Feng Zhang: The Future of Gene Editing - Schrödinger at 75: The Future of Biology

[Music] okay Knicks up is Fang Zhang who will talk to us on the future of gene-editing Fang is from the road and the McGovern Institute at MIT hi good afternoon it's a real pleasure to be here and to be a part of the celebration for schirling year at 75 I want to tell you a little bit about gene editing and what gene ID is and and where conniving is headed and how do we develop and apply technologies for modifying the genome with a potential to be able to treat many diseases in the future so one

of the really exciting advances in biology has been the completion of the human genome and the cause of human DNA sequencing has has dropped precipitously and so now we can sequence the genome of thousands or tens of thousands of individuals so by comparing the genome of these individuals we can find genetic differences that may underlie a disease so for example some of the mutations that we have identified cause sickle-cell disease or cystic fibrosis or IV generation but not alternative differences are necessarily harmful in fact some can even be potentially beneficial and so other ones including

mutations in the gene called ccr5 can confer protection against HIV or a decreased risk for Alzheimer's disease or diabetes and many others so as we understand more about how genetic differences contribute to human health and also disease one thought is why don't we take this information and go into the genome of ourselves and be able to remove the deleterious mutations in and put in the ones that are potentially beneficial so this is the the very idea that many folks have worked on for several decades now and one of the approaches is based on this this

idea this was developed by Jim Hebert and also Maria Jason and more than a couple of decades ago the idea is to use a way to break the DNA so think of the DNA is a very long string in order to be able to modify a specific letter within a DNA one thing that we need to do is make a cut in the DNA this is called a DNA that was trying to break if we know where the in the DNA we want to fix or edit then simply by making a double-stranded cut in that

location we can then trigger repair machineries in the cell to then go and be able to fix at those different mutations so then the challenge becomes how do you precisely make a cut in where you want to edit and to do that scientists have developed many ways to reprogram what's called DNA binding proteins these are molecules that can be engineered to recognize a specific DNA sequence and we introduce it into cells it can go and try to find the region that you want to edit and then be able to bring other proteins such as nucleases

to be able to cut the DNA so what we have been working on for the past number of years is to develop a specific molecule called Kasner or other variants of it to be able to edit a specific DNA sequences in the human genome Castine is from a bacterial immune system called CRISPR and it's a system that bacterial cells used to defend themselves against invading viruses the way it works is that it uses a small RNA to be able to recognize a specific virus sequence and upon recognition the enzyme cassadine will go and cleave that

virus sequence and that's to convince the virus infection but we can engineer a harness system to turn it into a way to be able to search for human DNA sequences and by introducing that into the human nucleus we can then program this to cleave a specific mutation locations and then be able to affect our genome editing so here is a short video that kind of summarizes the way that casts 9 from the CRISPR system it allows us to edit human DNA and so what you see here the blue glob is that cast 9 protein and

we can program it with RNA molecule that's shown read that that's supposed to recognize a specific human genome sequence and by introducing this into the human genome into nucleus this Cassadine protein with RNA guy can search along the human DNA and trying to try to find where the RNA matches the DNA and so that allows us to localize the enzyme and to the site that want to edit in upon recognition the enzyme then Cleaves the DNA which is in blue into two broken ends and that is what triggers the repair machine machineries in a cell

to be able to make a repair and so one way to repair it is called anomalies in joining it simply Rekluse the two ends together but it introduces the small change that can inactivate a deleterious gene even more powerful is you can provide a piece of synthetic DNA called a template DNA and that template DNA can carry almost any sequence you want to introduce into the genome and through homology or through similarity that new piece of synthetic DNA can get incorporated into the genome to replace the sequence or to fix a disease causing a mutation

and so this is the way that we go about using this DNA nucleus to make precise changes either to inactivate deleterious genes or to be able to introduce genetic sequences that are potentially beneficial for for improving human health in addition to using castes and nucleus there are other ways to use Kassner as well and that's done by inactivating one of the domains on caste 9 that's responsible for cleaving at the DNA and so when you do that then what will happen is that this molecule will now simply find DNA and the ones he finds it

it will sit on the DNA and not do anything more than that this provides us a way to be able to localize things to a specific location on the genome without actually modifying it and so then we can bring other types of molecules to that region to turn a gene on or turn a gene off or to be able to visualize how how the genome is dynamically rearranging itself and so these are the two really mean ways that we go about using the CRISPR cassadine system 14 editing either to cleave DNA or to use it

as kind of a localizer to bring different things into a specific location as we're working on cast 9 one of the things that we wondered is cast on is a very powerful molecule but are there more molecules that are like Arsenal or or related to cast 9 that may allow us to do other things to be able to study biology and to be able to treat disease so we went back to the drawing board and we look at the diversity of CRISPR care systems it turns out that CRISPR is a very diverse set of microbial

