Yeah, thank you very much Hopefully everybody can hear me So this is my conflict of interest slide It it does take a village to take ideas From a very primitive stage such as the stage that I typically am involved and get them actually out into the real world And and I'm indebted to these companies and I will say nice things about them For those of you who you know want to use your smartphone instead of listening to my lecture I would recommend. This is a distraction for a pged org where you can put your dot on the map here These are all sites where there are Genomically literate individuals and this is my wife teaching a class every where she goes She in addition to being an amazing researcher. She teaches these classes on genetics So hopefully you won't be disappointed by By a survey I found that this tends to work. It's it's rather like it's it's a rather like a tour But the there's articles behind all of this and I haven't found it useful to go through the articles Many of these are unpublished, but it's the same idea. Anyway, these are topics DNA is a material And in C2 method an NT2 sequencing method and then it's open cohort that we just heard about Then CRISPR which probably needs no introduction and then the brain initiative which may also but So DNA as a material and I give three quick examples Which is the super resolution example and then a drug delivery and then Information this is just kind of a small experiment that I did with Pung Yen and William Shee who the three of us and Pam Silver as well are Constitute the DNA nanostructure component of Harvard and And three of us three labs collaborated on making these these long rods that that allow you to barcode literally in multiple colors at High resolution sees about 42 nanometers between these dots the closest space dots So this is way beyond the resolution fluorescence microscope and unlike any every previous method for super resolution This the blinking of the fluorophores and I'm not going to go into the whole physics of super resolution But the blinking the fluorophores is here Regulated by the the binding and not binding a fluorescently labeled a ligand nucleotides to the barcode so that the labeling itself provides In a neat way of doing flickering for those who are interested in super resolution Now we call these DNA nanorobots. They are robots Only in the sense they have three of the key components of robots, which is sensors logic and actuators If you only have two of those you're you're a machine rather a robot and and and here what they do is they will sense either immune cells or cancer cells based on two optimers DNA DNAs that single strand DNAs that fold up and recognize surface proteins of T cells or cancer cells And then only if you have an and Both optimers are activated that makes it a the logic Will it open up and deliver the antibodies to either modulatory or killing this case killing And you can see this little animation of showing it opening up when it binds the little blue optimers bind And here's some electron microscopy showing that we where we replace the antibodies with gold nanoparticles showing the What we designed is what we got and DNA nanostructures actually are much easier than protein design which we also do pretty much all of these things work Without much ado and undergraduates do it in the summer competition called bio mount on so that's example number two of DNA nanostructures we think are fairly useful most of the history of DNA nanostructures has been toys But these two we think are potentially useful and here's another one and we don't know because it's still early days this is the Colbert that was earlier mentioned and Here he is hawking my book. We made it to number 57 on Amazon due to Steven And we also encoded this book all 5.2 million bits into zeros and ones from zeros and ones to ACG and T So all books these days are zeros and ones at some point or another and this is what we call archival storage It's not like your thumb drive where you're just like filling with all day. It's archival storage And it has the advantage for that. It's a very specialized field I mean, although it's a multi-billion-dollar business and most of you don't know about but DNA has the advantage over typical archival storage which we have to replace every three years or so in that this has a 700,000 year test run which is better than most other storage media and And we can store about 200 exabytes per gram so we can fit a zeta bite in the size of a foam Where an exabyte is 8 times 10 to the 18th bits or 10 to the 18th bytes And the way we did this, I mean what? Well, we'll come to a moment in exactly the chemistry and instrumentation But this is the landscape where we have in blue kind of standard Consumer storage ranging from the old-fashioned CDs up to modern flash memory and hard drives so we're getting up into the This is log 10 on the y-axis and For the density and then production scale on the x-axis kind of gives you some idea of How big the market is how cheap it is and that we know is you don't go out to lots of bits unless it's inexpensive And then purple are a bunch of physics based methods Which are hard to scale into consumer markets because they involve things like liquid helium which most people don't want to carry around liquid helium and And then in red or some biologicals this is this is where we we bumped up essentially six orders of magnitude almost seven or eight relative to most storage media in terms of density And then this this year we have some funding from one of the archival Companies to increase it by ten thousand fold so ten thousand fold in one year in terms in in this axis of cost So how do we and this by the way? This was published in science along with Sri Khasari was the professor in this case My perfect my postdoc pretending to be professor and me pretending to be postdoc. I did this mostly with my own hands So you can see why I'm giving a little extra time so this is the this is a The two main there are many different methods that we worked on with these companies from the very beginning inkjet and this Photo lithography which actually originated here in Madison and we had great I would Dedicate this slide to Franco Serena And anyway, these are these are two methods which are essentially based on analogous methods in the non biological world and here you have four different color four different Phosphoramidate chemicals GAT and C and here you have Their flow flow through a flow cell one at a time and where they're where you can get either photo deprotection You can get up to six million oligos per chip and the chips can be as little as two hundred dollars So this is really inexpensive and this brings us to an interesting topic, which is why is everything so inexpensive? so The as many of you may know so that's that was all about writing genomes. We have to read them as well and we read Everything we've written and this is a This example of Moore's law curve, which was which is pretty aggressive. It's 1.5 fold per year multiplicative so these are factors of 10 on the y-axis And if you predicted how long would take to go from the three billion dollars, you know to an affordable one meaning around a thousand dollars Which is right now at about a thousand dollar cost slightly higher price and it was predicted to take Take six decades that Moore's law rates like electronics, but it actually only took six years so don't believe it you don't don't feel limited by You know very long-term trends. I guess is a lesson here You can't say whether it'll take longer or or or shorter Okay, and that's for cost, but the Reading is not just about cost. You could also you could reduce cost by making worse Product, but it turns out the quality is improving exponentially as well and just like the previous exponent the exponent has increased at least in my Limited analysis here, so it's sort of in the point three to three percent range At the beginning of the genome project and now it's around ten to minus seventh as we'll see in just a moment That's one error and ten million and The appetite phase length, which we'll also see in a moment is how long you can read Where you know that it came from the mother or the father in the case of a diploid human, let's say Okay, so exponentially improving cost and quality So what's so what have we done for you recently say what's the unpublished sequencing? So first I have to introduce this this this super One way of getting super high accuracy and it's relevant We originally developed this we meaning Rob Mitra in the 90s and then Jason during 2005 and then with Roddy Dramonic and his colleagues at complete genomics with a series of papers that resulted in the clube fleet genomics system, which is Like a sequencing by ligation But we also helped with the sequencing by polymerase synthesis and here This is an ordered array most of the other methods are unordered their Poisson limited They're kind of random, but this is kind of pretty it shows that each of these dots was micro fabricated Which is coarse and then a single molecule and albeit a rolling circle amplified molecule Completely occupies each dot so the dot might be 200 to 300 nanometers But they're each completely dominated by one molecule and hence you get these pure colors You know yellow magenta blue and green and you just don't get any blueish green or yellow magentas That's that beats the Poisson distribution and it also In combination with another method gives us this really high accuracy So you dilute 10 cells of genomes are kind of broken into the 200 kb range into 384 well plates And now the probability of finding mom and dad's gene genes in the same well is very low It's about a tenth of a genome and then you amplify in sequences by this like Legation method in the previous slide and you can now tell you can get the haplotype phase Which is clinically significant because let's say you have good alleles g on a particular gene So this is one gene two mutations and the paternal genome chromosome gene had two C's as alleles Or you could have the same two C's if you kind of lose track of where you are as you're sequencing along Because everything's very similar Then you could have a see it from mom and a see from dad which would mean you have zero functional copies So one functional copy versus zero functional copies should matter to you If you're going to get your genome sequenced in the physician now. Oh, by the way, just audience participation How many people here have their genome sequenced? Okay We got some work to do. Okay, so that Was done with ten cells not because we were showing off but because ten cells is the right number of dilution so that you get enough copies so that you get high accuracy and And you get the separation that you need for getting distinguishing The maternal paternal copy and for that matter distinguishing other homologs in the genome But we can go to single cell and indeed we can go to subcellular and we kind of skip straight to there because there's a lot of