Advancements in Genome Editing Technologies and Applications

Philip Mars has been a proud member of the gardener community for 12 years, helping run the review panel for Garin Award winners.

### Gairdner Award Review Process and Advances in Genome Editing Technologies
* 🏆 The Gairdner Award review process involves two panels: a medical review panel of rising stars and a medical advisory board, which **selects top candidates from hundreds of nominations**.
* Dr. Feng Zhang, a Gairdner Award laureate, discussed the development and applications of **CRISPR-Cas9 genome editing technology** in his presentation.
* 🔬 CRISPR-Cas9 is a revolutionary genome editing tool that can make **precise changes in the DNA** of living organisms, with potential applications in **treating genetic diseases**.
* Dr. Zhang's team has developed new versions of Cas9 with **improved specificity**, reducing off-target effects that could cause **unintended mutations**.
* They have also explored other CRISPR systems, such as Cpf1, which has different properties and could be used for **specific applications, like multiplex genome editing**.
* 🧠 Dr. Zhang's team is working on combining genome editing with single-cell sequencing techniques to study **gene function in complex tissues, like the brain**.
* 🌱 The presentation highlighted the **rapid progress** in understanding and harnessing CRISPR systems from diverse microbial species for various **biotechnological applications**.
* 👩 Dr. Zhang acknowledged his team and collaborators for their contributions to developing these technologies, emphasizing the **importance of teamwork in scientific advancements**.

I've been a member of the gardener community for 12 years. My name is Philip Mars, and I help run the review panel that starts the process of deciding who our Garin Award winners are for the year. It's been a proud affiliation, and I must point out that working with folks like Dr. Dirks, his leadership, and Dr. Ross, and as the new leader, has been one of the most exciting aspects of my career. I wanted to give a few minutes on the review process because we are transparent about the review process. The nominees and the review themselves are confidential, but we want to be transparent and not opaque about how we select the finest. There are two panels: a medical review panel made up of rising stars, 25 to 30 of them in any given year throughout Canada. We get, on average, in the last few years, hundreds of nominations. Last year, for instance, there were 160 nominations, many that were new. The first process of the review panel is for these rising stars and Canadian science.

to say who should we put right to the top from that a short and a long list is developed. Sometimes the applications need a bit more finesse. As this is a great area, this is the right subject. We have this name, that's great, but we have all of the names. And that's a complex process of today's science because as our speakers in the earlier part of that morning of told us, science is a team effort. Nowadays there's a second panel, the medical advisory board. Many members are here this afternoon. Dr. Schenkman, for instance, a former Nobel laureate. And the purpose of that is that will narrow down this list of these 160 initial nominations and to come up with an idea as to who ranks right at the top at this moment in time. Sometimes we see nominations for a few years until the science is crystallized into something that the Gairdner family, when the board's first started in 1959, were keen on. It makes a difference in human medicine.

sometimes that's a hard judgment. Early technologies take a bit of time to develop into those that are on. Some things go right to the top very quickly. One can think of how quickly the RNA interference pathways were recognized and the guarantor foundation in o5 recognized fire and mellow and the Nobel Prize followed shortly after that. Review process. We're very proud of. We think it serves as well. It's often worth looking at what we're doing and how we do it. But we're all very proud of the process. This afternoon, we're going to have the opportunity to hear three of our laureates than this year following on in the theme of the day of a total five awardees in the field of Kris Pearn cast nine. Our next speaker is going to complete this story. Dr. Feng Jiang. I would have loved to have been on your promotion committee when you got promoted to associate professor this year. You know for those who sat on promotions committees, sometimes it's nice to have an easy package.

for the students in the audience, I just want to highlight that Dr. Feng is a bit younger than me. I got that disease of aging, but the ability to do chi quality signs to be recognized earlier in your career is out there. He emigrated from China to the United States des Moines in 1993, and what's really interesting is that he got his first interest in science learning as a high school student in someone doing genome editing in his home city. He quickly went on to excel in his studies, went on to a PhD work with dis Roth in optogenetics field exciting area that many scientists follow with avid interest. But in 2011, he opened up his own lab where he's a member of the broader McGovern Institute's, and today he's going to talk to us about the next stage of the gene Eddings paradigm and story we've heard about. He's got about 24 trainees in his laboratory, and it's amazing to see his a young career unfortunates have I would love to be on.