immune systems in addition a caste 9 which only constitutes a very small portion of the many many types of CRISPR systems there are in fact many other types of CRISPR systems but the other types that scientists have identified at a time or much more complicated unlike asinine which uses just a single protein to do everything these other systems use many proteins that have to assemble together in very precise ways to be able to facilitate and the recognition also destruction of viruses so we thought if caste 9 exists could there be other simple single protein systems

that may also be harnessed able to develop different biotechnological applications so we've been conceptualized as as follows we can group the CRISPR caste system into two types there's the class 1 system which are the complicated ones that use many different proteins to do what we wanted to do and then there's the class 2 system where cast line belongs and it uses just a single protein and so one question is are there other class 2 systems that can find from the natural diversity so by working with a collaborator Eugene Koonin at NIH we develop a computational

pipeline and to search it search through the bacterial genome make sequences to see are there new types of class - CRISPR systems and so by following through a series of steps where we begin with one of the molecules that's very well conserved in all CRISPR systems and using that as the bait to find other putative yet to be identified CRISPR systems were then able to identify a number of candidates that represent potentially new CRISPR enzymes with novel functionalities so at the end of that search we found a number of new systems and this is more

or less up to date a depiction of what we have found so there are three main types of CRISPR Class two systems there's the type 2 which contains cast 9 but in addition to that we also found type 5 which contains cast 12 this is also a DNA targeting system so you can also develop it for gene editing applications and then the third subtype is the type 6 system which carries a protein called cast 13 and unlike hash 9 cast 12 cast 13 targets RNA instead of DNA so RNA is the other molecule in in

living systems that transmits information from DNA into protein so we've developed cast 19 catch 12 for a lot of different applications in gene editing but I thought what I'll share with you today are some of the applications we've been developing with cast 13 to target RNA so cast 13 is is a very diverse family of proteins in fact there are there are four different members of casts 13 identified to date and they're called cast 13 a b c and d and they all target RNA with a guide RNA and so that means you can program

ibly target new molecules of RNA mRNA non-coding rnas in cells by simply giving the castor teen protein a short RNA guide molecule and upon recognition then castrating can facilitate modification of that RNA so one of the applications that we developed using cast 13 is to make it into an RNA editing system so rather than changing DNA sequences which can have long lasting consequences we can use RNA editing to transiently make an alteration in the cell to be able to achieve a change to the biology for example we can rescue a genetic disease causing mutation or

if you want to transiently alter the function of protein we can also do it at RNA level so the way to do this is we altered this cancer team all of you very much like what we have done with cast 9 you can introduce mutations into the into the regions that can certain molecule that's responsible for cleaving RNA to inactivate it so that it simply binds to RNA and so when we do that we can then attach onto this caste 13 molecule a sub-domain that's belonging to RNA editing enzyme called a dark a dark converts

adenosine in the inner scenes so that allows us to make changes at RNA level that that will affect single base changes so by taking cash 13 fused with Adar we can then program it with a short RNA guide to direct this molecule to a specific mRNA in the cell and when we do that then we can specify the conversion of a specific Dinesen on that RNA by misspelling that that base with a cytosine on the guide RNA and so when you do that you can then program the system to alter specific RNA molecules so this

is a more detailed version what that looks like so on top is the RNA that wants to modify and then in red and then extended with this her pain is the guide RNA molecule and so this guide RNA will find the target RNA through base pairing between the guys sequence shown here in red and also the target RNA and if we want to edit this specific dinner scene would simply miss pair that with the cytosine and so that we can make a precise change in that specific molecule so one application this of this could be

that this mutation would represent a specific change of amino acid that caused disease maybe in sickle-cell disease or or in a in a congenital disease and by editing that RNA we can then correct the protein product and so that we can rescue that the disease phenotype so then we tested to see how well this system works and in fact it can work a pretty robustly so when we design a guide RNA we can alter the target RNA with over ninety percent efficiency by the first version of the system that we developed was not perfect in

fact it introduces other unwanted changes within the cell and so by sequencing the cell even at its shallow depths of 12.5 times the coverage of RNAs in the cell we find that there are a number of our target activities and so what that means is that we're not only making the the desired change but we're also making changes that we want to within what we don't want to be introducing into the cell so to solve that problem within started to try to understand what's going on and so by using the same system with the RNA

guy that doesn't target the RNA that we're trying to modify we find that we still get very much the same off target modifications within that cell so what that means is that these are targeted changes are likely introduced not by the guide RNA but by the enzyme and that gave us a path to then go and try to improve the system so by looking at the crystal structure the atomic shape of this molecule and we can then design ways to be able to minimize the potential association between this molecule and also the unwanted changes and