interesting context in In transcriptomes now we're switching from DNA to RNA where a cell is not a Homogeneous object it actually varies down an axon or from one side to another of a secretory cell and so we want three-dimensional information in the context of multicellular and Typically in cell biology we feel limited by the number of colors they overlap in a spectral sense And we're limited to about four colors practically speaking But if we can reuse those four colors without really moving without so that we can find each pixel again We essentially get where in is the number of cycles of you reuse the colors and here cycles We're calling them next generation sequencing cycles Then we get four to the end colors and that blows up very quickly into a very wonderful large number of colors So how do we do this? How do we get 3d imaging four to the end colors where n might be you know? 30 or so at subcellular resolution limited only by the resolution light microscope Which we've already shown is sort of getting into the 10 nanometer range So this is You know we don't need to drill down on all the gory detail of the method But but basically we take a fixed specimen we fix with this odd peg Connecting this succinical Groups so these will cross-link any amino groups so their aminos in the proteins which are fixed And then as we make cDNA we add additional amino groups and they get fixed the protein So you essentially get something that's kind of like a porous gel made up of proteins cross-linking to Circular cDNA so we circulize with circle eye gaze and then we very lightly amplify just enough so that it raises the signal to noise But doesn't make the dots big so now you have a three-dimensional Array of dots and each dot can yield a sequence tag So you have all the advantages of in C2 hybridization But more colors and you have all the advantages of RNA sequencing, but you haven't lost your place in the cell each Position of each sequence read is meaningful now rather than random It's not on a okay So this is a postdoc Jay Lee and Evan Dardi mainly Have been pioneering this for many years Not published yet, but in it's in it's in review so So there it's rather crowded in the cell so the cell has about a mean distance depending on the cell type of 100 nanometers between polyadenylation pop any site in the RNA and So one solution to this is super resolution so I showed how we can go sort of into the 10 to 40 nanometer range and Molecular certification and deconvolution I'll show in the next two slides The idea here is you do a subset of the of the transcripts and Then it will be sparse And it will be easy to for the computer to read that the are the bar are the sequence tags It'll be sparse, but you'll be missing some RNAs You come back in and do another subset different from the first subset and now it will also be sparse But you can add the two together and now you've got higher density in the computer Even though you never had high density in the microscope So here's an example where you have you do the subset you could subset it in a variety ways But here you do a subset it ends in a and here a subset that ends in AC and here's a subset where has no preference You get basically all the all the priming and you can see as you go down this that it gets more and more sparse and easier to Read and the correlation coefficient gets better and better And then deconvolution is kind of something you do anyway I don't mean to be making a big deal about it But it's something that you computationally do to improve the sharpness of and this has been a big a very big impact here, and we have various methods for Quantitating the quality of these things in particular. Here's one that the peels Easily explained where we have we have a database of transcriptome that you're drawing from or or genome and it tends to be the case that that RNAs come from Even though there are some anti-sense RNAs it tends to be there's a dominant RNA in every place And so 97% of what we we see is on one strand the correct strand if there's a database and And and we think there's relatively low noise Now we'll come back to physique as a particular application in a moment about with the brain Now this what is what is it about this personal genome project? this is The only open and it's one of the few integrated and we argue that it's Inequal one, you know, so there's big data, which you usually think of as big cohorts here. We have Sort of cohort independent. We're treating each person as if they are an equal one even though we have ambitions to have a big cohort So we have the idea is not just going from personal genomes to traits and some hugely predictive Algorithm, but we leverage the million fold improvement and cost and quality to fill in some of the blanks in between here so we can fill in the Environmental components some of these are either naturally have a sequence tag or can be made to have a sequence tag like the microbiome is Extremely, incredibly important for this project and their immune response to microbiome tells us In fact our immune response to all sorts of environmental factors kind of tells us what what our body is thinking about the environment And so chemicals leave an impression either on the immunome or on the epigenome that is to say are there are RNA and chromatin reflect certain kinds of chemical insults and we We we also Biobank many different cell types including B cells and fiber blasts induced pluripotent stem cells and some and and we and derivatives thereof and Then we traits traits is a big open-ended thing and this is kind of intended to be like Wikipedia If there's a trait that's missing there, then you can recommend it and we'll try to get it in place This is now spread internationally in US Canada UK and a number of other about five other countries are in are in process And we have approval for fully open access, you know world's only open access source for this and There it goes and here's we have an annual meeting on DNA day in April 25th and Hope some of you will join us there. Oh, is there anybody here? This is a PGP It's more than people and you haven't had your genomes because that's that's a real pity. That's our fault. Okay So So this is the genomes environments of traits We require a hundred percent on an exam. This is so that we don't tell them later Oh, there's something really Bothersome we found in your genome. Do you want to hear about it? We don't that's not the time for them to say no We don't want to hear about it because at that point you've already kind of told them that there's something to hear about So we try to make sure they know what they're getting into And in terms of privacy as well, and then we have stem cells which can differentiate into a whole variety of human tissues here via the the Teratoma model where you get diverse tissues and here fully in vitro and some of the some of the Traits are quite a whole organism. This is a Functional magnetic resonance imaging of my my brain actually these slices are virtual not not actual as you might have guessed and This project has now Achieved Some governmental attraction in the sense that in an unprecedented collaboration between the National Institute for Standard Technology and the Food and Drug Administration they wanted to have genome standards for the for the world and And they looked around for properly consented individuals and found only one project worldwide which is ours And so they're starting with eight trios from the personal genome project meaning mother father and child And this will and you can learn about it genome in a bottle org and also the NIH in code project is now taken on the personal genome project the encode project is trying to get at all the Regulisis regulatory elements or every base pair in the genome finding functions for it, and it's been limited by access to Properly consented cell lines and more beyond consent. You'll see some other advantages technological ones we have Now I'm not gonna spend a lot of time on how but these are examples of Inequal one studies where you either discover a new allele and what you can and what you can do with it Or in some cases you rediscover or you you apply them But these are cases where people were not getting what they wanted out of medical care and they found it in their genome So, you know like the Volcker children's probably the most famous one where they sort of child Was three years old getting loss of intestinal surgery and they did his genome and found out it was an immune function And so cord blood and he's fine five years later John Lauerman was one of the PGP Early adopters and he was basically healthy although he had in his medical records These dark spots on his retina or scotoma and he had leg pain That was bad enough that he got hospitalized and they actually and this surprised me They did a genetic test due to his leg of thinking it might be a clotting disorder They did what kind of a standard genetic test not even they had no family history of a factor five lightens that clotting factor Polymorphism and he he had he did not have it now You can't conclude from that that he has no genetic risk factors and indeed when we did his whole genome We found that he had a jack-2 mutation Which was somatic it wasn't a germ line And and that is does explain the scotoma and leg problems, and he's taking aspirin, which is not such a bad lifetime price to pay So these are but the point of all these these are sort of in equal one studies some of the more researchy than clinical of Looking for Deleterious alleles and what you can do to solve them so in each of these cases the solution is on the far right But what about the opposite problem? This has been under Represented, which is what if you have a rare protective allele rather? So these are people that manage to live past 110 they're called super centenarians And the take-home from this is not that if you want to live past 110 You should smoke and drink to excess But that maybe they have the same kind of environmental problems all of us have but they have something else that Biological is protecting them that is allowing them to get past all the infections and the chemicals in their environment So what so we and others are in the process of sequencing many of these individuals And you'll have to stay tuned to whether we actually find any protective alleles or not But there are a few that we can look at as examples and it also illustrates more important than just this idea protective versus Deleterious it illustrates what we can do to move from a correlation based Human genetics research to causality based because in many cases the n equals one is quite clear I mean that is to say you know I'm arguing that all of us are an equal one and to do research properly we need to treat it as such But there's some cases where it's very clear that in the whole literature. There's only one child who is Documented as having a myostatin double null meaning both Maternal and paternal and there's one child who has a