your next promotion committee. Good afternoon. It's truly a tremendous honor and a tremendous pleasure to be here, and I want to first thank the Kiran Foundation for this tremendous honor. And I had the opportunity to visit Alberta young Edmonton earlier this weekend, and I'm really quite feel quite quite fortunate to be able to see Canada and interact with the scientists that's throughout the country me not only high school students but also researchers and learning about what they work on. It's truly truly being tremendous pleasure, and I hope that we'll continue to have many more interactions in the years to come. So I'll tell you a bit about sort of application of genome editing for for various areas, and just by way of getting started today, there are over 6,000 genetic diseases that have been identified, and we know the genetic mutation that underlie and many of these different disease so now you might think if we know what a mutations are in the genome that cause of these devastating illnesses why don't we just go into

these cells and then fix these genetic mutations. Why don't we just replace what is causing a problem with the wild-type sequences? Well, it turns out that the genome is microscopic and going into the genome, which is already quite complicated, 3 billion bases long, making precise changes in the genome is very challenging. And indeed, this is a challenge that has been worked on by many, many laboratories over the past several decades. One of the earlier approaches relied on the use of DNA binding domains and is a really nice idea. Where if we can build proteins and that can be customized to recognize a specific DNA sequence within the genome, then we can use that protein as an anchor to bring nucleus domains to specific sites and then facilitate editing. Maria, Jason, and also Jeff. Hey, Jim Hebert discovered over 15-20 years ago that if there's a double-stranded break within the genome, then those double-stranded breaks will be sensed by the cell as DNA, and they can trigger endogenous repair.

machineries belonging to either the non-homogeneous way or a homology-directed repair pathway to stimulate and increase the rate of gene repair. And so, using these DNA-binding domain anchors, we can recruit nucleus domains to specific sites and make double-stranded breaks and then be able to stimulate in hgj or HDR. And so, the key thing here is really developing an easy-to-use ability to introduce double-strand breaks. And so, here are two of the earlier technologies. So, one of them has been worked on by many labs for more than a decade. It's called a zinc finger protein technology where zinc finger proteins are naturally occurring protein motifs that recognize interact with DNA. But through directed evolution or targeted mutagenesis, it is possible to alter the binding specificity of individual zinc finger modules. And you can link that modules together to form what we call a zinc finger array to be able to recognize a long stretch of DNA sequence. So, each module a fitting finger recognized three bases. And if you want to recognize nine

bases you'll have three modules. 18 bases would mean you have six different modules. But challenge here is how do we go and engineer the specificity of these modules. Turns out that's a very challenging task. So even if you have someone who is very deeply experienced in engineering zinc finger proteins, it can still take several weeks. And even then, you're not guaranteed to have something that's easily inefficient and for the target sequence you're trying to modify. A few years ago, a second DNA binding domain technology surfaced and that's the tail technology which is in some ways analogous using finger because it also uses individual DNA binding domains that recognize single DNA bases that you can link together to form a tail array. And these tail proteins can recognize long stretches of DNA sequences 12 bases up to, you know, 30cm bases at a time. But still, tail proteins are highly repetitive. Each module is more or less identical to every other module. So you are working with polymer and in working with that long homo polymer takes quite

a bit of a complicated molecular biology manipulation to generate and new proteins and so because of these challenges one of the the knees was to see can we develop a new genome editing system that's not only easier faster but also cheaper so that we can really broadly apply it to to city many many different genes so earlier this morning we heard really fantastic introduction by by Fillion and rudolf von on the basic CRISPR systems and also by jennifer night menu on the the mechanism of CRISPR system so my foray into the CRISPR area began actually with the paper published by Sylvain muy new who is the Canadian scientists at Laval University and back in 2010 Sylvain published his paper where he showed that the CRISPR caste 9 system uses RNA guide to be able to base pair with the target DNA and then facilitate targeted DNA double strand break when I read that paper I got immediately very excited because as I was thinking about working on nucleases

