good afternoon. Hi, everyone. Welcome. Good afternoon, everyone. How are you doing this rainy afternoon? We're so glad that you're here with us. My name is Christina and I'm the family engagement coordinator for San Francisco Public Library. We'd love to know where you're tuning in from, where you're staying cozy in the rain and joining us for a wonderful afternoon of storytelling. Let us know in the chat. We'd love to know. And we'll get started just in a minute at two o'clock. All right, everyone. Well, we're going to go ahead and get started. Once again, my name is Christina Mitra, and I'm the family engagement coordinator for San Francisco Public Library.  Today we are celebrating storytelling as part of the San Francisco Public Library's more than a month program series, where we celebrate black history, culture and heritage beginning with Dr. Martin Luther King Jr.'s holiday, and then we go right into February. We call this celebration more than a month in an effort to emphasize that reflection, open dialogue and listening, interdisciplinary education and shared advocacy must take place in our communities, not just during Black History Month, but all year round. We hope that you'll join us for all the more than a month virtual programs that we have available for youth, families and adults. And to see our upcoming events, check out our book lists and our event page at sfpl.org. And our trustee friend in the chat will go ahead and make sure that you have that available. That website link. So I still want to welcome you all and I want to thank the friends of the San Francisco Public Library for their generous support of this program series. Check out friends sfpl.org for more information about how to support the library through the friends. And their support really makes sure that our youth and family programs come to life for you, even in the virtual world. So without further ado, I am really excited to introduce our performer today. This is Kirk Waller. Perhaps you're already familiar with his astounding storytelling skills, his ability to transform characters and bring stories to life with suspense, humor, empathy and compassion. You've seen him perhaps at one of our many locations over the years he is not only beloved by our young audiences, but by many of their parents and caregivers and people like me, our librarians as well. So with a lifelong love of story, literature and performing arts, Kirk Waller has been immersed in these art forms for over 20 years. He has won many awards for his work, including a parent's choice Foundation Gold Award for his CD, but why? Stories, music and songs. Kirk received his bachelor's degree in English and his master's in creative writing right here at San Francisco State University. So I would like you to welcome him with a huge virtual applause. And I welcome Kirk to join me here and take it away, Kirk. Thank you so much, Christina.  Thank you so much for tuning in. I'm really excited to be here.  Isn't this a perfect day for storytelling? It's kind of cold and dreary outside, but warm and cozy inside. So I have a few stories that I hope not only entertain you, but I hope you learn something new. I hope they pique your interest. And speaking of piquing interest, at the end of the stories, at the end of the session, we're going to have a question and answer period. So if you find something interesting, you say, hmm, I'd like to know more about that. Keep that in mind. Write the question down. Because it's YouTube, you can actually type them in, but I want to wait till the end just so we see them. Without further ado, get ready for your first story. It's called a Cow Tail Switch. Give me a second. I got to go change. Be right back. Once, many years ago, in the village of Cundi, overlooking the Caballet River in Liberia, there was a small village, and in Cundi there was this great hunter. He went by the name of Ogallusa. Ogallusa was the greatest hunter there had ever been in the village of Cundi. See, often he would go away to hunt. And when he returned, sometimes many days later, he would often come back with the most exotic, large, fierce animals. The men would gather around and they would sing his praises and admire his work. And the children would sing songs of him and raise their hands and circle him and cheer Ogallusa. They would say the mighty hunter has returned. And Ogallusa, he would not rejoice in the praises of the men or the songs of the children. He would rejoice in his own children. You see, he had seven of them and they were all boys. Yeah, seven boys, if you can imagine, and his wife was pregnant with an eighth child.  It was spoken over them by the village elders that one day all of these boys would have a gift, a purpose. And this gift, this purpose would bring healing, would bring blessing to all. Now, Ogallusa's wife did not know what this gift might be.  It warmed their heart and they kept those words close to them. But one day, Ogallusa left the village as he often did. He took a trail, though, that he had never taken before. And he left in a direction early, early before the sun rose and no one knew which way he had went or which hunting ground he had found or maybe discovered a new one. All they knew was Ogallusa had left. Well, that evening, when everyone came and sat down around the dinner table, there was one thing that was missing. Do you know what that one thing was that was missing around the table? But the food? A meal? Ah, yes. See, Ogallusa was missing. He had not yet returned from his hunting trip. And the people in the village began to wonder, where is Ogallusa? Where is the great hunter? He has not returned. We want to see what he has brought back this time. And his voice said, where is our father? Now, his wife was not too worried because she knew that often he would go away and he may stay a day or two and then return. But the day turned into two, turned into three and he'd still not returned. And people began to ask more and more, where is Ogallusa?  The days turned into weeks and people began to ask less and less about the great hunter. There came a time in the village of Kuni that no one mentioned the name of Ogallusa again. The time of the grieving, the time of the grieving had passed. And also enough time had passed for his wife to give birth to the eighth child. And it was, what do you think it was? Yes, another boy. It was another bouncing baby boy.  All the attention goes on the baby, everybody laughing and smiling and dreaming and hoping with that child. And so it was, they forgot even more about their loss and their grief and the pain of losing Ogallusa. And the baby began to grow and total and pull up and look around and be curious and wonder about his world, all about him. And he began to see other children and other fathers. They would put their children on their shoulders or he'd see them take them down to the river and teach them how to fish or teach them how to hunt. And he began to wonder. And when that little boy was old enough to speak his very first words, he said, where is my father? And it was at that moment that the other seven brothers knew that they must find out what happened to Ogallusa, good or bad. They must find out and bring peace to their family once and for all. But they had no idea where to turn, where to even start. Remember he left early in the morning before anyone else had risen. And one of the boys, one of the seven, he stood and he pointed with clarity. And he said, we must go that way. Well, the others kind of chided and laughed. Why must we go that way? And what will we find if we go that way? How do you know we will find our father? I do not know, maybe, maybe, maybe it is my, maybe it is my gift that was spoken of. But I do know in my heart that if we go that way, we will find our father. And with nowhere else to turn, they followed him into the bush. And if they turn to the left or right, he said, no, we must continue down this path. If we go this direction, we will find them. And they followed him to the path right out. And they went even deeper in the bush, but they kept going that direction. And soon one of the brothers pulled from underneath, hanging on a bush some cloth. It almost dissolved in their hands, but from the color and the cloth that was remaining, they knew that it belonged to Ogallusa, their father.  And even further, just underneath some leaves, they found the old, dry bones of their father. Ogallusa, the hunter to some, but to them all that was left of the man they called father. And they fell to their knees and they cried. They wept deeply.  