myostatin receptor double null if I've correctly interpreted the literature But and these so it's hard to get 10,000 of these Children to do a big study But you can do you can switch to causality and actually becomes more convincing than a Than many GWAS studies because here's three different animal models. You don't have to do three, but in this case there were This is the double-muscled cow and then on the right is the myostatin dog which is you know To scale and this is the the the intentionally made mouse On the right as well So that's myostatin double nulls and that's the top of this list and here's kind of a list I've collected of rare protective alleles in addition myostatin. There's LRP five Which is not a double null, but it's a particular set of there's a few alleles that result in in a heterozygous state of extra strong bones so so strong that they will they will damage surgical drill bits and saws and And these people are very heavy in water dense in water PCSK 9 is kind of stimulated the the pharmaceutical industry because these people have incredibly low LDL cholesterol levels In fact when I first heard about it just I said can they be alive and they're not only alive They are they have very low coronary artery disease CCR 5 and FET2 are viral receptors, which apparently are fairly normal health-wise In the double null state But they are abnormal in that they are resistant to HIV and norovirus Now a P P could be on another slide about deleterious alleles The reasons on this slide is decode genetics found although there's many alleles of a P P Which cause you to be at higher risk for Alzheimer's there are some that cause you to be a much lower risk Maybe delaying it by 10 or 15 relative to controls So I want some of that Okay, now how do we get some of that? How do us regular folks get the Good alleles the protective alleles that other people have so Timothy Ray Brown was one of the first Wasn't exactly gene and therapy But he had a he he had leukemia and AIDS and went from being one of the most unlucky people of the world to be the luckiest because his donor for the fixing the leukemia Was not only matched for HLA, but also was a CCR 5 double null, which is the co-receptor for the AIDS virus and This in part along with other biological facts inspired clinical trial of This company sangamo, which I played a very tiny role in many many years ago But don't currently not currently evolved in but they made zinc finger fusions with a Bacterial in the nucleus if okay one and only when they're near one of these two these two sets of four fingers each Are near one another will they bind well enough that the that the nucleus will cleave and make a double-strand break and Then make a mess of the CCR 5 gene both copies of CCR 5 This is now in phase two clinical trials and looks quite promising in terms of toxicity and efficacy although it does work better on heterozygous and homozygous and most people in the world are not Heterozygous for this but anyway, it's it's I suspect it will get approved by the FDA fairly soon But there might be something better you know I this is this is effective and low toxicity for for cleaving Targets targeted genes where you design the the the zinc fingers to bind to you to whatever gene you want wherever you want These are very challenging to do experimentally. They're very expensive to do And there is a better way we think now although it's still early days This is CRISPR for genome engineering and not just humans but many other organisms there are so we've Participated since of almost the very dawn of each of these methods in Seven different ways of doing recombination Maybe more but definitely they're two of them involve RNA at two at the top group two entrons Which was part of my thesis and CRISPR, which is more recently I mean and then they're five they're protein based and one of them It's actually DNA plus protein based the DNA is finds a homology region while most these other ones It's just the protein and I'll just quickly summarize these two in a couple of slides the the mage Multiplex automated genome engineering uses this lambda red single strand oligos. It's currently limited In its best form to E. Coli, and we have a little bit of Similar method in yeast And then CRISPR, okay, but let's but let's talk about what you can do with mage rather than exactly how it works What we've done is Applied it genome-wide to the coli genome So each of these little blue dots is or bars is a place in the genome where we've engineered a codon we've engineered every single instance of of us of the stop code on UAG into a stop code on UA a So 321 changes genome-wide we did this in little blocks, and then we conjugated in that the important thing was that this this was a case of Truly genome-wide engineering where we changed a function. We didn't just make a copy of a genome We engineered it so that and it wasn't something was gene engineering It was really genome-wide and we and this had three things that we were aiming at and three things that we found ice Metabolic isolation we can make the organism dependent upon a non-standard amino acid which is Topic number two we can put in non-standard amino acids now at very high efficiency Without any crosstalk with other machinery in the cell and finally we can get genetic isolation so that the DNA functional DNA can't go in and out of the cell and and our ultimate goal is to get multi virus resistance in this case this one was T7 virus but the idea is if you change the genome the genetic code enough It'll be resistant to all viruses a priori all natural viruses. This was done Farron Isaacs is now professor Yale or assistant professor Yale and Mark LaJoy is a graduate student And this was this we have three science papers come in two at one of them's come out and two more this month Oddly enough, I mean who would think that you'd get three science papers about E. Coli codons But there you go some things happen But CRISPR CRISPR is not mage. It can use mage as its input But it is a phenomenon that really traces back to E. Coli in 1987 at least this is the earliest one I can find But it was basically neglected as a technology It was it's actually junk DNA for a while and it was finally discovered that it was kind of a bacterial active immunity and then just in January it turned into a technology where we and others Showed that it could work in humans and then sent so there's a few articles This is actually just a tiny subset of literature now. It's a few months later humans mouse zebrafish rabbitopsis Drosophila seal against yeast and many others so it it seems to work in other people's hands in contrast to many of our other technologies Not that they don't work there's just there's hard and they require companies to get it to work This doesn't require a company Except maybe to make some short oligos and what happens is these short oligos go and go to into a guide RNA Vector so this there's a little bit of constant RNA and then the variable part here in green and and rather than the protein Recognizing the DNA this the protein helps the RNA recognize the DNA There's a little bit that's two bases that are recognized by the protein And you make a triple X where you have two DNA strands an RNA strand and so basically you really only have to program about 15 to 20 base pairs in here And this was published in science in January Prashant Mali as a postdoc and Luhan Yang just got her PhD a couple weeks ago and here this demonstration was Doing homologous recombination, but you can also use it for what I showed before with CCR 5 we can make it nulls But this homologous recombination we took two we made two targets right next to each other so showing how close together you can get these guide RNAs and And you can do one or the other or both if you do both then you typically get a deletion at the correct endpoints And we compared it to another recent technology that we and others worked on called talons I'm not describing most of these acronyms because they don't really make much sense to me anyway But anyway talons We're so it went zinc fingers talons and crisper and we compare them in the next slide So here is that that that case of correcting two different guide RNAs near each other overlapping a talon site so they're as close as they can be to one another and to You know to being at the same point in chromatin all the chromatin effects and so forth And this was picked to be a site that's not particularly talent and sensitive There are sites that really we cannot design a talent to work But this was a fairly typical site and there is a 9 to 22 fold improvement going from talons to the guide RNAs And you can see there's a fairly small range here about a factor of 2 In efficiency Between the two different guide RNAs that are near one another What else can we do a guide RNAs we can make deletions so you hold in one end So you make this is two guide RNAs so two two of these double strand breaks Where one hand the left end is held fixed and we move the right guide RNA along it And you can see it's fairly flat in terms of efficiency sort of in the 10% range for these particular cell types. We can get up to 40 50 percent And this we we've the biggest deletion we've we've attempted is 83 kb And it's about as efficient as a 1 kb deletion So this works over pretty long distances. This is the work of post-doc Susan Bern who also did this experiment where she replaced so almost to what we do is in human cells We're trying to develop gene therapies and so forth in this particular case We took the human thigh one which is interesting immunological locus and we made either one or two double strand breaks on either side of it in order to Replace it with a mouse thigh one. These are really convenient because they're great immunological reagents for faxing so here you see fax sorting with just the left guide RNA just the right guide RNA and With both and none and you can see what we're looking for is this box here Which goes it's about 0.1 percent in the control 0.5 percent In the just one end if you get both ends it shuts shut up to 16 percent So you can do this Replace little bits of genes large bits of genes deletions up to 83 kb Maybe more and they were not limited to double strand breaks We can make nicks and we can make variations on this where we've knocked out both nick both nickings So there's no cleavage whatsoever. So it's completely Nuclease minus and then we can add on domains either onto the protein or onto the RNA by coupling it to app to RNA binding domains so you can put things that modify chromatin or Transcriptional activators or oppressors on the protein or the RNA we've done both and Some of this is described in nature methods and some in nature biotech now specificity you This in principle should come up with the mega nucleases with the mage with zinc fingers the talons But it comes up a lot with CRISPR for some reason and that's fine And this is what we've done in our first paper We focused on computational and empirical