especially, I was working on Pelo nucleus at the time when I heard about the ability to use RNA to guide the specificity. I thought this could be a much simpler system. If we can get this to edit in the human genome, one thing to note about the double-strand break that's generated by Cas9 that Sylvain showed is that unlike the type 1 and also type 3 CRISPR systems where type 1 not only cleaves DNA but also does more damage on the target site, and type 3 system is a transcription-dependent DNA endonuclease, type 2 system makes a clean cut. And then you can use that cut to stimulate in HTJ or HDR process. So, at a time, there were many different applications that we heard about this morning, and a lot of it were focused on industrial applications, but I thought maybe we can use it harness Cas9 so that we can edit the genome of eukaryotic cells. So that we can have an easier, cheaper, and also faster genome.

editing so we work on exploring different Cassadine proteins and so we showed that you can take two different families of Cassadine proteins. One from Esther marvelous that the protein that she'll be my new had in his paper. And also another protein from s pyogenes said that Rudolf in and Emmanuel talked about in their studies. And by combining these Cassadine proteins with the tracer RNA and also with a CRISPR array and along with the host RNAs molecule, we can use it to be able to introduce double strand breaks. And so we engineered the CRISPR array with a small guy sequence that matches the target site on a human genome in the emx one gene. And then it's flanked by a short and motif called a pan motif. And they're using a decision we can use Cassadine and trace RNA and also CRISPR array to facilitate genome editing and within within human cells. And so when you have all four components or even without at the bacterial RNAs

three you can achieve genome editing within the human human cell. Another nice thing about the CRISPR system is that is naturally multiplexed, so each CRISPR ray carries multiple guide RNA to target many different viruses. And one application of that in human cells is that you can use this CRISPR system's natural multiplex ability to target multiple genes at once. And so what tested whether or not this would work. So we designed cast nine and trace RNA to work with a crisp array that carry two different guide sequences that target two different genes within the human genome, the emx one gene and also the parvalbumin gene. And then we show that using this approach we can simultaneously edit on both genes within human cells. And so here with the array continued mx1 and parvalbumin we can achieve editing at both of the low sign in the genome and then it's equally efficient as if we introduce the individual a guys to edit one or the other locus. So just to recap and we can we

can use cast 9 2008 genome editing, and we can use it in a couple of different ways. By taking these cast nine family members from thermophilus or from s. pyogenes, we can either use CRISPR raise along with trace RNA to facilitate editing, or we can use a single guide RNA that we heard about this morning to facilitate genome editing. Using either methodology, we can stimulate an hej or HDR by introducing double-stranded breaks within the specific target site. So, one of the really exciting applications that Chris Burke has 9 is maybe we can one day develop this as a therapeutic to go and treat diseases. And we thought, can we try to see it and how well we can use this in an animal model disease. Well, one of the challenges that we first had to resolve is how do we develop a cast 9 that we can deliver efficiently in vivo and we can edit with high efficiency to achieve a therapeutic benefit as pyogenes and also.

as thermophilus cassadine are to cast nines, but they are large and sometimes challenge the package into a single viral vector so that we can deliver both the catenary gene and the guide RNA. And so we thought, well, there are many different cast lines out there in nature. Maybe we can try to harness other cache lines that are even more compact that we can fit into convenient viral vectors and tested in vivo editing efficacy. So here is the phylogenetic tree showing the distribution of many different cast line family members, and you can see it's really widely spread through different bacterial species. If you plot the length of these cast lines based on their amino acid length, you can see that they distribute into two different clusters. There's a short cluster that's about the 1100 minnow asses long, and there's a longer cluster that's around 1350 net amino acids long. And we thought, well, maybe we can find some of the shorter ones, and that really falls at the shortest tail end of this distribution.

