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| kanzure | hail sagan. | 06:07 |
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| kanzure | .wik base excision repair | 06:11 |
| yoleaux | "In biochemistry and genetics, base excision repair (BER) is a cellular mechanism that repairs damaged DNA throughout the cell cycle. It is responsible primarily for removing small, non-helix-distorting base lesions from the genome. The related nucleotide excision repair pathway repairs bulky helix-distorting lesions." -- https://en.wikipedia.org/wiki/Base_excision_repair | 06:11 |
| kanzure | .wik strand invasion | 06:12 |
| yoleaux | kanzure: Sorry, that command (.wik) crashed. | 06:12 |
| kanzure | .wik strand invasion | 06:12 |
| yoleaux | kanzure: Sorry, that command (.wik) crashed. | 06:12 |
| kanzure | bah. | 06:12 |
| abetusk | http://arep.med.harvard.edu/gmc/genome_services.html . Of the DTC, there are whole genomes for $999 at 30x from Vertias (BAM file + VCF provided) and $1850 at 30x from Full Genomes.com (might be cheaper, don't know if they provide a BAM file) | 06:17 |
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| kanzure | ".... engineered zinc-finger recombinase (ZFR) fusion proteins are versatile alternatives to existing site specific recombinase technologies" | 06:26 |
| kanzure | "A programmable Cas9-serine recombinase fusion protein that operates on DNA sequences in mammalian cells" http://evolve.harvard.edu/123-RecCas9.pdf | 06:29 |
| kanzure | (recas9) | 06:29 |
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| kanzure | "We describe the development of 'recCas9', an RNA-programmed small serine recombinase that functions in mammalian cells. We fused a catalytically inactive dCas9 to the catalytic domain of Gin recombinase using an optimized fusion architecture. The resulting recCas9 system recombines DNA sites containing a minimal recombinase core site flanked by guide RNA-specified sequences." | 06:30 |
| gnusha | https://secure.diyhpl.us/cgit/diyhpluswiki/commit/?id=2fa9a753 Bryan Bishop: some gene editing techniques >> http://diyhpl.us/diyhpluswiki/gene-editing/ | 06:35 |
| gnusha | https://secure.diyhpl.us/cgit/diyhpluswiki/commit/?id=21db78b0 Bryan Bishop: also some protein parts >> http://diyhpl.us/diyhpluswiki/gene-editing/ | 06:38 |
| kanzure | oh yeah, wasn't there a photoactivated recombinase somewhere... | 06:41 |
| kanzure | "A photoactivatable Cre-loxP recombination system for optogenetic genome engineering" lice.btc.calendar.opentimestamps.org | 06:42 |
| kanzure | "Genome engineering techniques represented by the Cre-loxP recombination system have been used extensively for biomedical research. However, powerful and useful techniques for genome engineering that have high spatiotemporal precision remain elusive. Here we develop a highly efficient photoactivatable Cre recombinase (PA-Cre) to optogenetically control genome engineering in vivo. PA-Cre is b... | 06:42 |
| kanzure | ...ased on the reassembly of split Cre fragments by light-inducible dimerization of the Magnet system. PA-Cre enables sharp induction (up to 320-fold) of DNA recombination and is efficiently activated even by low-intensity illumination (~0.04 W m-2) or short periods of pulsed illumination (~30 s). We demonstrate that PA-Cre allows for efficient DNA recombination in an internal organ of living m... | 06:42 |
| kanzure | ...ice through noninvasive external illumination using a LED light source. The present PA-Cre provides a powerful tool to greatly facilitate optogenetic genome engineering in vivo." | 06:42 |
| gnusha | https://secure.diyhpl.us/cgit/diyhpluswiki/commit/?id=f81eb852 Bryan Bishop: also add integrases, homing endonucleases and fCas9 >> http://diyhpl.us/diyhpluswiki/gene-editing/ | 06:56 |
| kanzure | gah what. | 06:58 |
| kanzure | i meant: | 06:58 |
| kanzure | "A photoactivatable Cre-loxP recombination system for optogenetic genome engineering" http://www.nature.com/nchembio/journal/v12/n12/full/nchembio.2205.html | 06:58 |
| kanzure | "The findings reported here provide a foundation toward RMCE (recombinase-mediated casette exchange) on native genomic loci that would require two complete recCas9 target sites to be proximal to each other. The estimated 450 human genomic sites identified in silico for recCas9 might be expanded substantially by replacing the Gin recombinase catalytic domain with other natural or manmade small ... | 07:25 |