searches This is the one I showed you the very small one that I showed you but we generated computational For the entire human genome Guide RNAs for the whole genome Focusing on sites that were unlikely a priori to be off target So so we we took the whole genome and they filtered out the ones that were that might have a hit elsewhere and that allowed us to get 90% of the Of the genes and about 40% of the exons within each of those genes That's one there's two ways compute computation empirical We can also use nicks rather than double-strand breaks nicks greatly Two nicks are better than one double-strand break because each is independent And and we've done that That's item three and then for activation so this is not cleavage But activation we can get not just two but multiple sites and so here's an example of that where We we rigged it in such a way so that each individual activation CRISPR Cas9 is fairly low level of fold enriched fold Enhancement of expression with activation domain But as you get up into five and ten sites you get up to a 30 fold Increase over baseline of the transcription of this endogenous gene And this was published recently in nature biotech so so and these these are where the positions of those Natural so that we didn't in engineers we engineered the guide RNAs to match to natural endogenous sites scattered from Up up to 2.6 kb upstream for the transcription start site Okay, and we have other things in the pipeline for improving Specificity, but it's right now. It's already adequate for almost everything that we have in mind except possibly Human gene therapy which we're working on as well Now this is John Ock. He's He co-runs my lab with me and he's we've had Wonderful decades together and he's the one that designed these hundred that 90,000 guide RNAs that we published in January And as I said 90 percent of the human genes and 40 percent of the exons of those genes We're also targeting not only these messenger RNAs for in exons, but also activation upstream of messenger RNAs long non-coding RNAs micro RNAs And these are the number that we've targeted and we're using those in an interesting project of trans differentiation, which will be in the next slide Which is We hypothesize that or we wonder if any these are just cell types around the edge You don't need to read it But whether any cell type can be turned into any other cell type directly or indirectly and if so how many steps does it take? How many factors it take this is kind of a general question the one could ask and it could be fairly hypothesis Independent and and I talk about this a little bit in this like a systems biology opinion piece as an example of that here's reprogramming Not too different from what other groups have done in terms of turning IPS cells into neurons Including some groups here in Madison but but in this case we you can see this cluster this clump of Pluripotent stem cells they love to sit on top of each other and then With one of four different genes we found now this is shown Neurogenin one but but for three others we can get them to differentiate into a specific neuronal type What appears well by RNA seek analysis and morphology be very specific and they migrate out from this mass and they make this kind of this loose network of Bipolar cells we call them Where they have just exactly two processes coming out for them almost population-wide and this is a 98% Conversion maybe more in in merely four days this would if these are what we think they are in the in the brain This would be on the order of 70 or 80 days during normal human development, so You could call this an artifact or you could call it a technology okay, and we're and and This is kind of old data, but we have electrolytic activity and synapse formation in these neurons and the and the point of all this is not this particular Neural pathway, but with that we're trying to do this systematically using either the CRISPR method or other methods of of over producing or or not making nulls and Nulls something it's hard to do with RNA. I we did RNA sequencing on these and and we can I'm not going to go through all the details, but the point is we can characterize these cells and the problem with is there's not a lot of There there are Brain tissue in the databases, but they're not a lot of examples of single cell transcriptomes Where you have a pure cell type or a single cell type? So we we're hoping the in-situ sequencing will help us find out exactly what kinds of neurons these correspond to if any it Could be that this is not a natural these are not natural neurons. They don't but but they're interesting and that this is what? You can push Pluripotent stem cells to become and so we're they're telling us something now we've We've worked with Dunn-Ingber's group and William Poo and Kit Parker on organs on chips that all these different organs are represented by Model systems where you can have multiple cell types in a in a mechanically Significant environments for example here's the lung which has a air epithelium support layer endothelium blood and this behaves much more physiologically than any previous Culture condition rather like having cells on it's just a flat plastic With Kit Parker, we've done this contractile Assay for cardiac cardiac tissue and we can reproduce individual human Mutations we can test causality as I mentioned earlier not just animal models, but in this organ on a chip model Now I'm just going to end on a quick introduction to this brain innovative neuro technologies this was Something that my colleagues here listed here and I Somehow managed to get on to the political radar not that were any of us are particularly good at politics but this was announced by Obama in April 2nd and And I love the the acronym that he and his staff came up which was innovative neuro technologies Because it it's meant to convey something other than a juggernaut Unstoppable mega project but really to try to bring down costs and improve quality and maybe bring new modalities So what might those modalities be we've had a series of papers on this starting on some of them that predated that announcement in neuron and science and ACS nano and neuroscience Illustrating the various points, but the one I want to focus on just to wrap up here is this idea of integration or a rosetta brain we call it and the idea is you'd like to have a background information on behaving animal where you monitor perception and behavior and Then use four different kinds of fluorescent and T2 sequencing remember beginning of the talk fluorescent C2 sequencing We are developing a way of turning activity meaning brain activity of various biological activities like Calcium or other signaling molecules into a DNA ticker tape which we can read with fluorescent C2 sequencing We've got a little publication on this But it's by no means reduced to practice the connectome that we in Tony Zadar are collaborating on Where you can find the connectivity of every neuron to every neuron at the synapses that they make thousand synapses per neuron in some cases a Developmental lineage where we can develop use CRISPR to make barcodes real-time during development And then finally the transcriptome which is the what way I illustrated already you can do with fluorescent C2 sequencing The idea is not just to read the genome, but the model it and then write meaning that you you change the developmental biology you change the the Neuronal activity in order to test your ideas and the earlier you do that the sooner you know how far off you are in your knowledge So this is the big wake-up call a synthetic biology is And I'm not gonna go through all the bites that we get free season does it's not in my opinion Not daunting computational, but other people will almost certainly disagree But these are the different flavors of the rosetta the lineage to connectome the brain spike potentials or other activity that might be slower and so on Here's the example the brain Alan Brain Institute they did a serial section through mouse and human brains Looking for transcripts and they did them one transcript at a time.  It's easier to align the different images different brains have different Connectivity and so comparing different brains is problematic, so we'd like to do it all in one and that's the idea behind the Rosetta brain where we not only get all the transcripts in one brain, but we get all these other modalities as well and and that's my I'm gonna include on that particular fantasy and and this is people I want to thank on the Brain activity that map now renamed brain for innovative neurotechnologies and they're on our papers Sorry, I couldn't spend more time on it And these are people to thank for the personal genome projects and other people on the technology that mentioned all the way along And I'll just leave this up while we have a conversation with Questions, and I think there's still plenty of time left, but anybody's free to go. I guess so, you know, thank you so so It seems like they are more methylation sensitive than then Crispers Partly they they recognize a DNA from the major groove, which is where the five methyl C is there may be some new Amino Acid or some something that will help with the five-month-old C but there are other sites that that don't have any Cg dynucleotides in human that that essentially You know our my colleagues at Harvard to Chad Cowan and and others have found you just get zero recombinants out of hundreds of tries Or or NACJ and and we don't know what that is But and why and then they did they did ten of these and they did CRISPR side-by-side in every case CRISPR gave like 30 to 80% efficiency for things that we were getting 0% on the talent, so It's a little early to declare it, but I think that's the way it looks Well, I hope neurons aren't special But I'm certainly not naive enough to think that everything's going to be easy and I even said these may be Somewhat artifactual in some states and we won't know until we really get lots of trans single-cell transcriptome data You know we have reason to believe that it takes more than one factor to say for example Go from fibroblast to induced pluripotent stem cells, right and and I think to some extent we many of us felt that it well It's going to take four factors to do everything well But here's the good news is there are some cases where it's four and some where it's one But what we hope is that by finding all the cases where it is one we can have that as kind of a you know a starting point and Then we can do two factors at a time sort of semi systematically Maybe using the ones as starting points to seed it and then three and then four and five and go from there but it's it's basically it's a high it's a sort of a genomic Hypothesis independent way of finding out what the developmental landscape is starting from a cell that clearly wants to go a lot of different Directions based on the teratoma assay teratoma. You really it really goes all over the place Yeah, I didn't mention that yeah, but