so we began by choosing six different cast nines that represent this. This cluster is very very small cast nine and we eventually found one that can quite work quite well, and that's the one from Staphylococcus aureus. This protein is about slightly more than three kilobases long, and that means we can package both the staph aureus cast 9 as well as its promoter and the guide RNA within a single and associate virus, which is a V because it is one of the most events g3p vectors. So to see how well this would work, we then design the guide RNA that targets the pcsk9 gene, because we thought if we can use a viral vector that delivers into the liver and then knockdown or knockout pcsk9 expression, then we can reduce questro it's cute as pcsk9 is a very prominent drug target for reducing questro and reducing cardiovascular disease risk. So to see how well this works, we injected these into mice and systemically so you can prepare the

virus and inject through the tail vein. So we design two different guide RNAs to target pcsk9, and we build two different viruses to see how well does it work. On the left side is the editing activity, and so this is the x-axis, so that's the time course. Within seven days after the injection, we already saw quite good level of editing, so you get about approaching 50% of pcsk9 indels within the liver tissue section. When we sample the delivery tissue for subsequent time points, we saw that the editing activity is stable and it persists for the rest of the experiment. Now that we have Cassadine pcsk9 editing, how well this is translate into a therapeutic change in the animals. So we took serum from these pcsk9 knockout animals, and we measure the amount of pcsk9 protein residing in the serum. And the time course really agrees with the editing activity. So about seven, so this is before the injection, this is seven days after the injection.

across four different dosages. So, a full dose range. We saw that for each one of these injections, we no longer were able to detect pcsk9 in the serum of these animals. So, a reduction in pcsk9 should translate into a reduction in cholesterol. And so, then we next measure the amount of cholesterol in these animals. And what we found is that within seven days after injection, there's also a fifty reduction in the question amount in these animals. And so, these early data point to the possibility that maybe we can develop this as a therapeutic tool to directly modulate modify genes within a somatic cell tissue or into cells that we have taken X vivo and then and then modify them to achieve a therapeutic outcome. Now, one of the challenges of advancing caste 92 or therapeutic application has to do with how well can cast 9 modify only the target and not introduce any off-target and mutagenesis. So, over the past few years, we and also several other

groups have very comprehensively analyzed the specificity of Cas9, and what we found is that Cas9 indeed can introduce off-target activity. And so that's not ideal because when we go into a cell, we really just want to correct the on-target region, and we don't want to introduce other mutagenesis within itself. So we took an approach to try to improve the Cas9 specificity. And there were work by us and also by many other labs to improve the specificity, but most of those approaches are fairly complicated. They required modifying the Cas9 enzyme to recruit other nucleases, or modifying the RNA, or changing the inducibility of the enzyme. So we thought maybe we can develop a simpler solution. And so three of my teammates, Ian and Ling E and also Baron, they looked at the way that Cas9 will bind to DNA, and they thought maybe we can treat the way that Cas9 stabilizes nonspecific DNA binding to reduce alter key mutagenesis. Here is a schematic of how Cas9 would

bind to DNA, so DNA is shown here in red. It's naturally duplex. To order for cast 9 to bind, that DNA has to be unwound, and then the RNA can base pair with the target DNA. So RNA is shown here in green. When the RNA hybridizes with the DNA, the displaced DNA strand is negatively charged, and it's not stable. So in order for caste 92 to bind there for long enough to do out there and clear the DNA, and this displaced DNA strand needs to be somehow stabilized. And so we hypothesized that maybe there is a positive surface along where this displaced the NH rain would fall, and we can try to weaken that the nonspecific stabilization to make a sign more specific. So here is the crystal structure colored in the same way as the schematic, but you can also color it a different way by looking at a surface charge distribution. And this is how that looks blue is positively charged, and red is negatively charged. And so you can see this.

groove right here where the displaced DNA strand is supposed to fall is very blue, and so I thought maybe let's go into this region, inactivate some of these arginines, the lysines, and change them to neutral residues like alanine so that we can weaken the nonspecific binding. And then add the make has nine more specific. So it has stood about 30 different mutants with either individual arginine lysine substitutions or with combinations of mutations, and we found that we can indeed make new versions of mutant caste mutants that are more specific. And so here is an experiment where we begin with a wild-type cast night and a guide RNA that targets the veggie FA gene. We knew from previous experiments that this guide RNA has two prominent off-targets, and you can see here is the on-targeting, and these are the two off-target editing sites. Now with the hence cast nine, and we're able to reduce it down to levels where no longer able to detect using the specificity measurement assays. So both