| kanzure | ...serine recombinases that recognize different core sequences; many of these related enzymes have also been directed to novel sites via fusion to zinc finger proteins (19,63). Moreover, recent work altering Cas9 PAM binding specificity and the recent discovery of numerous Cas9 orthologs raise the possibility of further expanding the number of potential recCas9 sites (64-67). Extending the appr... | 07:25 |
| kanzure | ...oach developed here may eventually lead to tools capable of specific, seamless integration of exogenous DNA into the human genome." | 07:25 |
| kanzure | "The recCas9 enzyme operates on a minimal pseudo-core recombinase site (NNNNAAASSWWSSTTTNNNN) flanked by two guide RNA-specified DNA sequences." | 07:44 |
| kanzure | "The estimated 450 human genomic sites identified in silico for recCas9 might be expanded substantially by replacing the Gin recombinase catalytic domain with other natural or manmade small serine recombinases that recognize different core sequences; many of these related enzymes have also been directed to novel sites via fusion to zinc finger proteins (19,63)." | 07:45 |
| kanzure | er.... what? i think we need a programmable recombinase that uses guide RNA to match a programmable site on the genome. the pseudo-palindromic core DNA sequence recombinase matching stuff seems a little silly to me.... | 07:48 |
| kanzure | oh i see, i bet it's because the recombinases are bad at doing watson-crick base pair matching for arbitrary sequences. hmm. | 07:53 |
| kanzure | "Redesigning recombinase specificity for safe harbor sites in the human genome" http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0139123 | 07:57 |
| kanzure | "... we set out to identify mutations that enable unrestricted recombination between minimal recombination sites." | 07:57 |
| kanzure | " However, because of their strict recognition capabilities, recombinase-mediated genome engineering has been limited to cells that contain either pre-introduced target sites or rare pseudo-recombination sites [21]. To overcome this, numerous protein engineering strategies have been developed to alter recombinase specificity [22]. Yet despite several successes [23, 24], these approaches have r... | 07:58 |
| kanzure | ...outinely led to enzymes with relaxed recognition specificities [25, 26], stemming from the fact that many recombinases display an intricate and overlapping network of catalytic and DNA-binding interactions. In contrast to the SSRs described above, the resolvase/invertase family of serine recombinases [27] are modular in both structure and function, allowing the DNA-binding domains of these enz... | 07:58 |
| kanzure | ...ymes to be replaced without impairing catalytic function [28, 29] (Fig 1). " | 07:58 |
| kanzure | all of the "point a webcam at a lab bench" projects should be replaced by "generate accurate lab bench plans to label everything and keep track of work items, with checklists in a standard format for standard steps" | 08:02 |
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| kanzure | hm okay. what's needed is something more like the error checking proofreader behavior, instead of a quasi-palindromic core recognition site for recombinases. the proofreading should evict the recombination attempt if the sequences don't match. | 08:11 |
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| kanzure | i think you can do programmatic DNA assembly with the following- microbeads with attached DNA, each DNA has the recombinase target site, use guide RNAs and recCas9 (programmable recombinase), add DNA fragments which will get recombined into the growing DNA fragments attached to the beads. wash after each step. downside: need probably fragments of at least 100 bp length (really it could be plas... | 10:41 |
| kanzure | ...mids up to 1000s of kb in size, i think?), need 20-30 bp recombinase recognition sites (which would interfere with e.g. specifying a protein)..... each DNA fragment or plasmid you insert into the reaction would have two different target sites so that the guide RNA wouldn't accidentally use the wrong side of the fragment. every other reactionn step, you would alternate between guide RNAs (so th... | 10:41 |
| kanzure | ...ere would be a total of 8 guide RNAs you need to synthesize, actually). | 10:41 |
| kanzure | selection for improved performance of ecoli/yeast homologous recombination would probably be more productive of a pursuit. plus you don't end up with those weirdo recombination sites stuck in your output dna. | 10:43 |