but any question is fair. So go ahead What are the limits currently and the extinction? Yeah, so the limits Okay, so I would say so the extinction big big topic we have a little talk on that but It's it's a non-field so far. Although there's a lot of people Working on it talking about it whole meetings on it I think the first set of things is Bringing back things are in the freezer the second set of things are bringing out certain traits Well, which will enable species to survive in the modern world In other words address the issues that made them extinct and So for example turning elephants in the mammoths so they can survive in the tundra Which might be a better place for both them and the tundra and I could give a whole reason for why that Actually could be good for global Temperatures But you know it may not take changing the entire genome to achieve those ecological goals and you could get back quite a few traits with a fairly small number of Genetic mutations and that's our hope is to explore that space Initially as the technology keeps on its exponential curve by the time we get so that we actually want to change Every base pair for some compulsive reason The technology will be much cheaper. I think that's a project that could have big environmental Positives but we need to be cautious and we should wait until look or we should work for the cost down Yeah Yes Yeah, right Yeah, so Fortunately, so this is one case where I was Obsessing at the beginning about getting poly a adjacent tags as was the case in ESTs and sage early and fortunately my People working on this project completely ignored that and just went for randomly primed and that has the advantage that If you have something blocking the polyase site junction, you're doomed Potentially and while if you do random priming you you get multiple places You might miss an exon in a particular molecule, but unless that exon is systematically covered with a protein You shouldn't miss it in the cell as a whole That said if you really want to get all these things you should we should do more than we're currently doing to get rid of the Proteins and to get rid of the secondary structures We When we plot I didn't show the plot we have plot where we just saturate some certain RNA We looked at the saturation the RNAs and you can see every place we get a read is where the secondary structure is is lower But it's it's pretty well covered the whole transcript is covered, but you can see the spikes So anyway, it's like RNA-seq basically Randomly primed RNA-seq in principle you can get splice forms you can get allelic variations So if you have a heterozygoposition so you can get allel specific transcription, which is a cool thing But we're it's still a baby technology. Don't try to keep expectations low Yes You'd like to see what's your genome. Well, would you like to do it with your own hands? Or would you like somebody to do it help you? Well, so so there's different way so if you wanted to do it quickly and not with your own hands alumina offers their clinical service Cleo approved Then if you want to participate in the personal genome project It's getting better mainly because the international groups are far more efficient than we are Meaning the Boston group, but that's mainly because we're doing so many things They're kind of proof of concept things rather than pushing people through but this there is a line for the Boston part, but if you're Qualified for one of the other countries Or one of the other better yet one of the other ones that's starting up in the United States We'll get that there's there we have ways we're working on that will get the backlog and There are rumors that there may be ways of getting a million done. This is how rumors spread, okay Other questions Yes Coverage So the question just I'll repeat the question For the personal genome project. Are we getting good coverage of the sequence relative to other public genomes? There are very few public genomes other than ours meaning at any level even the thousand so-called thousand genome project Does not have a thousand high-quality genomes. It's a handful of high-quality genomes our all We have 200 high-quality genomes that are that are covered at 40x or better Which is very Well so so that so going public or private genomes, there's no human genome that's been finished There's none where we there's about two to five percent of the genome that's not known and it's and that section is not Something we should dismiss the cop my colleague Steve Carol McCarroll in my department recently showed that in some of those Regions that are not sequenced are many many human genes that have been making it very hard to interpret their Parallel logs which were in the parts of genome that have been sequenced they start look they look like snips But in fact, there's just a completely different gene or a related gene that's hiding in the in the center mirrors for example So one of our ambitions is to sequence through all the center mirrors and other gaps But we haven't done that yet, but but but the personal genome project are among the highest quality genomes Period as far as I know Thank you


All right, we are so excited to welcome everyone. We are here at American Airlines headquarters with Ronnie Skinner, who is the director of this entire facility. Ronnie, what would you like to tell us? Thank y'all for having me here. I appreciate the time with y'all. It's very exciting what y'all are doing and where y'all are heading. So wish you the best with that. Fantastic. We also have Kayla Scott, our amazing organizational development coordinator. She keeps all of our schedules straight and budget straight, and everything else. Everything straight. Everything straight, right, Kayla? Yeah, absolutely. So happy to be here. Thank you so much, Ronnie, for your time, taking the time with us today, and we're really excited. Absolutely. Wishing you the best. Thank you so much. Absolutely. And my first question is, what were the most common concerns that team members voiced regarding the move to the new headquarters facility? Sure, certainly. So a few concerns that I could share. Number one, probably the most importantly was there was a concern about a lack of personal space availability into what they had and what they were moving to. So we basically, the message went out that it was you in a box. And that was a very sincere message. So there was a lot of concerns, a lot of angst about it, because we are by nature hoarders. And in our old campus, we had memorial stuff that went back for 20, 30 years. And so people, they were very passionate about it. And so they were nervous about losing that. And the second question, were there any specific ways that American Airlines prepared their teams to address these concerns that proved to be beneficial? Certainly. So several things to add here. One of the things I would say that we got right is we started the communications very, very early. We were relentless with communications. So we didn't just send one email out and we were done. We had weekly communications about it. In order to make the move happen, we appointed two different classifications of people assisting us. And it was across different departments. We had what we called the change captains. And they were responsible for discussing the things that an employee would see that was different from the old headquarters, if you will, to moving over here to the Skyview campus. So every week, these change captains met with their own departments. They discussed things that the employees could expect coming to the new campus. And the other thing we had that was involved is we had a move captain. And the move captain was responsible for when the time came, departmental-wise, getting the departments what they needed in a way to their seating into the floor space and into the department space that they needed specifically for their items and storage. Part of the move over here was not only did the employees go to an employee in one box, everywhere we could, by design, we curved the departmental storage tremendously back. So what was some of the most surprising feedback that you guys received, either during or after the move from team members? So a few things that I could share is employees at the original old headquarters, if you will, they did not have electric set stands. So one of the big positives was everybody received one. And everybody loves it. It is amazing during the course of the day. You really don't even think about it, but it's amazing during the course of the day how many times you change that configuration up and down. And I personally, myself, included from a health perspective, I think it's healthy not to be sitting in a chair all day long and give you the flexibility to be able to stand up and motivate you during the course of the day as well. Are there any other topics we haven't discussed that you feel would be helpful for the city of Fort Worth as we prepare for our move to future city office? I do believe that communication is the best, not one in your done. I would certainly suggest start the purging concept as early as possible and not only start it, but assign somebody to go do walks and see if it's actually happening. We actually tracked our waste leaving out whether it was recyclable or not. And we tracked it because we knew there was a lot of stuff in the building that needed to go and employees were not gonna be able to do it. And we couldn't afford to get to the last minute and everybody then have to deal with it being forced. So the move captains in themselves, part of their job was to manage the purging and make sure people were doing that as they went along during the day. So at the end, as when we got to the move date, we didn't have such a backlog of all the purging items. So I would say that that's very important as well. What advice or talking points did American Airlines provide to their leadership for employees that were a little negative towards the move? A few separate things I would share. So for sure, make sure the leadership within your group is the ones sharing the message, show that they're bought into it, show that they're involved in it and let the message, if you will, come from the top down. And as much as you can, show that they're living it and they're gonna be a part of the change as well. A few of the other things I would say is we were bringing in renderings as the construction moved along so that we could help with some anxiety so that people could see basically what, if you will, they were getting into and some of the amenities that we were gonna have. So another thing that helped was talking about not so much what you're not gonna have but what you are going to have. So the leadership would explain a lot of times about the amenities and the things you were gonna have in these buildings that you didn't have before. So I think we mixed it up really well so that, yes, there were things you were gonna lose but also we spoke to the things you were gonna