of these are what we call enhanced cast knives with three different mutations, and they preserve the on-target activity while more or less eliminating the off-target activity. And so, this shows that we can take a rational structure guide in engineering and to make these enzymes more specific. We also did an experiment to see are we really requiring more stringent base pairing booting up between the RNA and the DNA to achieve editing. Here, this is what the wild-type last night, and the first bar shows a perfectly matching guide RNA. And then below that, each of the guy shows pairs of mutations along the length of the guide. And what we found is that in agreement with many other studies, the 5-prime region is less specific, so they can be tolerated if there are mismatches. The 3-prime region is quite specific, so it doesn't tolerate these kinds of mismatches. This is what the data looks like with the enhanced Cassadine now because we are weakening the nonspecific stabilization. That means the RNA needs to provide more stem or stringent.

binding to that to the complementary DNA, and so here, even the 5-prime region requires base pairing complementarity in order to have editing. And so, using both of these has nines, we can take advantage of full length of the guide RNA to achieve editing. So, we can use structure rational guided engineering to make Hasnain better, but we are no smarter than nature, and in nature, after all, has had billions of more years than us to evolve in an event at new technologies. And so, we thought maybe we can explore nature's diversity more. There may be other enzymes that Nature has evolved that we can harness that would make genome editing more powerful. So, one of the study we did is to explore previously uncharacterized CRISPR of systems. And if you look at why this might be interesting, we can look at the way that crispers are classified. Broadly speaking, there are two major classes of Chris resistance. There's the class one system, which utilizes and multiple proteins that come together and to facilitate the RNA.

guided recognition and also destruction type 2 class 2 systems are more simpler. They use a single protein like last night to facilitate recognition and also a target destruction. Now, there are multiple class one systems, but at the start of our study, the only understood system was cast 9, and so we thought maybe we can explore other class 2 systems to see if they are also used for genome editing. One of the uncharacterized has systems felt following in this organism called Frances illa. It happens to have two crisper systems, one encoding has nine and then another one that encodes the gene called CP f1. If you look at the domain organization CPF 1, you can see it's quite different from domain organization caste 9, and the domain organization where CP f1 is very similar to a lot of transpose xi. And so it wasn't clear whether or not a transpose of element just happen to have landed right next to a crisper a repeat system. I repeat array or CP f1 is yet another crispr RNA guided endo nucleus.

so we transplanted CPF1 locus from Francisella into E. coli, and we found that indeed there is an RNA-guided endonuclease system. In fact, it uses a different RNA targeting substrate, whereas Cas9 is a dual RNA-guided system, recording both its CRISPR RNA and also the tracer RNA that you, Manuel, told us about. CPF1 uses just a single RNA guide, and it has the direct repeat that's not a Phi prime in flanking at the target site. So we thought to see whether or not we can use this for genome editing, and we chose 16 different CPF1 family members. We knew from our Cas9 work that not all has lines can just be plucked out of bacteria and put into a human cell. Out of the 16 CPF1s, only eight of them were able to get reconstituted in vitro, and so they can cleave using a human cell lysate expressing these proteins. Out of these eight that work in vitro, two of them worked in vivo and to be able to

achieve genome editing, and we're happy to have found it, so we decided to focus on these two CPF ones from seed minute caucus, and also like no spear a CA. Next, we thought to see a health-specific CPF one, and so we designed a guide RNA that can be targeted by both CPF 1 and also by Cas9. We found, using an unbiased genome-wide specificity assay called BLISS, that whereas Cas9 exhibited a 2 off-target size, the CPF 1 system was more specific, so we didn't see any off-target cleavage. And if you choose a guide RNA that is unique in the genome, Cas9 can also be quite specific, so you don't pick up any off-target double-strand break activity. CPF one also can be quite specific and cuts strongly. And also, Jin Zhu Kim's work groups have also carried out analysis, and we have found a very similar specificity enhancements. Another unique thing about CPF one has to do with the way that RNA is processed. So, Emmanuel told us that

