumination of Chronos, a highly light-sensitive and fast opsin, can evoke spikes with submillisecond precision and repeated firing up to 100 Hz in brain slices from Swiss male mice. These results pave the way for optogenetic manipulation with the spatial and temporal sophistication necessary ...
06:49 < kanzure> ...to mimic natural microcircuit activity."
06:50 < kanzure> "Two-photon computer generated holography (2P-CGH) recently demonstrated three-dimensional optogenetic control of selected pools of neurons with single-cell accuracy in-depth in the brain. Here we show that exciting the fast opsin Chronos with amplified laser 2P-CGH enables cellular-resolution targeting with unprecedented temporal control, driving spiking up to 100 Hz with submillisecond ...
06:50 < kanzure> ...onset precision, using low laser power densities."
06:51 < kanzure> looks like this was the only other mention of Chronos channelrhodopsin in the logs,
06:52 < kanzure> .tw https://twitter.com/Addgene/status/888431974012129281
06:52 < yoleaux> New Viral Vectors available: #optogenetics Chrimson & Chronos channelrhodopsins AAV5 serotype via @eboyden3 http://hubs.ly/H0880zV0 https://pbs.twimg.com/media/DFRYK-yXgAAUdjf.jpg (@Addgene)
06:54 < gnusha> https://secure.diyhpl.us/cgit/diyhpluswiki/commit/?id=3a82a7cd Bryan Bishop: mention LwaCas13a and casposons >> http://diyhpl.us/diyhpluswiki/gene-editing/
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07:05 < kanzure> Opekktar: greetings.
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07:44 < kanzure> PEI == polyethylenimine
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08:27 < kanzure> i wonder how many "crispr associated proteins" they are going to let themselves number. cas13a is already a high number.
08:29 < kanzure> "RNA recognition motifs: boring? Not quite" http://www.sciencedirect.com/science/article/pii/S0959440X08000584 (2008)
08:29 < kanzure> "The RNA recognition motif (RRM) is one of the most abundant protein domains in eukaryotes. While the structure of this domain is well characterized by the packing of two α-helices on a four-stranded β-sheet, the mode of protein and RNA recognition by RRMs is not clear owing to the high variability of these interactions. Here we report recent structural data on RRM–RNA and RRM–protein ...
08:29 < kanzure> ...interactions showing the ability of this domain to modulate its binding affinity and specificity using each of its constitutive elements (β-strands, loops, α-helices). The extreme structural versatility of the RRM interactions explains why RRM-containing proteins have so diverse biological functions."
08:30 < kanzure> α-helices and β-sheets are some of the easier types of protein structures to design. it's sort of good news that this is what RNA recognition motifs are based on.
08:37 < kanzure> "Challenges in DNA motion control and sequence readout using nanopore devices" https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4710574/ (2015)
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09:03 < nmz787> back
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10:33 < kanzure> reviewing scalingbitcoin.org papers. bleep bloop.
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10:37 < nmz787> derrrr dee doo blehhh fffzzzz booo beee booo
10:48 < kanzure> nmz787: sounds like you need some GCTA https://www.youtube.com/watch?v=ID6KY1QBR5s
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11:03 < nmz787> awww yeaaah
11:04 < nmz787> perfect for the mood
11:07 < nmz787> where's that minimal primer length paper?
11:07  * nmz787 checks locally
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11:12 < kanzure> we need a librarian
11:13 < kanzure> ... other than me.
11:15 < nmz787> seriously, I was even remembering back to when I tried to use Mendeley on windows (I think)
11:15 < kanzure> use a bookmark manager with tagging
11:16 < nmz787> hrmm, maybe we can use the pdfparanoia parsing code to index for us?
11:16 < nmz787> hook that up to generate wikitext or something
11:32 < nmz787> hmm, a local CAD simulation/modelling engineer looking for work just friended me on linkedin... sent him my fenics to CAD and fenics navier stokes links, along with a list of open-source CFD software
11:32 < nmz787> https://en.wikiversity.org/wiki/CAD_to_FEniCS_example
11:32 < nmz787> https://fenicsproject.org/pub/tutorial/html/._ftut1009.html#ftut1:NS
11:32 < nmz787> https://cfdandheattransfer.wordpress.com/category/cfd-softwares/
11:33 < nmz787> and of course my programmatic BRLCAD solution: https://github.com/nmz787/python-brlcad-tcl
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12:06 < nmz787> .tell yashgaroth kanzure: what about using a ribosome that can accept single codons at a time, without having the growing peptide fall off? any evidence that could work, or work towards finding/building that functionality?
12:06 < yoleaux> nmz787: I'll pass your message to yashgaroth.