He looked at his other brothers who were grieving and still on the ground. He said, I believe that I could put these bones together again. Why would you put the bones together? And what good will it do if you put the bones together? I do not know maybe that, maybe that's his mind gift, but I know, I know that I could put these bones together. And so, like putting together pieces of a puzzle, a boy put every single bone in its proper place. And then another brother, inspired by his older brothers, said, I believe it's my gift then to put flesh on these bones again. And like a spider weaving its web, flesh and muscle and sinew began to appear on the bones, bringing them together as they began to gather themselves. And still another brother stood up, inspired by the last, and said, I believe it is my gift to put blood back in that body again. And he reached out, and he grabbed the cold ankles of Ogallusa. And as he held on to them, blood began to rush through the body. Heart was beating, blood was pulping. And another said, I believe it is my gift to put movement in his body. And as he touched his outstretched fingers, the hands began to move, the feet began to move, and this cold, still body stood for the first time. I believe it is my gift to put breath inside of him. And he reached out, and touched his chest. And rose, and fell, and rose, and fell. And Ogallusa was a breathing, living soul again. But as he stood there silently looking at his boys, the last one said, I believe it is my gift to put words in his mouth again. And he reached out, and he touched his face. And Ogallusa looked at his boys and said, it is time to go home. And the boys began to sing, and laugh, and cry, and dance all around Ogallusa. All the way home, it was like a procession. He led the parade of joy, of hope, of life. The people in the village saw them passing by in their mouths, hanging open. And eventually, they made it back to that little village. The wife embraced him, and the villagers gathered around, but Ogallusa went inside his home. It was his custom. It was the custom of the village that if anything like this were to happen, that someone were to die, and somehow come back to life, it was the custom to stay inside their home for seven days. It was also the custom to shave their head clean, a symbol to show that they had been to another world, and returned again to this one. And when they had returned, and the seven days were complete, a great feast. A great feast was ordered, and there was food, and music, and singing, and dancing. And the elder of that village brought out what they called a cow tail switch. There was a switch made from a cow's tail, and it was woven with cowry shells and beads, and it was beautiful, it was the symbol of honor, something like a scepter. And he presented that cow tail switch. Ogallusa. Ogallusa accepted it with pride. He held it close to his heart. But then he rose it up in the air, and he said, I will give this cow tail switch to the one of you who was most responsible for bringing me back to the land of the living, and others they began to immediately argue who it was that was the greatest, who it was that was most important, who it was that most significantly brought him back to life. Who do you think it was? The one who pointed the way? Yeah, if I hadn't pointed the way, we wouldn't have known where to found him. And the other said, but I put flesh into his body.  And finally Ogallusa raised the cow tail switch, and he silenced the crowd. I have made my decision. Everyone looked at him, mouths open, eyes wide. And he approached his sons as they all stood lined up, trembling with anticipation and hope. And he raised it up and he said, I will give this cow tail switch to my youngest son. For had he not said where is my father, I would still be lying cold in the land of the dead. And so this story, it accentuates a proverb that says a man or woman is never truly dead unless they are forgotten. Oh, that was a friendly, fun way to start. So thank you for letting me get ready, get back here to tell you the next story. So that one was called a cow tail switch. There's one quick question I can answer more at the end if we have time, but someone did ask what kind of instrument is that. And so it is called an Ngoni. An Ngoni. It's a West African instrument is the instrument of a griot 13th century. It is made out of goat skin strings, and this is a gourd. So that's just the short answer, like I said, we may be able to talk a little bit more afterwards. But I have another story I want to share with you this one is is not a new story is not as old as the cow tail switch. But it is still pretty old older than some of you older than some of your parents maybe older than your grandparents 1960 is when this takes place. And this is here in the United States of America. It's called the Greensboro for I got to go get on my shirt and tie so give me a second and they're going to come back with this story. Let us break bread together on our knee. Let us break bread together on our knee. As we walk hand in hand, as we pray to heal our land. Let us break bread together on our. Breaking bread together. Sitting and eating a meal has been around since we've been here since there's been food and since there's been people. So what's your favorite restaurant? What's your favorite food? Pizza hamburgers cake. So imagine this with me for a minute. You want to go to your favorite restaurant and eat your favorite food and sit with everyone else. But one thing. You're not welcome there.  There's a sign on the wall that clearly says colors served. In fact, all you wanted to do is have a meal. We hold these truths to be self-evident that all men are created equal. Unless you were black in 1960 in America. Segregation was in restaurants and schools, drinking fountains, buses, just about everywhere, especially in the south. There were these four young black men. College students. I want to call their names. Ezell Blair Jr. David Richmond Franklin McCain Joseph McNeil. These four young men, the youngest was 19. So they were students. And they had talked every day about the things going on in the world and society and race relations and equality. And they wondered why things were the way they were. And more to the point. How they could make a change. How they could somehow make a difference. And something had happened that previous Christmas, so December of 1959, that changed everything that pushed them into action. One of the young men, Joseph McNeil, he had went on a bus trip and went out of town. And when he came back at the bus station, he was tired. He was hungry and he wanted something to eat. So he went up to the window and he said, I'll take a cup of coffee and a boy, you know we don't serve your kind here.  Now go on. And he could feel rage rising. He could feel his heart beating in his throat. Heat rising in his face. He was humiliated. And he went and he told his other three friends about this. And it was at that time that they decided something had to be done and not just talked about. Franklin McCain was kind of like the leader of the four, the natural leader. And so he challenged them right there in that dorm room, right there. He said, are you willing to do something about it?  And he challenged them one by one if they were going to do something or just talk about it. So they came up with a plan. So in Greensboro, North Carolina, there was a Woolworths. Now Woolworths is what you call a five and dime. Everything in the store, you could get for either a nickel or a dime. Now that's some good shopping. Now it was kind of like our dollar store today, but a lot better. A lot more quality in the items and a lot more variety to choose from. And so they decided that they will go into the store and they would purchase things. Just to show that they were honest, good paying customers. And then they would sit down at the lunch counter and order something to eat and demand to be served. They were committed to this plan and they were committed to nonviolence. The day was February 1st, 1960. The four of them met in front of the local library and they were dressed in their best. They made sure they had their shirts, their ties, their overcoats and their hats.  Our Father, who art in heaven, how would be thy name? Thy kingdom come, thy will be done, on earth as it is in heaven. Give us this day our daily bread. They also prayed for protection. They also prayed for change. When they were finished praying in silence, they walked to the local wars. And according to their plan, when they got inside, they bought just regular items, comb, toothbrush, toothpaste. They paid for it and they made sure that they kept their receipt. And then they went and sat down at the lunch counter. Each of them took a stool. The waitress, a white woman, came up to them, what are you boys doing here? We want to be served. You know I can't do that. We don't serve you here. Well, why not? We pay for our items. We're paying customers and he showed her the receipt. We don't serve color to you.  But they didn't leave. They wanted to be served. So they took a deep breath and they sat in silence. Frustrated the waitress went and got a black worker. Her name was Geneva Tisdale. She thought maybe she could talk some sense into the boys. What are you all doing here? You know you ain't supposed to be here. You got to leave now and don't come back. It's people like you that make our race look bad. She didn't know that was people like them that was trying to make things better for everyone. And they still didn't move. So they went and got the manager. The manager came and tried to make him leave. Tried to talk him into leave. They didn't leave. So he felt he had no other option but to call the police. So the police came and they with a look of intimidation just kind of hit his billy club in his hand as he paced the floor back and forth in back of the seats. And the boys they braced themselves. Not knowing what was going to happen was he going to beat them or hit them or haul them off to jail. But they just sat there. And the police really didn't know what to do because they really hadn't broken any laws and they were sitting there peacefully. He had never faced anything like this before. So eventually people started coming on lookers curious seeing what was going on and the world started filling up. The only other thing that happened that first day was at the end of the counter. There's this old white woman. She had been staring at the floor the whole time just staring at him and she got up and she start striding over towards him slowly. And they didn't know if she was going to spit on them or curse at them or what she was going to do but they braced themselves for an attack. She came up to him and she put her hand on one of the shoulders of one of the boys.  