Cassadine uses tracer RNA to process CRISPR ray into the individual a dual RNA guides. And then you men you also found that CPF one process is the CRISPR array into the individual RNA guys. And this is advantageous because this means that we can even better achieve multiplex editing. We can just use a single promoter to drive a long CRNA array that carries multiple guide RNA sequences. So we tested to see whether or not this would work. And we build two different AVS. One that expresses a CPF one using a neural no specific promoter. And a second AV that uses a use exposure to drive a a triple array a guide RNA that can get processed by CPF one. And also a GFP marker just so that we know which neurons car transduce. After we injected it into the brain, we can get very good transduction in the dentate gyrus advice. These are red fluorescence that are stained from CPF one expressing cells. And then the green fluorescence shows cells expressing the expression of the second guide RNA vector and so.

there's very good colocalization, and if you dissect out that piece of brain tissue and measure the level of editing, you can see that there is very good editing at all three genes. And so, for MU tv-tuner neurogenic under ligand 3, and also the Rd one, we can get editing. And in these post mahalia neurons are just through a single pane memory expression, we have continued to mine these CRISPR systems because CPF 1 is unlikely to be the last CRISPR system to be found. And so, we set out a really fantastic collaboration of Eugene Koonin and as constant in severe enough where we use the computational pipeline to identify a novel CRISPR assistance. We began with a protein called Cass 1, which is the most conserved Kristopher enzyme involved in the acquisition of immunity, and then by doing blasts, we can pull out all the bacterial sequences of the contacts that carry the cat one gene, and we can annotate open reading frames so that we know what are the nearby genes.

because CRISPR systems are encoding operands, we had good expectation that the nearby genes are likely to also be CRISPR associated enzymes. We can then annotate each protein as either unknown or a known gene, and we just focus on unknown genes because they would point as to novel CRISPR proteins. And so, using this pipeline, we were able to identify a number of different and new candidates, and so each one of these candidates without you to characterize them more and see whether or not they have interesting potential as new novel technology. So here is a cast 9 in CPF one. Using this pipeline, we were able to pull out three new CRISPR candidates: that's class 2 candidate 1, class 2 Canada - in, class 2 candidates 3. And the most interesting one to us was the class 2 candidate 2, because unlike all the other enzymes, it contains nucleus domains that are found normally RNases or in toxins. And so we thought maybe this is the RNA guided RNA targeting system that we can use to target a different set of experimental.

applications. Based on that hypothesis, we then set up a screen. We chose the RNA-only virus MS2, which is a phage that only exists in RNA form. And the way it's a small genome, only 3,800 bases, so we can design guides that pile along the entire length of the genome, shifted by one base at a time. And by cloning these guides designed us and synthesized in vitro, you can put them into vectors and transform E. coli. And now each E. coli carries a single guide RNA that complements with the MS2 phage. So by challenging these bacteria with MS2, we can see do any bacteria survive. And if any survive, then that means some of the C2C2 system might be active, might have defended the E. coli against MS2 infection. And moreover, from sequencing that the remaining guide sequences, we can figure out how the C2C2 target is target RNA. So based on that experiment, we found that for the latter trickier shocky C2C2, the targeting requirement just that the guide's sequence target.

site has to be flanked by a single base. It can either be a C or a or a U. So this is great. Now we have a RNA guided RNA targeting system, and we're trying to now explore its various applications. So one of the things we thought to see is can this enzyme have activity in eukaryotic cells. And so we modify this enzyme so we can express it in human cells, and then we compared it with using shRNA to knock down a specific target gene. And so for two different target sites, this is the first guide, and this is the second guide. The CRNA conditions compared favorably with shRNA conditions, and we can use it to be able to knock down gene expression. In addition to using a CRNA for knockdown experiments, we can also use it to just bind to RNA and try to visualize things. And so very much like a DNase, we can inactivate that nuclease domains to turn it into a generic RNA guided RNA binding protein. And so here we fuse this

inactive c2 c2 with em cheery so that we can bind to mRNA in cells, and then visualize the localization of these mRNA. To test this, we'll use the AC RNA that targets the beta-acting gene. So, in collaboration, we transfected all these into cells and apply it a stress condition so lean arsenite on these cells. And when the stress was applied, we saw that MJ ray fluorescence localized to stress granules perinuclear. And if you don't add a stress, then the venture expression is in Hardy cytoplasmic. And this shows that we can use this as a way of monitoring and tracking RNA localization in living cells. So now we have a lot of different tools we can perturb DNA, we can perturb RNA, but we also need to develop Drita methodology to figure out what are the perturbations doing without reading it out we won't really advance our understanding of the biological mechanism. And so, to that end, we've been developing a single-cell single nuclei.