get as an addition in regards to your amenities. So definitely highlight that. Don't let it go all negative. Make sure you're speaking to some of the things you're going to get as a part of this. So every chance that you can, bring the product out, show the product, show the demo, involve the people, it goes a long way. And just a follow-up on that. It wasn't just the one box, it was also you couldn't bring a space heater, you couldn't bring, there were other things along with that, correct? That is correct, that is correct. Because of the city for fire code, we reminded everybody of the things that are not allowed in the buildings. And we moved into a building that everything was functional, if you will, because we didn't have an older building. And so I think people learned right away they could count on the steady temperatures and I think that went away as well. But yes, you're absolutely right. There was a lot of people that had things that they weren't supposed to have and they didn't get to come with them. And they didn't get to come with them. So were there any hindsight things that American Airlines learned after the move that they wish they had incorporated? So I was trying to think about that. I don't really think so not on a big scale. Part of the, because of the way the building was built out, we moved into this building one time at a time. And I would say because of that, we did take advantage of a few misses. And so because we moved into it one tire at a time, then when the second tire moved in, we did change a few things up. And so each time we brought a tire online and moved those people into these towers, there probably was a few things we changed. I don't remember specifically what they were, but that allowed us not to move the mass at one time. We wound up moving 5,500 people into this facility over the four different tires one month at a time to kind of give you an idea of scope of how we moved. And there was a lot of excitement, because I remember we had a huge team here. We had lots of, go ahead and talk about that. Yeah, absolutely. So we had a welcome committee. We had a welcoming bag. We put the essentials in that you could think of that somebody would need in a new building that they didn't have before they might have forgotten. We did some giveaways for the food programs that created a buzz. So every chance that you can, bring home something that gives some buzz and gives people something to shift their focus to. It worked really well. How did y'all address individuals that love to play music or individuals that maybe would have a sports game going while at work and they'd be glancing over or watching a news program? How was that handled? So a few separate things that I would share on that. So when we did the design of the building, there's a noise reduction system referred to as white noise. And by design, we put it in place so that it has the ability to be adjusted in many areas within a landing on a floor. So number one, that was one way we disguised the site, if you will, and buffered it out. So that helped a lot. The other thing we had is we actually had training where we talk about building ethics, is what we called it. And it was the building ethics of you moving into an open campus. So as an example, as you pointed out the noise, we spoke to that, we reminded people, that's why you have, we call them huddle rooms. So if you need to take a phone call, if you think you're gonna have something that may be contentious, go to a huddle room. It gives you the ability to show at the door you have 100% privacy. So this may sound funny, but just as much as you're thinking about the noise, all the kitchenettes are the same.  So we actually went down to the point in the building, in the ethics piece, we started talking about food. And we mentioned to people, be mindful of what you're breaking in to have with you at lunch today. There's a hundred people on the floor and be mindful of your neighbors. And so we actually did that. And people are intelligent people and they understood what you were trying to say, if you will. And we didn't have any pushback with it. And I will tell you so far, and myself included, every now and then you get reminded, hey, hey, keep it down or something like that. But on a big scale, we've been here and I've not seen or heard any issues on a big scale from a noise problem at all. We wanted to cut down on trash and we thought it was the time to implement it. And so in this building, we chose not to give everybody a personal trashcan. I actually, myself, I thought that was gonna be more of a concern, but it really never slowed people down at all. So what we do is, within a common space, we probably have one small trashcan to probably 30 people. But we also said that if you wanted to bring in your small trashcan, you're more than welcome to do it. And it was amazing because you can see in the building at the end of the day, you see a VP empty in their own trash, you see a manager empty in their own trash.  So I think that was a positive that came out of that. And again, from a big pushback perspective, we didn't have any pushback at all. And I thought we were going to, but we didn't. All right, what types of communication and or training did American use to prepare employees for the transition prior to the move? Sure. So one thing we were lucky with, our architects had done this before and they provided a lot of different tools that we could use to communicate this out. The move captains were provided a lot of tools that they could speak to their own departments with. We started what we call a one campus, one team email distribution list. So everybody that was going to be moving, they got a weekly email. And in the email, it talked about what we were going to achieve by the next week. It talked about the moving dates. It gave pictures of the construction. And it also gave you an email that was a one station email that was addressed during the day that you could send an email to and ask questions about or concerns, anything you had about the move. So that was probably one of the most popular and the best ideas we've had given the people a stream for that. And then a weekly communication that went out. And even to today, we actually still use that. We call it OCOT email distribution. And now it incorporates our entire campus. One of the concerns that we're hearing about is parking. And I remember that came up here as well. Because people are concerned about how are we all going to get in there on time? Our building is lead gold. And by design of that, there's no reserve parking with the exception of the obvious, which is handicapped. So outside of that, there are parking spots that's designated for fuel efficient cars. There's parking designated for electric car, charging cars. And what did American do to help build awareness around that open etiquette? The open concept etiquette. Well, there was several times where we let the architect that was building our facility get in front of our people and speak to that. They did, in the beginning, they did several Q&As. There was a lot of questions and answers as to why we were moving to that. Collaboration obviously was one of the biggest things at heart. And that was one of the key drivers of the design of the building. On purpose, we have on the northern ends, we have common space that's readily available to everybody. It is a fantastic place to meet up with. One of the things that I'm amazed at every day is how many people choose to go meet up at the coffee place. And it's very cross-department and it's more about people that know each other. But by doing that, it's amazing how much, how many times and how frequent people talk about shop, just because they're in passing, it was not scheduled. And it just happened to be because you're going from one end to the building to the next.


The challenge of the Yukon The swiftest and strongest lead dog of the north country Blazing the trail for Sergeant Preston of the northwest mounted police In his relentless pursuit of lawbreakers On King! On New Huskies! Who discovered in the Yukon a stampede to the Klondike in the greedy race for riches Now back to the days of the gold rush when Sergeant Preston and his wonder dog King battled through storm and snow to preserve law and order as they met the challenge of the Yukon Every spring in the Yukon country Jim Green and his friend Dan Peters were returning to their homes on the outskirts of Selkirk after a successful day of hunting Peters was carrying a small animal wrapped in a scarf that wriggled in the squirm like a cat in a bag As they approached Jim's cabin Jim's young son Bob came running to meet them Poor Dad Yes he was looking for you How was the hunt Dad? Fine son, we got a big caribou hanging on a tree limb to go back after with a dog sled and we got something else too Now we might be glad to give it to you What is it?  Are you gonna let me keep him?  Do you think we ought to keep him in the cabin?  I wonder if you have time to stop a minute I want to show you something Why, yes I guess I have What is it? It's in the back of the cabin Say, isn't that a new lead dog you have? Yes it is, isn't he a beauty?  Yes Bob, he's rather young to be a lead dog He's hardly more than a puff But he's smarter and stronger than the rest of the team in spite of it Maybe a better time for your sled sergeant You see, he might not like the surprise I'm gonna show you Don't worry about King Bob, I've been training him ever since I got him He'll do anything I say Well, alright, come on Fine King, side me fella Dad and Mom went into town for supplies I thought I better stay here and watch Blackie Blackie? He's a surprise, here he is in this pen What? A bear cub? Well, where'd you get him?  