I only wish it had been done years ago. And so you could say that first day was a success. The second day the Greensboro 4 became the Greensboro 6 as two more friends came with them. By the end of that day it was 16 people sitting at the lunch counters. And word began to spread and on lookers and police and people from the local news began to come. And so day 2 turned into day 3 and by the end of day 3 hundreds of on lookers, supporters, black and white were filling up that Woolworths. Day 4 took a turn. A gang of local white men to counteract these peaceful protestors. They went early, early, early before the protestors got there and they occupied the seats. So they couldn't sit in the stools. But it really didn't work. The four still came and they stood behind them and they asked to be served. The other on lookers and supporters came and they filled up the Woolworths. The young men, the white men, they did not take kindly to that. So they began threatening them and cursing at them pouring hot, scolding coffee over them. Let's cigarette butts, stuffing them in their pockets. And so they still did not budge. They did not leave. And they demanded to be served. On February 6th, a bomb threat was called in and everybody was cleared up. It turned out to be a false alarm, but it really got everybody's attention. So they backed off the protest. It was a two week cooling off period. And the Greensboro Four and their supporters, they talked to the management at Woolworths and they talked back to them. But nothing was really resolved. So after two weeks, the protest resumed. And people began to come in from all over the place, not only support that particular protest, but other lunch counters. Protestants began to show up. By day eight of the protest, there were eight cities, 54 Woolworths that had protest there. Now all of them were not peaceful. Some violence did break out in some. It wasn't by the protestors, but by those that didn't want integration, that didn't want the protestors there.  And then the police would come and arrest the protestors for disorderly conduct. But eventually, after all the protest, after all the loss of income, after all the bad publicity, Woolworths decided that they would desegregate all of the lunch counters. All across the country. Now, a lot of historians believe that it was this moment. The Greensboro Four and the protest that sparked a civil rights movement. It also sparked the development of the SNCC. That's the Student Nonviolent Coordinating Committee. Now that was the organization that organized the Freedom Rides. That was the organization that helped organize the march on Washington, where Martin Luther King Jr. gave his famous I Have a Dream speech. That was the organization that teamed with the NAACP, the National Association of the Advancement of Colored People, and then helped push the legislation and get the Civil Rights Act of 1964 passed. Who would have thought that just wanting a meal, sitting down and eating with everyone else would have led to so much sweeping change. Let us break bread together on our knees. Let us break bread together on our knees. As we walk hand in hand, as we pray to heal our land. Let us break bread together on our knees. Thank you so much for listening to that very, very important story. You know, there's so much work we still have to do as a people, as a society, but there's some change that has been made. And that was just one example of how one change led to others, led to others, and led to many others. The Greensboro Four, if you want to learn more about them, please ask me. Definitely look it up yourselves. So much more to that story, but I wanted to give you a little snippet into some things that happened. One last story for you. This is what I think you're really going to enjoy. It's one of my favorite stories because it's a lot of fun. If you like laughing, you'll like this story. If you ever had an itch somewhere where you couldn't reach and it was really hard to get, then you'll understand this story. Please enjoy Scratch That Itch. Okay, I got one last story for you. Now, this one's a little short, but it's really fun. This goes in the line of those long, traditional, old, old, black African American folk tales called Brer Rabbit Tales. Now, if you know anything about Brer Rabbit, you know he was always tricking somebody. Brer Wolf, Brer Bear, Brer Fox. And it was Brer Wolf who had finally got tired of Brer Rabbit. And he had made up in his mind. He said, I'm going to get that rabbit if it's the last thing I do. And so he had made up in his mind. And the very next morning, he saw Brer Rabbit and took off running after him. Brer Rabbit ran and ran and ran as fast as he could. And finally gave that wolf the slip. And he came to this opening, this big old clearing. And in his clearing, Brer Rabbit saw Brer Elephant. Now, Brer Elephant was rubbing his backside up against a tree trunk like this. He was like... And Brer Rabbit was like, huh? And then he took some branches with this big old trunk and he threw them up in the air. He let them land on his back like... And Brer Rabbit was like, what? And then finally, Brer Rabbit, he couldn't stand it. He said, uh, good morning, Brer Elephant. Elephant said, it's morning, but was good about it. I got this itch that I just can't scratch. He said, uh-oh, oh no, scratch that itch or I'm gonna blow. Like that. And Brer Rabbit said, I'll help you, Brer Elephant. I'll scratch your back. Oh, oh, you'd be such a good friend. So Brer Elephant picked up Brer Rabbit, put him on his back, and Brer Rabbit was just about to scratch his back. He said, but wait, wait, wait, wait, wait.   Yeah, yeah, yeah. All right. So Brer Rabbit, he went to scratching his back. Oh, he was scratching it good and chomping up and down and even biting his thick elephant skin. And Brer Elephant was loving, he said, he said, uh-oh, oh yeah, a little to the left, right there. And Brer Rabbit said, no, no, no, no, no, no. Remember, you're supposed to say, oh no, Brer Rabbit, you're killing me. Oh yeah, oh yeah, oh yeah. Oh no, Brer Rabbit, you're killing me. Yeah, yeah, yeah, yeah.   And when Brer Rabbit saw that wolf, he really started pouring it on. He saw like, like scratching elephant's back real big like that and like jumping up and down. And Brer Elephant, oh, he was loving it, but he remembered it. And he said, oh no, Brer Rabbit, you're killing me. And Brer Rabbit said, yeah, Brer Elephant. And when I get done with you, I'm gonna get that wolf. He been chasing me all morning long and I'm tired of it. And Brer Wolf, when he heard that, he tucked his tail between his legs, turned himself around and he got himself out of there. And that was the day that Brer Elephant made a new friend with Rabbit and Brer Rabbit finally got away from Brer Wolf. Thank you. All right, way to end a nice cool evening with a nice warm story. You know, thank you all for coming. If you have enjoyed the story, please look them up. You know, all those stories you could find, at least some version or so in your local library, online, Greensboro for Caltails Switch and his last one about Brer Rabbit. Christina is gonna come back. Oh, hey Christina. Hey Kirk. And if there's any questions that people have, we're gonna start out with those. And if not, we're gonna say goodbye, but I think we have a few of them. Absolutely. Well, first, I just want to personally thank you so much for bringing your gift of story with us. I loved all of those stories. I know that we've been seeing a lot of love in the chat. People have been sending us hearts and just hands of gratitude for what you brought with us today on this beautiful, rainy, cozy afternoon. So I just want to thank you Kirk and thank you on behalf of our audience for sure. Thank you. Thanks for sharing that because I'm not seeing the YouTube link. I might go back and look at it later. So thanks for sharing that Christina. Glad people are enjoying the stories. Absolutely. And I love how the library made a cameo in the Greensboro for story, actually, in terms of them gathering in front of the public library. I thought that that was really great. So there's a lot of appreciation for that story in particular, I think, in these times. So yeah, I just want to remind folks, please go ahead. If you have a question, just put it in the chat. We have some that will share right now just from the beginning. But if you have questions, this is a great time. Oh, let's see. Here we have just one that just came through. This is from Lawrence. And this is not a question but a comment that that Lawrence was five years old in 1961 Greensboro for happened. So, Wow. Yeah. So he remembers it first hand sounds like. Sounds like that too. And then someone else says thank you we're really enjoying this program. And Renee star also says thank you Mr Waller and SFPL. So a lot of people sharing a lot of gratitude. Well, I'll go ahead and ask you a question. I just would love to know, you know, where did you learn your stories and where did you really develop this love of storytelling. That's a good question. I've been asked the first one I don't think I've been