12:07 < kanzure> uh how are you delivering only a single tRNA?
12:07 < nmz787> umm, nanopore?
12:07 < nmz787> dilute solution moving super slow?
12:07 < nmz787> so ass they pass the ribosome they interact, and then continue on out of that area
12:08 < nmz787> as*
12:08 < kanzure> so you need a fluorescent event to confirm?
12:08 < nmz787> uh, maybe something with impedance detection
12:08 < nmz787> since the tRNA will lose a big amino acid
12:09 < nmz787> it should look different, maybe
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12:35 < nmz787> does anyone here worry about using red text when collaborating, for fear of color-blind people missing your text as invisible? Or do most color-blind people use color-shifting display software?
12:42 < kanzure> is this your first time using red text
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12:54 < nmz787> no
12:54 < nmz787> I just think about it at work
12:54 < superkuh> Nah. No worries.
12:54 < nmz787> since there are so many new people I interact with pretty often
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14:19 < streety> are color-blind people unable to see red on a screen? I thought they just couldn't distinguish from green
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14:33 < nmz787> hmm, not sure... I used to live with a guy who was colorblind, and he said red traffic signals looked vaguely brown
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15:17 < nmz787> sup yashgaroth
15:17 < yashgaroth> yo
15:17 < yoleaux> 19:06Z <nmz787> yashgaroth: kanzure: what about using a ribosome that can accept single codons at a time, without having the growing peptide fall off? any evidence that could work, or work towards finding/building that functionality?
15:18 < nmz787> yashgaroth: also, did you see Josiah's self-CRISPR thing
15:18 < nmz787> ?
15:18 < nmz787> self meaning himself
15:18 < yashgaroth> haha that dumb josiah bullshit
15:19 < yashgaroth> I skimmed the "blah blah blah, here I am injecting a plasmid with PEI into myself, it will transfect two cells, woo look at me"
15:20 < nmz787> so I was thinking, if it modifies, does it mod just one strand?
15:21 < yashgaroth> if you get both gRNA and the protein inside the nucleus, if it hits one strand it'll probably hit both
15:21 < nmz787> then leaving a mismatch, and some other replacer enzyme comes along and chooses to excise and repair either, right?
15:21 < nmz787> ah, so two gRNA
15:21 < nmz787> targeting both senses
15:21 < yashgaroth> crispr just repeatedly cuts until you get an error that mutates the recognition site, right?
15:22 < yashgaroth> crispr-cas9, I should say
15:22 < nmz787> and presumably if the plasmid doesn't have an ORI or whatever they call it in mammals, it and the enzymes will eventually degrade and should be relatively transient, aside from genomic mods
15:22 < nmz787> idk, actually
15:22 < nmz787> re crispr-cas9
15:23 < yashgaroth> assuming it reaches the nucleus, yeah it'll eventually fade away as the daughter cell that carries it runs out of telomeres and dies
15:23 < nmz787> reading NEBs description now
15:24 < yashgaroth> you don't really have an ORI for mammalian plasmids unless you're co-expressing e.g. SV40 or EBV protein that will replicate the plasmid
15:25 < nmz787> ah, right, cas9 just cuts at the gRNA specified site
15:25 < nmz787> then it's up to homologous recomb I guess
15:25 < yashgaroth> well, an error therein to be specific
15:25 < nmz787> hmm?
15:25 < yashgaroth> requires a replication error during the homologous recombination
15:26 < nmz787> why, otherwise the existing gRNA-cas9 will just re-cut?
15:26 < yashgaroth> exactly
15:26 < nmz787> ok, so then my question earlier still holds
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15:27 < nmz787> it's 50:50 chance that the either side of the mismatch will get excised?
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15:27 < nmz787> *"chance that either side"
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15:28 < yashgaroth> I guess it'd have to be the side complementary to the gRNA that needs to get cleaved/error'd
15:28 < yashgaroth> since the recognition is only on that strand
15:28 < nmz787> maybe I need to also review homologous recombination
15:30 < nmz787> yashgaroth: the recA-like protein, assuming it never found it's match, would it just keep scanning say a plasmid forever?
15:30 < yashgaroth> 'til it runs out of ATP, pretty much yeah
15:32 < yashgaroth> also side note, PEI transfection in vivo is super inefficient, crispr'ing only inhibits myostatin in the cell it mutates, I know people are frothing about crispr but that shit was just a dumb spectable; ok I'm done
15:33 < yashgaroth> what was the ribosome stuff, you want to feed tRNAs to a ribosome sequentially?