| kanzure | other downside of stepwise programmatic DNA assembly through recCas9 is that you would need to synthesize all those DNA fragments. and all the RNA guides. you could probably do one pot if you had unique RNA guides everywhere, though. so that might be nice... | 10:48 |
| kanzure | er, i think yashgaroth's recombinase assembly scheme described on napkin last night might be v. similar to existing casette exchange protocols? | 10:49 |
| kanzure | "Iterative in vivo assembly of large and complex transgenes by combining the activities of ?C31 integrase and Cre recombinase" http://nar.oxfordjournals.org/content/33/22/e189.short | 10:51 |
| kanzure | cool "A phage protein that binds ?C31 integrase to switch its directionality" | 10:52 |
| kanzure | "A motif in the C-terminal domain of ?C31 integrase controls the directionality of recombination" | 10:52 |
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| kanzure | yashgaroth: in your diagram yesterday, wouldn't there be completely random recombination as a result? | 11:04 |
| yashgaroth | how so? the recombinase scans nearby dsDNA with the ssDNA it's loaded onto until it finds matching homology, then holds it in place until other enzymes come in and/or it dissociates | 11:18 |
| kanzure | "matching homology" sure.. so either "have lots of different recombinases", or "only load one other dsDNA into the mix at a time" ? | 11:19 |
| yashgaroth | recombinases aren't sequence-specific | 11:20 |
| kanzure | they have that palindromic core site thingy that they match against | 11:20 |
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| yashgaroth | nope, they take up 6 basepairs physically but the sequence doesn't matter | 11:20 |
| kanzure | i am very confused then. you sure? could you show me something that agrees with this? | 11:21 |
| kanzure | "... This 34 base pair (bp) loxP recognition site consists of two 13 bp palindromic sequences which flank an 8bp spacer region" | 11:21 |
| yashgaroth | nono it's not a sequence-specific system like loxP; normally when they're used for double-stranded break repair the break could happen anywhere so they aren't tied to a specific sequence | 11:22 |
| kanzure | so there should be a non-specific recombinase somewhere? | 11:23 |
| yashgaroth | yeah like recA isn't sequence specific, or the UvsX protein from T4 and other related phages | 11:23 |
| * kanzure looks | 11:24 | |
| kanzure | .wik recA | 11:24 |
| yoleaux | "RecA is a 38 kilodalton protein essential for the repair and maintenance of DNA. A RecA structural and functional homolog has been found in every species in which one has been seriously sought and serves as an archetype for this class of homologous DNA repair proteins. The homologous protein is called RAD51 in eukaryotes and RadA in archaea." -- https://en.wikipedia.org/wiki/RecA | 11:24 |
| kanzure | cool: "The RecA-ssDNA filament searches for sequence similarity along the dsDNA." | 11:24 |
| yashgaroth | mhm | 11:25 |
| kanzure | yeah that is very useful. okay. | 11:25 |
| kanzure | thanks | 11:26 |
| yashgaroth | e.g. in isothermal amplification where it's mostly used, you can just throw in any suitable primer sequence and it'll seek out a matching site in dsDNA, then shove the primer in there; in that case, until a polymerase comes along and extends off it | 11:26 |
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| kanzure | there is a guide RNA programmable recombinase "A programmable Cas9-serine recombinase fusion protein that operates on DNA sequences in mammalian cells" http://evolve.harvard.edu/123-RecCas9.pdf | 11:28 |
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| yashgaroth | only really good for deletions between somewhat restricted sites so far, but looks interesting | 11:33 |
| kanzure | "we attached an inactivated cas9 and then some magic happened" | 11:34 |
| yashgaroth | "The recCas9 enzyme operates on a minimal pseudo-core recombinase site (NNNNAAASSWWSSTTTNNNN)"; not wherever you want, so probably not great for genome editing unless you get lucky | 11:36 |
| kanzure | yes that was the source of my confusion, then i looked up cre recombinase and such and.. yeah. | 11:37 |
| yashgaroth | yeah when you say recombinase everyone thinks of sequence-specific cre/lox, or like phiC31 | 11:38 |
| kanzure | recA looks very useful. | 11:53 |