sequencing methodologies so that we can pair that with editing to figure out what is happening in these cells. And so it's an IO me and also inching worked on developing a technique called nuke seek that we have applied to neuronal tissue because neurons are intrinsically very elaborate and have complicated morphology. And so by taking out only the nuclei from neurons, we can more easily extract out cells and then carry out individual cell sequencing. We found that the transcriptome from a nucleus and transcription from a hotel are fairly representative of the transcription state for the cells. And so from doing this type experiment, we can carry out functional classification of cells. So by doing completely unbiased clustering analysis on the individual nuclear transcriptome, we can group hippocampal tissue from the brain into different cell types from different brain regions, the entire iris were CA 2 or CA 1 or inter neurons within the hippocampus. And we can also do step clustering too. So you can take the GABAergic cluster and then

divide it into part of human cells, somatostatin cells, and many other different kind of cell types. So we apply this to see, can we use this to study biological process, and we combined it with edu-based labeling, and to look at proliferation of newborn neurons within the adult hippocampus. And so using this methodology, we can label and then and then collect cells at different time points after the initial labeling, and then look at how does transcriptome change at different time points post differentiation of these meta cells. And so we use fax sorting to extract out these label nuclei. By doing the same analysis, we can cluster them along a very nice temperature ejection. So, this is all the cells, and different colors represent different groups of cells extracted at different time points after the initial labeling, and you can see that they fall along this continuum. So, the blue cells are 1 to 2 days, and then these really dark red and in kind of orangish cells are from the later labeling time points. So, based on this time

trajectory. We can then identify what are the marker genes that are expressed at specific time points. So, for example, by looking at expression for Sox 9, we'll find that it expresses in the early post-differentiation cells, whereas neurod1 expresses in the later post-differentiation cells. And so, by getting a time course of how the transcriptome changes, we can then use some of these marker genes to potentially drive the maturation of cells into specific fates. And so, we can especially combine that with individual cellular models that were generated using genome editing to be able to study functions of specific mutations. So, this is pretty much it. Just to recap, we are really excited to continue to explore the natural diversity in these interesting microbial systems to be able to develop novel technologies. And we are also committed to developing the applications and also making sure that these tools are easily accessible and distributed to other laboratories. And then, as the tools become mature, we're also really excited to start to apply it to the neurological questions that the

motivated us to develop the tools in the first place, and to really see if we can advance our understanding of some of these diseases. So, as we continue, we're continuing to take census, and this is the latest census from Chris persistence. So, in addition to the class 2, and also the CPF 1, C 2 C 1, and also the C 2 C 2, I started the C 2 C 3 and C 2 C for assistance, and also the type, the type 6, we most recently computationally identified yet another RNA-guided RNA targeting system that we call a caste 13b. And we are now just starting to functionally study what these systems are doing in back Oh sells. But this is really the most exciting and inspiring thing. This plot shows the number of genomes, bacterial genomes, that have been completed over the past 15 years. And you can see in the past five years, the number of genomes that have been completed has really grown exponentially, and this really represents

and present opportunity to go and harness and understand. I explore these diversity and harness them to be able to further advance our ability to study a biology and also to be of the tree diseases in the future. So finally, this is the last slide but also the most important slide, and this is my team at the development of these technologies has been a team effort, and it's really thanks to many of the students and postdocs who I have worked with shown here, and also our long list of really fantastic collaborators that we have been fortunate to work with and to develop many of these technologies and applications, and last but not least many of the funding agencies both governmental and also private foundations that have supported our work over the past several years. Again, thank you, thank you again. I want to thank the governor foundation again for this opportunity, and it's really a great pleasure to be here. [Applause]

Genome Editing Using CRISPR-Cas Systems:  Development and Prospects-Feng Zhang

CanadaGairdnerAwards

The Gairdner Award review process is transparent and involves two panels: a medical review panel of rising stars and a medical advisory board including a former Nobel laureate.