so by identifying all these different residues on this molecule we can then alter them so that we can we can try to get it to recognize RNA in a more specific fashion so it has stood a large number of them and for example some of these restaurateurs like to 375 G can significantly improve the recognition of the of the desired molecule while ignoring the molecules that we don't want it to modify and this is the result of that change so the first version of the system you can see that in addition to making the desired

change we also get a bunch of other changes in the RNA whereas once we engineered this protein to be more specific we can get it to be much much more specific so we then also profile the whole cell to see in addition to making changes on the RNA that weren't trying to modify is it also changing other RNAs that are not recognized by this molecule and so this is the original version of the system this is the desired change but you also get many many other undesirable changes but then once we start to engineer the

protein we can really clean this up so now we get the desired change and very few unwanted modifications in the cell and in fact now most of these mutations they don't really they don't really have a functional change to those specific transcripts so this is part of what we're trying to do to develop this into a system to be able to deliver into the body to be able to treat a specific disease causing and mutations so to take this further forward and one of the things we've been doing is to develop a way to deliver

it and one way to do that is to package this into a virus based vector so that we can deliver use this virus to deliver into cells in the body it's the way deliver this into neurons and try to see can we make precise changes and at an efficient level in in the neurons that we try to modify and so what we find is that indeed if you want to modify the specific DNA RNA base in this neuron we can we can do it fairly efficiently so this is one way that we're trying to advance

the technology forward but another way is to expand the capability what we can do the Adar enzyme naturally converts a to I and that's only one form of a change but it would be good to be able to make other changes and so we can engineer Adar to do that and so one of the things we did is the original system is called repair which converts a to AI and if we if you use this to convert see it doesn't really alter this cytosine residue but if we introduce engineered mutations into this enzyme we can

then get get it to change C to a you rather than a to R I and so now we can develop a second system called rescue that then converts the C into yourself and that will allow us to make even further number of changes within the RNA of these cells the finally as we start to engineer the system to make it more robust more specific and also more efficient one of the things that's really helpful is to be able to know how this protein looks so that we can take the structure and then use a

rational approach to further engineer the system and so of all the family members of cancer team ABC and so forth we found that that the B family the Cancer Team B molecules are the most robots in the in in human cells and so so if you compare their activity the ones that on the bottom left corner are the ones that are most robust and most of these are the blue ones so castor Team B and so to solve the structure would then use x-ray crystallography and we solve the crystal structure for for cancer named B

so now we know this is how it looks and we can go and start to tweak many of these different positions on this molecule to make it more specific so that we can edit RNA in a precise way and also make it even more efficient so we're continuing to further improve on the system make it more specific more efficient develop delivery methodologies so that we can use it to edit cells in a very proof in the efficient way in the body and also beginning to test this using animal models to see how well we can

really use the system in a therapeutic context so we have been able to do a lot with CRISPR systems using cast 9 cast 12 cast 13 then one of the questions that we ask is can we generalize this idea even more can we broaden it to looking at other systems in the microbial world that may be quite interesting and so one of the things that were continuing to work on to further push these gene editing technology is also beyond is to look for other biological systems that are putative Lee programmable so if you think about

the ones that we have worked with so far cast nigh antibodies or even at the microbial pale proteins most of these belong to immune systems and these are naturally occurring but rapidly diversify systems that that nature uses to be be able to defend against different forms of invading species and so we are now asking a question are there even more programmable systems beyond the ones that wipe that identified so far and furthermore by looking at microbial based adaptive immune systems who may be able to identify some of these new putative reprogrammable systems so by taking

a computation approach where I began to scour the microbial diversity and so using computation you can identify many many different candidates I'll just show you a few interesting examples this is example 47 out of a 9,000 of them and you can see that this is another repeat containing protein that carries regions with variability that that's linked to a protease domain and so one hypothesis is that maybe these repeat residues allow us to develop new proteins that can recognize altered protein substrates to be able to degrade them insults another example are these secreted proteins that may

act as immune molecules in the bye bacterial world and so these are all highly conserved proteins and you can see if you line them up they are largely similar except for a fixed region that's very very variable and so by trying to understand what these variable regions may be doing we can then further try to understand how we might be able to harness in this molecule and to develop interesting applications and to recognize diverse substrates so this is probably just the tip of iceberg because as we sequence the bacterial diversity we're now accumulating large numbers

of genomic sequences and so this is a plot a to date so as of now we have a 140,000 bacterial genomes and as sequencing technology further advances we're going to accumulate many many more bacterial genomes and from that will undoubtedly be able to identify many more interesting microbial physiology and microbial mechanisms and some of them who might be able to harness and develop into a powerful gene editing systems or even for additional applications so that's where we are but it's really a very exciting future but finally I just want to acknowledge my team at MIT

and the Broad Institute who have worked with me to develop many of these ideas and also our collaborators in the funding agencies that generously supported our work so thank you very much [Applause] [Music]