Well, let's try it, Bob Now, let's see, how do you open it? Right here, this wire Oh, alright King, go in and see Blackie Careful now, don't hurt him Look, King is looking like he's afraid They're both young, they'll be playing together soon Dogs and bears don't usually get along together, do they? Well, any animals will get friendly if you get them together when they're young enough, Bob Please, Sergeant, will you bring King back here once in a while and let him play with Blackie? I hate to have Blackie get lonesome It would be nice to have a friend, an animal friend, I mean Sure, Bob, I come to see your father often When I do, I'll bring King We'll see that Blackie has a friend Gee, that'll be fine And so, through the following months King and the Black Bear became friends Blackie disliked other dogs but always accepted the big lead dog that he'd known since he was a cub As the months passed, the Bear grew fast and Bobbie's affection for him grew too Blackie was almost full-sized when one day, two strange men stopped beside the cabin where the animal was chained Bobbie was feeding Blackie as the men approached That's something that's quite a pet you have there Yes, sir, I raised him It was just a baby when I got him Here's some more fish, Blackie Hey, but he eats a lot, don't he? He sure does, but I can get all the fishing needs He likes berries and things I should think he'd be lots of trouble for you Oh, he's worth it That Bear would make good fur blankets You quit talking like that Charlie's an Indian I guess he don't think it's very practical to keep a pet like that Well, I don't care what he thinks Like, he's the best pet anybody could have I suppose you wouldn't think of selling him I should say not There isn't enough money in the world to buy Blackie That Bear, him, lots of trouble Maybe it would be to you, but I like it Well, I ain't interested in buying him I get troubles enough Is he gentle? Blackie wouldn't bite anybody Nobody's ever been mean to him He doesn't like dogs very much, though Doesn't like the most bearest dogs Well, come on, Charlie We gotta get back to town Goodbye, kids Bye Here, Blackie That big bear Yeah I could use an animal like that You mean you want Bear? How'd you like to earn some money, Charlie? How much money? Maybe it's gotta be paid off in fire water What you want me to? I might need a little help tonight I'm leaving town When I go, I'd like to take a big black bear in my wagon with me Me not help Steve Bear I didn't say anything about Stephen anything I'm sick and tired Maybe I won't need any help A job like the one I'm thinking of should be pretty simple About five weeks later Sergeant Preston returned from a northern patrol He was on his way to headquarters in Dawson City when he heard young Bob's voice Sorry, Steve, Sergeant Preston Oh, Bob, it's good to see you It's good to see you I've been waiting for you to get back Everything all right at home? No, Blackie's gone Somebody stole him about a month ago Stole him? We found his chain file Someone took him during the night But why would anyone want your pet bear? He's about full size now, isn't he? Yeah, he's awful big But he was gentle and I missed him He was a nice pet Remember how he and King used to play together? Yes, they always liked each other King was the only dog Blackie ever liked Well, Bob, the only thing I can do is keep my eyes open for him Gee, Sergeant, I knew you'd help me You've grown a lot since you saw him last But you can always tell him about that white spot over his left eye No other bear will have a marking like that But don't plan too much on my finding him, son This is a big country, you know But you're always going on long trips to different towns I know Well, I'll certainly try to find him Maybe I can pick up some information I'm sure I'll know Blackie if I see him I sure hope you'll find him I'll do my best, Bob See you later I have to report to headquarters Thanks a lot, Sergeant Bye Bye Come on, King Good morning, Inspector Oh, how are you, Sergeant?   Why, yes, sir, I have I know that tribe, they've always been peaceful We've never had any trouble with them I know we haven't But someone is selling them liquor, I believe We can't seem to find out who it is I've used the young Indian guide from that tribe His name's White Eagle I believe he's the son of the chief I might be able to get some information from him Good, that's what I was hoping for Just what have the Indians done, sir? Well, as yet, there hasn't been any murders They have raided a trading post Belonging to Pierre LaSalle Yes, sir, I know Pierre They did it during the night when Pierre wasn't there A few trappers have reported being robbed And a couple of drunken Indians pulled knives on a prospector And stole some gold dust Now, before things get too violent We have to find out where they're getting their liquor I'll do my best, sir You'll start tomorrow then? I'll start in the morning, Inspector I'll see Pierre and try to find White Eagle Good, they're sooner the better South of Dawson, a big covered wagon Stood in the center of the clearing Huge letters on the side of it read Beaver Bill by Beaver Oil The magic medicine Near the wagon stood Beaver Bill himself He was a big, powerful man And there was a wicked grin on his face As he teased a big black bear that was tied to a stake The full beast was bewildered and angry As Bill poked it with a sharp stick The man was so preoccupied with his cruelly That he found a notice, a young Indian Who had come quietly on Marcus and his feet And stood behind him, watching Come on, you lazy cuddly This will teach you not to be a leftover Ah, that's better Get your temper up, ah Wait the chain was longer so you'd get at me, ah You're plain and filthy Unless you get gentle, ah Most cuddly at that time, is ya? Why you keys bear like that? You come from? Me from Indian Village Why you hit bear? You got to keep me in It might not fight Anyway, it's none of your business And don't come sneak it up like that again You'll have to get shot Ah, bear leg hurt The dog done his last night Before he got the dog What are you doing here? What do you want?   What are you doing here? Where are you holding that knife? Me We not know it you You were waiting for someone else, weren't you? Me not tell Was it the man they called beaver bill? Him go to town Him not in camp And you thought he was coming back, is that it? Him bad Now why do you say that? Why were you planning to kill him?  Him hurt my people Why they go? You know I'm your friend, don't you? Yes You friend Soon you'll be chief of your tribe And you'll be a good brave chief Me try I could arrest you for this But I'm not going to I want to help your people There must be a reason for you doing this Your tribe's in trouble That's why I've been sent up here Now I promise you I'll help them If you'll help me Father say no tell I don't like to ask you to disobey your father Maybe You come to village You see I intended to go to your village from here We'll go back there together but Wait for me here I want to have a look at the camp of beaver bill Me wait A short time later, as white eagle led sergeant Preston To the cabin in which his father lived The Indian village was very still Except for some dogs that barked at him from a distance When the two men entered the cabin They found the old chief lying asleep On a bed of spruce boughs You see My father He was sick No can talk You're right He's dead of the world What's that on the floor? Here You look A bottle of beaver oil He hasn't been drinking this has he All brave drink it A little bit of it left in the bottle Well This isn't medicine, it's bad whiskey Did he get this from beaver bill Me not tell You'll find out But this isn't the same thing that pier has The bottle he bought is some kind of liniment White eagle You must help me So far you haven't told me anything But I found out for myself You'll find fire water in bottle An old beaver bill is selling it to you I'm going to arrest him Now will you help me?  No it's fire water Me pay with gold Fine I'll stay away from him To last for the show I'll call you when I want it You're going to arrest him now? Not yet white eagle I'm going over there now Come on King Me wait Anybody else want beaver oil?    You got a piece of bear in these glasses Look, they're touching over They like each other They're buddies Well, David Bill I guess I win the prize Looks like my dog can stay in there As long as he likes It sure does When you look at the critter He still pays off Show them nature of the curse Drop that paper or I'll break every bone in your body I won't Give it here Hand over that prize money It ain't fair I won't pay it You better like to go into the pit or the pit Hand it over Here you are Two hundred dollars gold pieces Two hundred Three hundred Thanks, Bill Now don't go away We have a little more business to attend to How'd you know that bear wouldn't hurt your dog? Strangely Well, I just had a hunch When I saw that white spot over his left eye Well, by darn, I never heard of anything like that Me either Let him have business You got me Just a minute Wait, Eagle Meet here, Sergeant Sergeant Maybe I should introduce myself I'm Sergeant Preston of the Northwest Mounted Police A Monty A Monty without his uniform And I'm putting you under arrest For selling liquor to the Indians You can't prove that Oh, yes, I can I have the evidence right here Show him those bottles, White Eagle Here, bottle That isn't medicine It's bad whiskey, Beaver Bill You know there's a law against selling liquor to the Indians Right, Eagle You filthy wretch No, Bill I'm taking you with me You'll spend the night in jail in Dawson City But do I can My bear A bear will be another charge against you You stole him from a small boy in Selkirk You can't prove it As for the furs and blankets in your wagon Most of them belong to Pierre LaSalle I'll take them back to him You won't leave them where you're going The next morning at Pierre LaSalle's trading post Sergeant Preston put a stack of gold coins on a counter As he talked to Pierre and White Eagle This is yours, Pierre But Sergeant, I cannot take that money You'll bring back most of what Indians steal Well, after you take what's owed you Use the rest for supplies for White Eagle's tribe Food enough to last through the winter Blankets, whatever they need Well, that money is yours and King's No, White Eagle King and I aren't allowed to accept prize money When we're on duty Oh, this is a fine thing you do, Sergeant I don't think we'll have any more trouble With the Indians now No, I thank you for my people You will come now and see them Some other time, White Eagle King and I must be on our way You see, there's a small boy down in Selkirk We'll be very anxious to see that bear All right, old fella, let's go It'll be quite a trip with a prisoner And your old friend, Blacking Yes, King, the case is closed A challenge of the Yukon A copyrighted feature is brought to you Each week at this time And all characters, names and incidents Used are fictitious Let's go