The process receives hundreds of nominations each year and aims to recognize individuals making a difference in human medicine.

The Gairdner Foundation recognizes the work of young scientists, such as Dr.

Feng Jiang, who is making significant contributions to the field of genome editing.

Jiang's research focuses on the application of genome editing for various genetic diseases and he is honored to interact with scientists and researchers across Canada.

The Evolution of Genome Editing Technologies

Genome editing faces challenges due to the complexity of the microscopic genome and the difficulty of making precise changes.

Earlier approaches focused on using DNA binding domains to recruit nucleus domains and stimulate gene repair.

Zinc finger protein technology and TALEN technology were developed to recognize and modify specific DNA sequences, but they were challenging and time-consuming.

The discovery of the CRISPR-Cas9 system offered a simpler, faster, and more efficient genome editing method by using RNA guides to facilitate targeted DNA double-strand breaks, allowing for easier and cheaper editing of the eukaryotic genome.

Exploring Different Cas9 Proteins for Genome Editing

Researchers combined Cas9 proteins from Esterella maris and S.

pyogenes with tracer RNA and a CRISPR array to introduce double strand breaks in the human genome.

The natural multiplex ability of the CRISPR system allows for targeting multiple genes simultaneously within human cells.

By harnessing a compact Cas9 protein from Staphylococcus aureus, they were able to achieve efficient in vivo editing for potential therapeutic applications in treating diseases, as demonstrated in a mouse model targeting the pcsk9 gene.

The Effect of Cas9 Mutation on Gene Editing

Researchers conducted an experiment to assess the impact of Cas9 mutation on gene editing efficiency and off-target mutagenesis.

The study showed that the mutated Cas9 enzyme was able to increase the specificity of gene editing, reducing off-target effects.

By introducing about 30 different mutants, the researchers were able to develop a more specific version of the Cas9 enzyme.

Engineering More Specific Enzymes for Genome Editing

Researchers have developed enhanced Cas9 enzymes with three mutations, maintaining on-target activity and reducing off-target activity.

The study shows that a rational structure guide can be used to enhance the specificity of enzymes for genome editing.

By exploring uncharacterized CRISPR-Cpf1 systems, the researchers discovered a new RNA-guided endonuclease, Cpf1, which uses a single RNA guide and has a different RNA targeting substrate compared to Cas9, offering potential for genome editing.

This research demonstrates the possibility of using rational engineering and exploring natural diversity to develop more powerful tools for genome editing.

Advancements in Genome Editing Technology

Researchers have discovered that the CPF1 system shows high specificity and strong cutting ability, making it a promising candidate for genome editing.

The CPF1 system's unique RNA processing allows for better multiplex editing, demonstrated by successful transduction and gene editing in the brain.

A computational pipeline has also identified a novel CRISPR candidate, class 2 candidate 2, with potential RNA-guided RNA targeting capabilities, opening up new possibilities for experimental use.

The Development of CRISPR-C2C2 for RNA Manipulation

Researchers developed a CRISPR-C2C2 system to target and defend against the MS2 virus in bacteria.

The system showed promise for gene knockdown in eukaryotic cells, comparable to traditional methods like shRNA.

The CRISPR-C2C2 system was also repurposed as an RNA-binding protein to track RNA localization in cells, showing potential for visualizing stress response.

Additionally, the development of single-cell sequencing methodologies, such as nuc-seq, allowed for functional classification of neuronal cells, providing insights into cell types and transcription states in the brain.

Studying Biological Processes and Cell Differentiation

A new methodology is being used to study the proliferation of newborn neurons in the adult hippocampus.

The process involves labeling and collecting cells at different time points to observe changes in the transcriptome.

The goal is to use this research to advance the understanding of diseases and drive the maturation of cells into specific fates, ultimately aiming to apply it to neurological questions.

Additionally, a new RNA-guided RNA targeting system called Cas13b has been identified and is being studied in bacterial cells, showing the exciting opportunity to further advance biology research.