asked the second one before so so that's the kind of how I started in storytelling. Just I think over the year well literally I literally started I used to work with children like in an after school program kind of summer program and you know how you read books to them all the time and if any you parents know and children know that they like to hear the same stories like over and over again. And then one time I was reading the story for the 11th 1000 time that I'm like, you know what, I know this story so I just kind of put the book aside, and I just like from memory just told him the story it was like, something like like a light went off at that moment because it was kind of like, I could see them instead of like, you know, looking at the book and the pages, and they could see me and her laughing and talking and kind of creating this magic and bringing up things and adding things to the story what went along and that was kind of like how it was born to me for me, you know, of course, storytelling has been going on forever but for me. That was my big discovery so I started from that point, just really developing my own particular style. I had already kind of done some movement and some mind in my life so I kind of incorporated that. Over time I started adding like a little singing here and there and and music as you saw with the angoni and a few other instruments that I bring into it. That's kind of how it kind of nurtured itself over time and now I can't. I can't live without our store our lives are stories and I just see them everywhere. And so it's so much a part of my, my emotional DNA that I can't get away from it. I love that we are continuing to get so much love from people watching someone did ask again and maybe they didn't they missed at the beginning. Can you share again about the instrument that you share during the first story. I did just kind of rush past it goes in between stories, but this instrument really pretty it's called an in Goni it was handmade for me by someone who creates in Goni and it's from a family of instruments string instruments from Africa West Africa, and this particular instrument, the in Goni is an instrument of agree oh and if you're not familiar with that term agree oh is a French word, but it was those those historians those storytellers. They were like ambassadors to the king, and they would know the history and the lessons and the religion and the culture and the music of that particular group, and they would know it all by memory they knew the birthdays and people passed away they knew everything, and they would sing they would recite they would do it in poetry form and they would pass those on from generation to generation, and this is one such instrument that they would use in their craft and I should say use as a past tense, there's actually still grills. Today, if you look, look up grills in West Africa that still hold that office and still do that and storytellers are a form, you know much different form a form of grill because we're passing on that culture from generation to generation. So it's made out of goat skin. A gore and the string. And of course you know you can see that the wood and it's tuned. This one is a very simple one is just a six string in Goni. There's eight there's 10, and then there's a very involved Cora, which people may know it's many many strings in the same same shape, but it's more like an African harp. And so yeah that's kind of like the little expanded answer of what an in Goni is. Thank you for that. It really adds such a wonderful layer to your storytelling to hear the live music. Someone is just sharing that you're a great historian and kids from the precede after school program have enjoyed these stories today so we've got lots of different viewers from around really just thanking you for your gift.  I think so much one of your sub themes is like you know more than a month that black history is more than one same thing with any culture you know sometimes I think it's great that we take time to celebrate, you know, different holidays and different months and one you know women's month and different cultures, but it's really important to that we they're not just marginalized into just, you know, okay so we did that you know we celebrate black history now let's you know. But that this is who we are as a society we all have different things to contribute, and that black history is only a part of that, but black history is American history. You can't really separate it, this is the time that we've chose to emphasize it I think that's what I want to take away that our lives our culture, who we are is a part of us, and the part of the greater society. Always remember that share that and never be ashamed to pass that on. Thank you Kirk, and I know that our audiences are really appreciating that answer thank you so much for being with us today. Thank you everyone who watched us and who will watch us will have this up for the next 24 hours. And then we will actually bring it down but you can catch it again starting our March 1 after black history month so we hope that you will make sure that continue to share these stories continue to share your own stories and develop your own craft. Maybe in the same way that Mr Waller did here today so I hope that you continue to connect with us at SFPL in the virtual world. We will continue to be bringing many different events for you, including next Thursday we will have Puna Solomon who has been studying this classical steel pan, the steel pen instrument since age five her dad is actually the inventor of the modern steel pan and she will give a live performance for us so but once again thank you so much Kirk and everyone here. Take good care be safe and we look forward to seeing you either in person on screen soon. Bye everyone.


There's one item out there that is so versatile, that it can be used in almost any industry, in any niche, or in any business. Can you guess what it is? Guess what it is. Doctors use them, they give them out, teachers have them, parents use them. There's one for pretty much any occasion. There's one for almost any show out there. There's literally one of these for anything. Have you guessed it yet? If you guessed stickers, well, then you're right. This video is gonna be all about some of the best stickers that you can start selling and where you can source them. Trust me when I say it, some of these suppliers are specific for stickers, so you're gonna wanna stick around for these, because I'm pretty sure that you haven't heard of some of these. Now, I know you're probably thinking, Mario, stickers, seriously? Yeah, stickers. When I say that stickers can be used in any industry, I mean it. You can use it in literally almost any industry. Like I said earlier, doctors give them out to kids, teachers give them out to their students, parents have them for their kids. There's different stickers for different occasions, there's some for graduation, for Valentine's Day, for Christmas, for birthdays, celebrations, and much, much, much more. Now, individually, stickers might not really make you a ton of money, but a lot of the times, people don't just purchase one sticker. Stickers are usually purchased in larger packs, whether they be five, 10, 15, 20, 100, 1,000. Stickers are usually purchased in larger amounts. Now, when it comes to stickers, these are the two most common kinds that you're gonna find. So these two terms are gonna be used interchangeably, primarily because most people don't really know the difference. But when it comes to stickers, there's typically gonna be decals and stickers. Now, what's the difference? So a decal is this right here.  As you can see here, it doesn't have any background, it doesn't have any paper around it, it's just the design itself. For the most part, these you're gonna find at businesses, you're gonna find them on the business windows, it's gonna have information on the business, or these are also very commonly used on cars. These are the ones that people use to put their Instagram names, some funny designs or just whatever it may be. Now when it comes to stickers, these are typically gonna be full color and they are gonna have some sort of background. It's gonna be one large solid piece. As you can see here in this C is for coffee or this teacher sticker. It's one big piece with no negative space in the middle and the design is either printed on there using a basic inkjet printer that you can use at home or a specialty sticker printer. Now for the most part, stickers are gonna be cheaper than decals. The reason is because since there's so much negative space on decals, it takes longer to produce. On top of that, the material for decals is a lot more durable, it's a lot more stronger and it's a lot more waterproof. But the more generally used type of sticker is gonna be this kind of sticker, the basic stickers that you find at a store or that you can find in your online orders that you receive. Now when it comes to sticker categories and sticker niches. So as I mentioned earlier, there's literally a sticker for any category, any niche, any show, any book, anything that you can think of. They have some that are motivational quotes, some funny sayings and a lot more. So when it comes to these different categories, you can niche down, niching down will really help you attract a specific audience, but you're also able to have a generic store selling generic stickers. Now, if you do decide to go that route and start a general store for stickers, I