15:34 < nmz787> I was thinking about if you could cut a plasmid with diameter D, then on a membrane with 2 nanopores D length apart wash the plasmid fragments and electrophorese partially-through the nanopores, so they are stuck about mid-way, then add ligase on both sides. I would expect that some of these two fragments would have ligated to become a 2x-sized plasmid circle again
15:34 < nmz787> and you could then add in some rec-A like protein on just one side, and get the circles to rotate like a ferris wheel through the 2 nanopores it was laced through
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15:35 < nmz787> start and stop it by starving ATP
15:35 < yashgaroth> that first part sounds in the realm of possibility
15:36 < yashgaroth> you're counting on the recA nucleofilament to run into the nanopore and kind of pull the plasmid through the pore toward itself, not sure if that'd happen
15:37 < nmz787> then you connect the nanopore electrodes up to a pre-amp, and out to a stereo system... and use a laser/AFM/SEM to make the worlds first nano DJ
15:37 < yashgaroth> I'll get iarpa on the phone
15:37 < nmz787> yeah basically that was what I was thinking
15:37 < nmz787> I guess if two recA got on in opposite directions, they'd tug each other
15:37 < nmz787> I wonder if they'd snap the DNA
15:38 < nmz787> * nano DJ turntable
15:38 < yashgaroth> I think they'd try to shove themselves into the nanopore and get stuck and/or dissociate since the plasmid has far more inertia
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15:38 < nmz787> hmm, like kilodalton wise?
15:39 < nmz787> atomic mass?
15:39 < yashgaroth> either/or
15:39 < nmz787> I wonder if it would change things if they were tethered near the nanopore
15:40 < nmz787> meh, I guess not
15:41 < nmz787> how are things down where the sun shines?
15:42 < yashgaroth> it's uh...oh it's like 90 out, what the fuck is this
15:42 < nmz787> dang
15:42 < nmz787> it was partially sunny up here today
15:42 < nmz787> fall is kinda late this year
15:43 < nmz787> or at least it hasn't been as overcast as I think it would have been
15:43 < yashgaroth> yeah it's been fucking hot here, normally there's no real need for AC the entire year
15:44 < yashgaroth> anyway from what I can tell from oxnano's information, they're able to reverse the electrophoretic effect through individual pores, so your nano DJ is still doable
15:45 < yashgaroth> I don't know how far apart their pores are for that though
15:48 < nmz787> yeah
15:49 < nmz787> I am more into solid-state pores anyway
15:49 < nmz787> found a nice paper describing a high-volume manufacture technique for making a nice 3-electrode sandwich through the nanopore too
15:49 < nmz787> 2 for electrophoresis through, and 1 for sensing
15:50 < nmz787> they were theorizing/aiming to finally do single-bp electrophoresis, sort of like a stepper motor
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15:50 < nmz787> I think that paper only proved the very reproducible fab of the pore and sandwich layers
15:51 < nmz787> (i.e. with TEM cross-sections)
15:52 < nmz787> yashgaroth: this one dx.doi.org/10.1039/c3nr06723h
15:53 < nmz787> "A double-stranded DNA of length 1 kbp was translocated through the multi-layer structure RIE-based nanopore demonstrating that the pores were open. The ionic current through the pore can be modulated with a gain of 3 using embedded electrodes functioning as a gate in 0.1 mM KCl aqueous solution. This fabrication approach can potentially pave the way to manufacturable nanopore arrays with the
15:53 < nmz787> ability to electrically control the movement of single or double-stranded DNA inside the pore with embedded electrodes."
15:53 < yashgaroth> I biasedly would prefer protein-based pores since eventually we can modify them to be more useful than a hole, but that's a while off
15:53 < nmz787> 99% yield on their fab
15:54 < nmz787> I am also thinking about system complexity, like during running it
15:55 < nmz787> assuming electrodes are more stable than enzymes, or yield higher number of single uses/actuations... then less enzymes means less risk, or longer stability/life
15:55 < nmz787> also yes, magic proteins are a while off
15:56 < nmz787> at that point though, we should be able to revert to oxnano's method, or some improvement of that
15:56 < nmz787> (of seeding nanopores on membranes)
15:56 < yashgaroth> true, I'm imagining a protein-based ratchet with azobenzenes or something, but those are some nice looking pores
15:57 < nmz787> brb
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21:34 < kanzure> protein-based rachet..... probably the only ratchets in this area are going to be from some of those multi-step motor proteins that walk all up and down the cytoskeletons.
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21:46 < kanzure> .title https://www.youtube.com/watch?v=NujlXgBmUoU
21:46 < yoleaux> Sea Wall (Blade Runner 2049 Soundtrack) - YouTube
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--- Log closed Sun Oct 08 00:00:40 2017