| kanzure | holds some ssDNA and searches a large amount of dsDNA. that's very polite of it. | 11:53 |
| kanzure | ah, it was used in the "in vivo assembly cloning" reaction i saw yesterday, "IVA cloning: A single-tube universal cloning system exploiting bacterial in vivo assembly" http://www.nature.com/articles/srep27459 | 11:54 |
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| kanzure | recA recombination mechanism is "still unknown" | 11:56 |
| yashgaroth | I recommend T4 UvsX, it's from the e. coli phage | 11:56 |
| yashgaroth | yeah we don't know how it does its thing but that's true for like 90% of proteins | 11:56 |
| kanzure | oh wait. i am wrong. they are *not* using recA. they are complaining about recA and the 150-200 bp homology requirement. | 11:59 |
| kanzure | "The presence of a recA-independent homologous recombination pathway in E. coli was reported more than 20 years ago19,20,21, but has been neglected until recently, except for sporadic use in specific high throughput applications22,23. The pathway is mostly uncharacterised but is most efficient at recombining linear DNA fragments, likely acting through an annealing mechanism20,24, although alte... | 12:00 |
| kanzure | ...rnative mechanisms have been suggested25,26. Conveniently, the recA-independent pathway is responsible for the recombination of short overlapping sequences19, whereas the recA system requires longer homologous DNA stretches (>150-300 bp)." | 12:00 |
| yashgaroth | uvsx and reca have a similar mechanism but people prefer the phage, I presume since it doesn't require long sequence homology like that | 12:00 |
| kanzure | ah. | 12:01 |
| kanzure | er i think this paper is literally just using pcr? | 12:07 |
| yashgaroth | and then shoved into a bacterium which completes the recombination? hmm "The number of colonies produced (yellow) and the percentage of correct clones (red) decreased with more modifications (n = 3-5)" | 12:10 |
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| kanzure | the requirement for primers is a little strange. isn't this pcr assembly or extension pcr? | 12:16 |
| yashgaroth | I think it's just traditional pcr/primer-based mutagenesis, but you end up with a bunch of fragments with homologous blunt ends, and hope a bacterium picks them all up and recombines them all correctly, or else it dies | 12:19 |
| kanzure | .wik polymerase cycling assembly | 12:19 |
| yoleaux | "Polymerase cycling assembly (or PCA, also known as Assembly PCR) is a method for the assembly of large DNA oligonucleotides from shorter fragments." -- https://en.wikipedia.org/wiki/Polymerase_cycling_assembly | 12:19 |
| kanzure | weird. ok. | 12:20 |
| yashgaroth | in PCA you're still just relying on dumb sticky-end basepairing, and every fragment has to be nice and linear ssDNA, plus pretty much the entire oligo has to be homologous to its neighbor | 12:23 |
| kanzure | why are we still using primers for generic dna amplification? aren't there a bunch of dna-binding proteins. you'd need one that scans until the end or beginning of a dna fragment, then activate/attract/bind polymerase. | 12:38 |
| kanzure | biology is really annoying | 12:39 |
| yashgaroth | I mean there's stuff with random hexamers for like whole genome amplification, otherwise polymerases want a primer real bad | 12:41 |
| kanzure | sure--- if cells had such promiscuous polymerases, they would end up copying everything (lots of noise and wasted genetics on random nonsense) | 12:42 |
| kanzure | which is genetically unsustainable | 12:44 |
| gnusha | https://secure.diyhpl.us/cgit/diyhpluswiki/commit/?id=f4770083 Bryan Bishop: some notes about recA and recombinases >> http://diyhpl.us/diyhpluswiki/gene-editing/ | 12:45 |
| yashgaroth | mhm, you work with the tools you have sadly | 12:45 |
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| kanzure | https://soundcloud.com/professorburial/hung-like-carl-sagan | 13:38 |
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| kanzure | hrm | 15:06 |
| kanzure | "On the overwhelming importance of shaping the far future" https://docs.google.com/viewer?a=v&pid=sites&srcid=ZGVmYXVsdGRvbWFpbnxuYmVja3N0ZWFkfGd4OjExNDBjZTcwNjMxMzRmZGE | 15:06 |
| kanzure | from http://www.nickbeckstead.com/research | 15:06 |