highly suggest that you take the time to really design and make your store look good and be easy to navigate. Like I said, when it comes to stickers, the categories and the niches are absolutely endless. So you're gonna have a lot of different categories on there. So make sure that your website or your product page is easy to navigate, it's easy on the eyes and most importantly, it's well-organized. This will all contribute to a better customer experience, making the possibilities of them making a purchase a lot higher. Now, before we continue, if you want access to everything I'm talking about in an easy to reference cheat sheet, all you have to do is go ahead and comment down below hashtag stickers and let me know which one of these kinds of stickers you're planning on selling. Are you gonna sell individual stickers? Are you gonna sell them in bulk? Are you gonna sell customs? Are you gonna sell generic? Let me know your thoughts down in the comments below. I would love to know. Once I see that you went ahead and did that, I'll go ahead and reply back with a link to the sheet. All right, now the moment that you've all been waiting for. What are the best stickers for you to dropship? Well, since there are so many different ones to choose from, these are some of the most popular ones that people are actually actively looking for. And to kick off this list is probably one of the more popular types of stickers. Everyone loves to rep their city, especially people in places like Miami, Las Vegas, New York, Los Angeles, and that's city stickers. So as you can see here, just searching up Los Angeles stickers on Etsy, you can see quite a few different ones. So you have this one right here that just has the basic LA with the palm trees. This is a classic LA look. Looking at the reviews, this item itself has 10 different reviews, all five stars down the line. People love city stickers. Now aside from that, you also see some NBA ones, which I'm gonna tell you right now. Do not sell these. Don't even try. Sports organizations will crack down on you if you're using copyright items. These people, these organizations, they do not play. Don't sell their items. But aside from that, they have a vintage Los Angeles travel sticker. This one's really nice and the store actually has a lot of reviews and a basic Los Angeles sticker. Now this one's a sticker because it actually has a white background. That's one of the ways that you can tell the sticker from a decal. But check this out, reviews for this item. This item has 38 reviews. That means a minimum of 38 people have purchased this item. Guaranteed it's been more people since not everyone leaves a review, but that just shows you that a simple sticker that just has the basic city name on it actually sells. Next up we have stationary stickers.  They're what a lot of people use to put on their stationery. A lot of students use these to put on their notebooks, to put on their laptops, on their books. So as you can see scrolling through Etsy, they're all pretty similar in size. And one thing that really stands out to me is that nobody is selling single stickers. Everyone is selling them at a minimum, a pack of five. But aside from that, you can see that they have some pretty unique ones. They have some vintage Halloween stickers. They have some snack stickers, which I would advise against selling something like this because you have a brand name on their Kit Kat.  In a third spot we have art related stickers. Now these stickers are pretty self-explanatory, but they're stickers that are simply going to have some sort of art piece on them or they're going to resemble some sort of art piece. So looking through Etsy once again, you can see that these stickers all are of paintings, of fish in particular. You have one of a little plant over here. You have the Starry Night sticker with a little bit of editing in it. You have here some people from some pretty famous paintings. Scrolling down you can see a few other ones. Here's a parody on one with sleigh on it. That is, that's not what I thought it was, nevermind. You have one here that's actually, this one's really cool. This is pretty unique, a color palette as a sticker. This is really nice. And if you keep scrolling, you see a few others that have to do with art. They're all related to art in one way or another. Up next is one of my favorites and that is Glow in the Dark stickers. I absolutely love Glow in the Dark anything. I think it's super cool, especially when it's on something like a t-shirt. But the next best thing to a t-shirt in my opinion is stickers. So when it comes to Glow in the Dark stickers, there's pretty much something for absolutely everything. But some of the more popular ones and some of the ones that are more relevant to the Glow in the Dark theme are gonna be things like outer space, planet and star stickers. Some that you can stick on the roof, on the walls or around the entire room. So that way whenever you turn off the lights, it looks like you're in space. Kids absolutely love that. And so do millennial parents because these really resemble the stars that we used to put up on our roofs all the time. Ah, nostalgia. Those were the good old days. Next up we have label stickers. Now label stickers can come in a few different options. So you have some that the customers can use to print by themselves. So these are typically sticker sheets that you're just gonna place in any home printer and simply just print out your stickers on there. A lot of the times these are used as labels to label different things around the house, around the office or just things that people need to remember. But aside from that, you can also offer things like these right here which are also label stickers but that the customer doesn't have to print on. They can simply just write whatever they need on there. You can also offer a holiday version of these kinds of stickers. So when it comes to Christmas time, whenever people give out presents, a lot of the times there's gonna be a sticker on there that says to whatever your name is from Santa or from mom, dad, uncle, whatever it is. These are holiday label stickers and during the holidays, of course, they do absolutely fantastic, especially if you can have some unique ones. Car stickers are up next and it's actually car decals, mainly because people typically use decals for their cars rather than the stickers. Although some people do use stickers, decals are more widely used. Now when it comes to different car decals, some of the more popular ones have to be the social media or social handle tags. So these are typically going to be just an Instagram or Snapchat or a TikTok logo with your customer's social media username. Now these of course are custom, but there are different ways to do this and still dropship. One of them is by simply still using Etsy. Now for this, you're not gonna really be able to use automation, but what you're gonna do is simply just offer custom decals tell your customers to let you know what platform it's gonna be for and what the text is gonna be. Then as soon as you get that information, just go on over to Etsy and place the order yourself. Now as a quick tip for success, try running hybrid dropshipping stores when it comes to stickers. While generic stickers are still widely sold, people are always looking for them. People are also really wanting custom stickers or custom decals, but split your store into two different categories pretty much. The custom and the generic side. This will really help you reach a way broader audience and help bring in more customers as well as profits. Also when it comes to custom decals, a lot of the times people are only gonna order one or two, which at the end of the day you can charge a little bit more for since you're not gonna be offering bulk pricing. Okay, so this next one is not necessarily a sticker or a decal, but I mean technically it is because it sticks to you. Anyway, so we're talking about temporary tattoos. Now temporary tattoos are hugely popular, especially amongst kids and during some holidays, such as Halloween. These obviously you can find in almost anything that you can think of. So looking through some of these, you can see that they have some pretty interesting ones like these right here for your face. These are really interesting, these are unique. These could probably make some pretty good money during Halloween. And you have some other ones that actually resemble actual tattoos such as these right here.  