Welcome back to the channel everybody here again in South Africa. I hope you're loving this series It is extremely windy today And we're getting ready to move camps and head to another region of South Africa That's gonna have a much different terrain than this I wanted to share another story with you guys before we head out and a learning lesson for me that I wasn't even gonna post but I feel like I need to because it's a good learning Lesson for any bow hunters out there on the very first day we hunted here Rob was filming me and We went out on so many different stocks and we were just learning how these animals were behaving and Really trying to dial in what we needed to do to get an animal with the bow and by the last stock I was just a little edgy. I was like man. This is gonna be extremely hard. Even though it's the first day is just Getting turned down by all these animals and figuring out it's gonna be very tough with this wind Swirling and able to shoot with the wind But one of the guides told us that there was a read book that had been hanging in this particular Valley was a small valley and it had these these shrubs in it and he had been there Somewhere around that area the whole time That they had been there just roaming around so it was almost a for-sure thing that he was gonna be in that area So we went ahead and went over to that spot It was late in the afternoon and that stock ended up going on farther than what I thought it would we kept creeping and creeping around these bushes and finally One of the read buck does came out Soon as she came out. I thought to myself that is a Texas white tail dough and then the buck followed He ended up running out to 61 yards and I had to dial out on my bow And as soon as I drew out to that 61 yards something I've practiced a lot. He runs off Right there. I probably should have put my bow down because the 61 yard shot would have been great That's well within my you know my comfort zone Even though it was uphill and it was a little windy. I feel like I could make that shot What he ran out to it was 80 something yards. I think it was 83 yards Then I said I Don't know. I think I just want to take this shot because I'm set the end of the day I've been stalking all day. I haven't got anything. I don't know how many opportunities Tuned ease I'm gonna have I want to take this shot and I don't have an 80 pin and I ended up guessing and that was another mistake right there as I guessed on that shot and I completely missed the animal my third mistake was I did not test my broad heads when I got here. I've shot rage trypans Everywhere I go, you know, there hasn't been anything I've encountered that won't handle a rage trypan White tails and hogs and I've never hunted big African game Not but these aren't big But everyone says use fixed broad heads use fixed broad heads So I did not practice with those fixed broad heads and I did not know where that arrow was going to travel completely then my fourth mistake y'all The reed buck ran out to a hundred and three yards And our guide Patrick gave me the range on the binos and I said I'm gonna just bracket it And kiss again and fling an arrow and when I did it actually hit the reed buck and it hit him in the back leg That was a terrible mistake on my part. I should have never taken that shot y'all I should have paused at the 83 yard shot and let off and just let him go. It's on the first day You know, we got more time left to hunt if any of y'all have ever wounded an animal. It feels Terrible as I thought well that animals gone. I wounded it. It's probably gonna get eaten by jackals or something but then luckily we came across that reed buck again and The guys were able to get it with a rifle and get it down at least and that made me feel So much better. It was just a it was a huge learning lesson for me and I will never I will never draw back like that Unless I am a hundred and ten percent confident and really check Check my entire scenario that's going on all the little factors and after that y'all I was questioning my existence as a bow hunter entirely just after that day I was like, man, you know, what am I doing? I'm thinking I can take shots like that You know getting cocky like that and you know, I practiced and practice me around practice a lot obviously practice throughout the year, but That was just out of my league. So going out bow hunting again. I did not have confidence, but Luckily, y'all I was just able to get a great opportunity and get a 30-yard shot I didn't think we were gonna be able to get a 30-yard shot He had to play everything right conditions had to be perfect for the wind in the right direction and Just the noise level these Nihilas and these water bucks through their ears the way they can hear It's just incredible all these animals incredible eyesight.  So Don't stop at the South African buckies otherwise known as the Nananga I decided to branch out and get lamb and mint this time Nanaka Nanaka Sorry Patrick's not gonna let me mess that up. You have to learn three different languages here in South Africa when you grow up in these parts Lamb and mint my looks like yours So mine's lamb the lamb here totally different than it is y'all It is I don't know what the the lambs eat, but it doesn't have that like gaming goatee flavor It's more like steak. It's delicious Lemon mint was excellent. I will have one another one of those before we go for sure So it's getting a lot more arid now. Yeah, it's dry It's a bit nippy out here and into the desert Big okay, you can tear your face off Probably not what you thought right The area we're heading to now is like what you would think of more as Africa, but still to me this looks like South Texas Painted rocks. Yeah, they even got the painted rocks. I mean, it's beautiful But it's gonna be very difficult to try to Get something with the bow may have to switch it up end up using a rifle So we are here at another town about halfway to our next destination, but we're stopping at the taxidermist first I do have that read buck. I'm gonna do a euro amount on that on that read buck mainly because I don't want to look at that thing and Just have the memory of the bad miss that I had but I did shoot a very very nice Nala and that was with my bow. So big trophy for myself and We might have a few more before we leave but Ryan who's with us. He has racked them up and Rob's Rob's already got a Nala and he has a Sable which is one of the most sought after like true trophies Of Africa, so let's go see what else they got in here. This is all salt in here This is all hides Possum All these are animal skins Wow Yeah, so Feel how hard that is What oh my goodness Yeah, check the pool Oh, that's a whoop That's gonna be crazy like four or five months from now when we get these things back We're gonna totally forget about it. Then we're gonna show up and it's gonna be like Christmas So so here's my two animals right here got my reed buck which you guys From Texas and pretty much looks like a white tail with with horns and Then the absolutely beautiful and Yala skin OSG is gonna be Surprised by that one Oh Smell in here it smells like your taxidermy place like for fish very pungent Just look at all these amazing animals y'all So these are some that are being done this right here is actually a gem spot on the next place that we're going to they have a lot of these So this might be the next thing It was like it was like nail polish times like 45. Yeah These are I think dick-dicks David or these dick-dicks or these dikers a cliff-springer, right? That's a cliff-springer Tiny little deer look at that little guy So you guys the whole paper that they're talking like ears broken both spitting Cudu are massive There's your Nile crocodile That's creepy see a lot of examples in There had that had pedestal bounce in regular shoulder mounts. They had full-body mounts crazy stuff like crocodiles lions baboons all sorts of tiny deer species that I don't even know the names of them. I just had a What would be the equivalent of a whopper here in South Africa? What is that place called? Hold on. Let's get this on. We got sound like a steak. It was a chain Steak stickers Patrick. What was that burger place? Steers Steers and beers I love this truck here by the way It's land cruiser truck. I would love to have one of those Ladies and gentlemen, look at these Huge trees and a giant basket seat thing. Look at this. Look at this. It's like a big old Birds nest over here. It literally looks like South Texas Heavy heavy cover brush rubs not not much trees. There's some trees right here at the lodge That's about it the rest of this area. It's about 10,000 acres that we're in we drove through like these different preserves that had all sorts of animals so everybody that doesn't know pretty much Africa split up in a bunch of High-fence ranches like it's not the kind of high-fence ranches you think of like in Texas and Other parts of the states. These are so big that it oftentimes you don't even see a fence and this number one to keep those animals in of course and you know the running Business of hunting out here and people coming out to hunt these exotic animals But it also prevents any disease That the animals could get from crossing over into other ranches and things like that. It's also huge for The preservation of different species For example like the the sable that was almost hunt hunted into extinct Extinction and because people are interested in the hunting sables now all that money that has been brought in from hunters And the preservation because it's now important to preserve that for a financial Incentive it helps grow that population back up and they've come up significantly and that's been that way with a lot of other species so It's a it's kind of a weird circle like if there wasn't hunting here The economy would be a lot lower and the species there would be a lot more poaching and things like that So there's a more of incentive to protect them and it's just like in the States too, you know, we want to Practice good conservation preservation of our species and that's what's going on here and because of that we are seeing just Literally hundreds of animals a day at least on this other place. We were out here It's gonna be a lot harder to see them from what I've seen so far, but it's a lot more planes game in this area So we can run into Kim's buck springs buck spring buck all sorts of antelope species and just really see some cool stuff I just don't know if we're gonna be able to get it done with the bow But hey, it's all about the experience bow hunting was the jam on the last spot And if it's for rifle hunting on this one, I'm not gonna be disappointed. These rooms are super nice I was not expecting this at all. I thought the last place we were out was really nice and now like me Rob Ryan we all have our own Little lodge rooms and then there's also a main Big cabin as well. That's out here and it's got a bunch of really Really neat mounts and it's just just amazing y'all We actually have a little bit of time left in the day a couple hours So I think we are going to kind of mount up and go see the terrain And maybe even pick up a rifle and go hunting. So There is a gums buck out here, which kind of looks like an oryx, which we have back in Texas You see all a lot of exotic ranches. They have really long spiky horns Really the animal I had on the top of my list when I came here was a water buck Just because they look like it's just a majestic mule deer white tail elk species And they're very hard to kill and I was unable to get a proper stock on one at the last place Just just got busted really hard to kill with a bow They have something here that is called a lesh way. It's a red lesh way and it looks similar to a water buck Again, very lucive all these animals out here are gonna be very elusive. They have extremely good eyesight So say to because we're about to get a whole new experience in a different part of South Africa Oh, it doesn't hurt nearly as bad like I like beforehand. I couldn't close it like that You want to like scream I was almost going home. It was it was almost like you were going home Not for sure. So I would let you know This one in the back is a nice