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| kanzure | from TeMPOraL "Reminds me a bit of one of the points of the book Death's End (third part of the Three Body Problem trilogy by Cixin Liu). A small spoiler follows. The point raised in this book is - the Earth looks much different thanks to life it has than it would without it. Consider e.g. mountains that erode slower because there are forests and foliage that disperse the wind. Would it not be... | 17:56 |
| kanzure | ... the case that advanced alien life existing in the universe also affects its evolution? So what if some of the physics we study is actually not how the universe looked at the start? Could the speed of light have changed because of aliens weaponizing physics in past wars? Could the curled up dimensions string theory postulates be actually be another consequence of powerful entities doing wide-s... | 17:56 |
| kanzure | ...cale manipulation of the universe? Maybe initially, before life, the universe actually had 11 full, expanded spatial dimensions?" | 17:56 |
| kanzure | to which i replied with some links, https://news.ycombinator.com/item?id=12996544 | 17:59 |
| kanzure | "... part of the trilogy is his answer to the Fermi paradox (minor spoilers ahead): because of the universal limit of light speed, and exponential growth in technology, by the time you see evidence of advanced alien life, chances are they have developed to the point of being an existential threat to your own civilization. So the only logical behavior is to remain hidden and immediately destroy... | 18:07 |
| kanzure | ... any civilization you detect" | 18:07 |
| kanzure | also, whenever genome synthesis becomes extremely cheap, we should make sure to name it "the really big bang" or something. yep. | 18:10 |
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| kanzure | .title https://news.ycombinator.com/item?id=12995125 | 18:36 |
| yoleaux | EmDrive study officially published | Hacker News | 18:36 |
| ebowden | I saw a clickbait saying that the peer review and publishing totes proves it works. | 18:44 |
| ebowden | *clickbait article | 18:44 |
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| fenn | does emdrive work? | 19:52 |
| FourFire | Ketogenic/Avoid grain diet has made me have sudden insights (intelligence effect), but also I am suddenly very sick, which hasn't happened to me in three years. | 19:55 |
| fenn | maybe your new diet is missing a mineral (zinc?) | 19:55 |
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| fenn | "It's so unbelievably inefficient right now though, it's hard to imagine it improving by orders of magnitude." is like "nobody drives in new york because the roads are full of cars" | 20:02 |
| fenn | would emitting a gravity wave be a way around the "but it violates conservation of momentum" refrain? | 20:04 |
| FourFire | fenn, Read the research paper yourself: http://arc.aiaa.org/doi/10.2514/1.B36120 | 20:07 |
| FourFire | They claim to have ruled out thrusting against earth's magnetic field, among other things. | 20:07 |
| FourFire | I'll try addind zinc somehw | 20:08 |
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| kanzure | how about a virus focused on causing accelerated general aging targeted for the general population at large | 20:31 |
| kanzure | "forcing function" | 20:32 |
| kanzure | i guess the trouble is that the virus would be easier to treat than aging itself | 20:35 |
| kanzure | so it would only work if people didn't know that early aging was caused by a virus | 20:35 |
| kanzure | and if they didn't investigate whether it was of viral origin | 20:36 |
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| kanzure | but otherwise, that might put sufficient fire in everyone's pants to actually investigate anti-aging | 20:37 |
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| FourFire | kanzure, dangeriously veering apon moral virologist territory there | 20:42 |
| kanzure | or maybe memory loss would be more effective (dementia, amnesia, short-term memory loss, working memory problems, long-term consolidation issues, or even temporary problems) | 20:42 |
| kanzure | FourFire: i am so far veered that not even a map would get me back to earth | 20:43 |
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| kanzure | "At the last community meeting, we announced that I was stepping down from the BioCurious board." - tito is kill | 21:42 |
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| FourFire | politcial reasons? | 21:50 |
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| kanzure | who knows. | 21:57 |
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