And then you have a few other ones such as these that are full color and pretty large pieces. And you even have some glow in the dark ones. Next up we have custom stickers. Now just like with custom decals, a lot of the times you are gonna have to place these orders individually by yourself through a supplier that you find on let's say Etsy. But there is a way that you can offer these in bulk orders or order larger amounts through a supplier like Aliexpress. So the first thing you need to do is find a sticker provider. So pretty much just go to any sticker listing and send that supplier message. Ask them if they can make custom stickers and what their minimum order quantity is. If it's something reasonable, maybe like 10 or 20, then you can simply go back to your customer and tell them that you can do custom stickers for them. The only thing is, is that you are gonna have a minimum order of whatever it may be, 10 or 20. And of course, from there just hike up the price. This next kind of sticker is one that has actually been making waves fairly recently. I've been seeing them a lot on TikTok lately and I've been seeing people actually make these themselves. Of course, when people make these themselves, they are gonna be charging a little bit more. So as drop shippers, we can take advantage of our suppliers over at Aliexpress, Etsy or Amazon and sell them at a better price. On top of that, we can also give some pretty good price when it comes to higher quantities. So just looking on Aliexpress and typing in nail stickers. Now, if I didn't mention this before, which I don't think I have yet, I'm talking about nail stickers. Now, when it comes to nail stickers, these are very, very small. So you're not gonna just sell one of these. These are gonna come in sheets and those sheets are gonna have multiple quantities on them. They're gonna have different designs or they're gonna have the same designs. So scrolling through a few of the different listings here, you can see that this one right here is a few different sheets and they all focus on a different theme. 42 cents, you can offer some avocado nails, some cherries, a mix of the different fruits. What I think looks to be lemons and bunnies, whatever it may be, you're gonna be buying different sheets and those sheets again, are gonna either be in the same theme or they're gonna mix and match. Now, one cool thing that you can do with this, one tip for success that I'm gonna give you is, if you're on Instagram, which if you're not, why are you not on there yet, you need to get an Instagram and you need to have a TikTok to show off your different products. But aside from that, if you're on Instagram, look for different nail salons. Look for different people that offer nail services, whether that be painting, cutting, manicures, pedicures, whatever it is. If they offer services for nails, you can message them and offer them these different products. You can offer it to them at both prices so they can order more because of course, they're gonna be using them all the time. Next up, we have 3D stickers. Now these come in a few different types of designs. You can offer different kinds of 3D stickers. So one is these right here.  This is a pretty cool sticker, I like it.  It actually makes it look like there's something in there. It makes it look like there's an actual hole in the wall where a mouse is doing its business. Aside from that, you also have some like these, which are holographic stickers. It's actually a lot easier shown through a video rather than pictures. So you can see here how it looks. Then aside from that, you also have these which are a bit more true to 3D because they actually pop out. They have a texture on them. They have different curves.  Now when it comes to sourcing your different stickers, there's a few different places that you can do so from. While some of the more generic places to order more generic stickers are gonna be places like Amazon, Aliexpress, Alibaba, DHK and Walmart. There's also a few other specialty sticker stores that you can use to offer some more unique and custom stickers. Now of course, each supplier has its own benefit. For one, the first batch that I mentioned, you can benefit from automation. Now what I'm talking about when it comes to automation is simply the fact that you can automate your entire drop shipping store. So you can automate everything from the product importing. You can simply log into your account over at autoDS.com, click on add products, put in single product and type in the link to the sticker you wanna sell through there. And it'll be automatically imported into your store ready for you to sell within minutes. Aside from that, whenever somebody places an order in your store, it's gonna be automatically fulfilled using AutoDS. Also, any revisions you need to make, any revisions to the title, the description, if you need to edit any of the images, you can do everything from this same platform. As you can see, the benefits of automation are absolutely amazing. It really does make running an e-commerce store practically hands off. But aside from the more generic stores, you also have, like I mentioned earlier, more specialty stores. Now while you're not gonna be able to benefit from automation from these, you are gonna be able to offer different and unique products. And the first supplier that I'm gonna mention to you is StickerU. Now StickerU.com offers a little bit of everything. So they offer different types of stickers, they offer labels, they offer decals, and they even offer temporary tattoos. And to give you even an extra bonus, they offer iron on transfers. What's an iron on transfer? It's basically just a sticker that you can stick on a t-shirt, apply some heat to it with maybe like an iron or something like that, and it'll stick to the shirt. It's pretty much all you need to do, and you have your own custom shirt. But aside from that, they do offer a few other products, but primarily speaking, we're talking about stickers, so they do have custom stickers as well.  Like look at this one right here, it has the nurse sticker, and that is, as you can see, actually cut out. It's cut out to the shape of the sticker itself, or the sticker design. This type of cut is called a die cut, simply meaning it's just cut around the image itself. It's cut to the shape. Aside from that, you have the kiss cut stickers, which are basic squares, rectangles, circles, triangles, and you have a few other different options. So they have the holographic ones, they have glitter stickers, and best of all, the last thing I do wanna mention is that you're not just gonna find small stickers on here. You're gonna find some basic tiny ones to some pretty large format stickers that can take up a whole car door, or that you can stick on a wall. Next up, we have one of my favorite sticker providers, and that is Sticker Mule. Now Sticker Mule offers pretty much every single type of sticker that you can imagine. They offer everything from regular stickers to labels, magnets, and they actually have a unique one that our last one didn't have, and that is packaging tape. Now packaging tape can still be considered a sticker because it does have a sticky backing. Now this is something pretty unique that you can offer a lot of places don't offer. Custom packaging tape is something that a lot of businesses use. Now another supplier that you can use when it comes to printing out custom stickers is gonna be a print on demand supplier. For these, you have a few different ones to choose from, but two of the most popular ones are gonna be Printify and Printful. Now when it comes to Printify and Printful, of course, you're not limited to just stickers. You can print pretty much anything you need.  They have magnets, they have sticker sheets, so you can offer sheets with multiple different stickers on them. And they also have vinyl stickers, holographic stickers, and pretty much specialty stickers. Now the cool thing about using a print on demand service is that you're not gonna have a minimum order quantity. If you want to just order one sticker for just one or $2, you can do so. And the last provider I'm gonna give you for this video is gonna be Merchise. Now Merchise is another print on demand supplier, but they're not as big as, let's say, Printify or Printful. Their quality control is a bit more centered, it's more in-house, since they do all of their printing themselves. They're not outsourcing it to different printers around the country. So because of that, they do offer some more simple controls, but they do offer some pretty high quality stickers. So as you can see, these right here come in a few different sizes. They have from three by three all the way to 12 by 12. They have three different tiers. And if you scroll down a little bit, you can see a few different shipping times. So in the US, they ship pretty quick between four to seven business days to Europe, 10 to 20 business days. And to the rest of the world, it's anywhere between 10 to 25 business days. For those of you that don't know, Rowe stands for rest of the world. I didn't know that myself. I literally just Googled that. So FYI. Aside from that though, they do offer some larger stickers. So they offer a large, medium and small, ranging from 7.8 by 11.8 inches to 15.7 by 19.6 inches. Now I know that a lot of people watching this right now are not in the US, so you all don't work with inches. So for those of you that don't know how big these measurements are, Mr. Video Editor, please put the conversion right here. We totally appreciate your work, sir. Thank you. And those were some of the best stickers that you can start selling, along with some of the best places that you can start sourcing your stickers from. Huge thank you to all of you for watching, especially if you made it all the way to the end. As always, it truly means the world to me that you watched the entire thing. Huge thank you for watching. Huge thank you for sticking all the way to the end with us. Remember, if you want access to that cheat sheet, all you have to do is simply just go ahead and comment down below, hashtag stickers, and let me know what kind of sticker you're planning on selling. Are you gonna sell decals?  Are you gonna sell stickers for certain occasions? Or are you gonna be selling generic stickers? Let me know down in the comments below. I would love to hear your feedback. Once again, huge thank you to everyone for watching. My name is Mario with Out of the S, and I'll catch you all next time.


For the governor-elect of Abia State, Alex Ooty has given his acceptance speech moments after his victory was made official by INAG. Appreciated in indigents and non-indigents who participated in the polls, he noted that his victory came after three attempts to become governor. Ooty saluted the industry and the resource-owners of all residents, both religious and traditional institutions, among others. Even as I come out of a bruising and long-run-out campaign, I know that this is just the beginning of the journey. There's a lot of work to be done and we must get our hands on deck to ensure that we take back our state and bring the much sought-after dividend of democracy. You voted us to serve you and not to be served. You voted us to alleviate your sovereigns and not to compound and multiply them. This is a servant that comes with an unusually high sense of devotion and commitment, giving a check hard history of the people. During my election-hiring campaign, I traveled all nooks and crannies of Abia State, and that enabled me to have first-hand assessment of the needs and expectations of Abia. These are the basic things expected by the people who are responsible government to provide in order to unleash the potential inherent in the typically hard-working Abians. Based on my findings and those of my team members, it's on the nooks that an immediate hands-on approach is required to hit the ground running and to ensure that we alleviate the sovereigns of all Abians. I promise you that I will serve you as governor of all Abians and all citizens living in Abia, irrespective of land, religious situation or political living.


So thank you very much for the opportunity to present my work in this conference that I've really enjoyed so far. So I'm going to show you what we've been doing with system CRISPR in bacteria. I know we're using them both to develop tools to study and to fight pathogenic bacteria. So I first want to give you a bit of history about these CRISPR systems. So they basically were first discovered but not understood in 1987 by this Japanese group who was just sequencing this IAP gene and just downstream of the genes. They found this interesting sequence where you had these repeats that were interspaced by this sequence that looked sort of random. And the size of the repeats and of these variable regions was very well conserved. At the time of course they didn't understand at all what these sequences were. And you should look at the citation report for that paper. Basically it was published in 1987 but it got almost no citations until 2007. So in 2015 it hasn't been updated but it's probably up there now. So what happened in 2007 is basically this paper. So that's a landmark paper in the field of CRISPR. It's basically the first experimental evidence that this CRISPR loci actually provides acquired immunity against bacteriophages. And what they did in that paper is they worked in this strain of streptococcus samophilus. The interesting thing also to note is that this paper didn't come out from an academic group but rather from the industry and from a company called Danisco that was later bought by Dupont that was developing ferments for lactic fermentation, for yogurt production, cheese production. And these people as other speakers before have noted have big problems with viral contamination that can ruin the batch production. So they always try to select strains that are resistant to bacteriophages. And when doing so basically they noticed that the CRISPR locus was changing and that it was capturing new sequences coming from the phages that were using the challenge. And so when CRISPR captures a specific spacer so the sequence coming from a phage it becomes resistant to the phage that matches this sequence. So that was a very interesting discovery. So since 2007 the field has moved at a quite incredible pace and we now understand a lot about this CRISPR systems. And this is basically the picture of how they work. So first I should also say that CRISPR itself stands for clustered regularly interspaced short palindromic repeats so that's very complex acronym and that was coined actually by bioinformaticians. So that explains very well the structure of this low-cyber, you have these clusters of repeats that are regularly interspaced and repeat themselves frequently contained short palindromes. So hence this long CRISPR name. And the idea is that when you have a bacteriophage injecting its DNA in the cell at some frequencies a CRISPR system thanks to some proteins known as CAS for CRISPR associated is able to capture a piece of DNA from the phage and integrate it along with a new repeat in the CRISPR locus. Once it's captured this information in this adaptation phase it's then able to use this information to fight infection by phages, similar phages. And the way it does that in this immunity phase is by transcribing the CRISPR locus in the first precursor RNA that's then processed into some small CRISPR RNA and those small CRISPR RNA are actually in complex with CAS proteins that are going to be able to cut and degrade sequences homologous to the CRISPR RNA. So basically the CRISPR is building a memory of past infection and then using that memory to fight infection by similar phages. I want to go just a bit more in details about one specific CRISPR system. So that's a Type II C CRISPR system from Streptococcus pyogenes and there's a good reason for that is this system is the one that is the most used for biotechnological application of CRISPR. And you see that this CRISPR system contains actually four CAS genes, CAS9, CAS1, CAS2, CSN2. It has a CRISPR RNA with actually six spacer and repeats. And the interesting part here is that it also has another small RNA involved in this process that's called a tracer RNA that stands for transactivating CRISPR RNA. And these three CAS genes here, CAS1, CAS2, CSN2 are actually involved in the capture of new sequences for the CRISPR system. And the CAS9 protein itself is able to carry out the whole immunity step from these systems. So the role of this tracer RNA is actually also very important in this system. What it does is that it's complementary to the sequence of the repeat. So when you transcribe the primary transcript here from the CRISPR array, the tracer RNA can then hybridize making duplex RNA with each of the repeats. And that duplex RNA is then recognized by CAS9 and by the host RNA3 and that processes the primary transcript into the smaller CRISPR RNA. And after this process, you basically end up with this complex as CAS9 and these two RNAs, the CRISPR RNA and the tracer RNA and all the elements of this complex are absolutely essential for the function of the system. So once you have generated this complex, this nucleoprotein complex, what happens is that it's going to basically look for possible targets. So it acts as a surveillance complex that's constantly scanning the DNA in the cell looking for homologies with the CRISPR RNA. But first what the complex is actually scanning for, is it's looking for a small motif, in this case the motif is NGG and that's known as the PAM motif for protospacer adjacent motif. And once CAS9 finds this GG motif, then it starts to unwind the double helix and pair it with the CRISPR RNA. If there is some match, then it can completely pair the full length of the CRISPR RNA and that triggers a conformational shift in the protein in the catalytic domains in contact with the double helix, introducing a double strength break in the target DNA and that can then lead of course to the degradation of the target DNA. So the way this system works has basically been very well described by the group of Emmanuel Charpentier and Jennifer Dodna and that got them to get this breakthrough price last year and mingle with some Hollywood stars like Cameron Diaz and the CEO of Twitter here. So it's good to know that if you do exciting science you can meet your movie stars. So what have we been doing with this system? So you said we're a microbiology group and we work on both understanding the ways this CRISPR system works in bacteria but also developing technologies and today I'm going to mostly talk about the technological aspects. And the first technology we developed using this is the strategy to edit the genome of bacteria. And the strategy here that we employed, so I'm showing here what we did in E. coli, we introduced a plasmid carrying Cas9 in the cell and then we can electroporate together another plasmid that is programmed with a CRISPR that will target here a gene we want to modify and at the same time we introduce a specific mutation we want by electroporating a single-stranded oligo that carries a mutation we want to introduce. And we don't do that just in any strain, we do that here in this HME63 strain from the lab of Donald Cort that expresses a lambda-red recombination machinery that allows to basically recombine this oligo at a high frequency and then the CRISPR system can basically select this mutation by killing all the cells that did not introduce this mutation. And that works relatively well. So at the same time that we were doing that work, there has been a lot of groups starting to work on CRISPR systems and people have called that the CRISPR craze and this is just a tiny, tiny sample of all the papers that have been published describing the development of CRISPR tools for genome editing in a very broad range of organisms. The global idea of all this tool work is, in all cases, you start with programming Cas9 to cut out a specific position in the genome that you want to modify. And once you generate this double-strand break, then the cell can deal with it in different ways. So either you can provide a template for homologous recombination and that's where you can control maybe the exact point mutation you want to produce, gene insertion, deletions, etc. If you don't provide a template for recombination, you can maybe rely on endogenous Ripper systems of the cell such as non-homologous and joining and HEJ. And in that case, the cell is able to just join together the broken ends, but it frequently makes mistakes doing so. And like this, you can introduce small indels at the target position and that's used a lot to introduce knockouts. And the last possible outcome is if the cell is not able to repair the break, then this will, of course, lead to the death of the cells. And when you look at the literature, the picture that we have is that this basically two pathways seem to work pretty well in eukaryotic systems, actually very well in eukaryotic systems to the point that the technology has spread super fast and now CRISPR editing has become really a standard technology to introduce mutation in eukaryotic systems. And in bacteria, it looks like CRISPR system is actually pretty good at killing the cells, and you can still use that for editing purpose but more as a selection tool than a way to trigger the introduction of a specific mutation. And the fact that you can actually use CRISPR to kill the cell, you can think that maybe you can even use that as an antimicrobial strategy. And like the very basic experiments you can do is if you have a plasmid carrying Cas9 and a CRISPR that you program, for instance, here to target the kinamycin-resistant gene, so that's work done in staphylococcus eras, that plasmid, you can transform it very well in the cell if the target is not in the chromosome. But if the target is in the chromosome, then you recover very little transformments and the idea is that the CRISPR is actually going to kill the cell. So like this, you can specifically kill bacteria that carry, for instance, antibiotic-resistant genes or virulent genes. But of course, you need to be able to deliver the system to an old population of cells. And the way we're doing that is by using phage as a vector. So phage are naturally able to package not only their own DNA but other DNA present in the cell, and that's called transduction. It turns out you can actually increase the frequency of transduction really a lot by simply cloning, for instance, a packaging signal present on the phage DNA. If you put that on a plasmid, then you can have the plasmid being packaged at a very high frequency in the phage capsid. And then you can use this system to inject a plasmid in the old population of cells. So we're using this strategy to inject a CRISPR system consisting of Cas9 and a CRISPR that we program to target an antibiotic-resistant gene present in the chromosome of Staphylococcus aureus cell. And the idea is that this should kill the bacteria. So this is just to show you the specificity of it. So what you see is loans of cell on a plate and it's actually either a strain that carries a kinamycin-resistant gene in the chromosome or that does not. And then we program the CRISPR either to target the kinamycin-resistant gene or with a spacer that doesn't target anything, and then we just put a drop of our CRISPR phage-med preparation on top of the bacterial loan and we see that it's clearing the loan only when the CRISPR is programmed to target the gene and the gene is present in the chromosome. So this is a very specific antimicrobial. So you might wonder why do we want to make antimicrobials that are so specific? What's the purpose of this? And the idea is that if you're able to specifically eliminate strains that carry antibiotic resistance, for instance, then you can take advantage of the competition with other strains that do not carry this resistance gene to more effectively eliminate the threat. And to demonstrate this idea, we did this very simple experiment where we just make a co-culture of two Staphylococci strain, one that is resistant to kinamycin and has this APH gene in the genome and another that's sensitive to kinamycin. And what we did, too, is that we put GFP-plasmid in the kinamycin-resistant so that we can easily follow both populations in a co-culture. And so this is what happened in the control experiments. So the cells are mixed one-to-one. We follow both the optical density and the dashed line is the fluorescence in the culture. And so in the control experiment, you see that about half of the population is the kinamycin-resistant cells. Now what would happen if you use, for instance, kinamycin as an antibiotic? So if you had an infection like that, it would be a very bad antibiotic choice.  If you make a slightly better antibiotic choice, so, for instance, streptomycin, here what happens is that you basically will kill both populations of cells. But after a little while, you would select resistance in both populations and you would still end up with about half of the population being the bad guys. But now if you actually treat with a CRISPR system that specifically targets the kinamycin-resistant gene, so that the purple lines, you see that the fluorescent signal here is not recovered. In fact, we killed all the kinamycin-resistant cells, but what it means is that we killed enough of them and we let the other population grow in the culture. You see that by following the OD curve and these cells don't occupy the niche and prevent any possible survivors from coming again. So in this specific scenario, using a CRISPR antimicrobial can actually be more efficient than using antibiotic treatments to eliminate bad bugs. So we showed that we can not only do that in a laboratory strain of staphylococcus aureus, but also with some really pathogenic and problematic strains, like the metisline-resistant USA-300 strains that are a big problem in the US right now. And we showed that if we make a mixed population with this MRSA strain and some non-pathogenic staphylococci, we can specifically eliminate the MRSA strains by targeting the MAC-A-resistant gene. So I also want to mention that there is actually a very interesting side effect to speak of this strategy, which is when we treat a population of bacteria, we inject the CRISPR system not only in the bad bugs, but also in bacteria of the same species that might not carry the target. They're going to survive, and if our CRISPR is carried on a replicative plasmid, they're going to keep that CRISPR and know they are basically immune to horizontal gene transfer of the targeted genes. So in this case, we show, for instance, that we can immunize a population of staphylococci against the acquisition of the tricycline resistance by injecting this CRISPR system, and then we show that if we control that basically a non-working CRISPR system, we can do a transduction experiment to recover the tricycline resistance, but if we immunize, we cannot do it. So that's also an interesting possibility. So we went to do also some animal experiments with this ID, and this is a skin colonization model in the mice. What we do is we shave the back of the mice, and then we colonize with a mixture of resistant and non-resistant staphylococci, and then we treat with our phagemid preparations that target specifically the canine mice and resistant ones. The resistant ones are also here fluorescent, so we can nicely follow them after treatment, and here we show that we're able to specifically decolonize the antibiotic-resistant bugs. So it's not as efficient as in vitro, but it also works in a more complex environment like the skin. I also want to mention that this work was pursued by a startup company called Eligo Bioscience, of which I'm a co-founder, and that's hosted at the Institut Pasteur. So basically, we had this picture right now where Cas9 is very useful to make genome editing in your carrots. In bacteria, it tends to rather kill the bacteria, and you could ask the question, why, and is this really always the case?  And not your carrots. So some experiments that we've done recently to address this question is simply to do a very easy assay where we still have Cas9 in the cell and we transform a CRISPR that we program to target many different positions across the genome of E. coli. And then we see what happens. We basically just count how many colonies do we get when we do this transformation. And this is the result that we got that was very surprising at first, because we really expected the CRISPR to kill E. coli at any target position, but what we realized is that some target positions we don't recover colonies, meaning that the CRISPR killed the cells, but other positions we actually recover just as many colonies as a non-targeting CRISPR control. And what we saw, so that was interesting, is that for some of them, the one where I put a star, the colonies that we recover are actually some pretty small colonies, indicating that the bacteria here are pretty stressed. And this points to the ideas that probably, first, not all targets are equal, some targets are better than others, and some target position might be tolerated by E. coli, and E. coli might be able to constantly repair the breaks introduced by Cas9 at these targets. And we could confirm this idea by simply repeating this experiment in a RecA mutant, so I think you might not see very well the white bar here, but that's a control if we don't target anything. Of course we cover a lot of transformants, but all of those, there is no white bar, basically, because if you're in a mutant of the repair systems of E. coli, RecA, then all these targets are going to kill the cells. But that still doesn't tell us why does it kill the cell when it actually does. And the idea is that it probably does so because when CRISPR is going to cut in the genome, it's going to cut all copies of the genome at the same position at the same time. And it turns out that most bacteria strictly rely on homologous recombination to repair breaks. So if you don't have a sister chromosome to do homologous recombination with, then basically you're dead. And we believe that this is the kind of damage that CRISPR does, and to demonstrate this what we did is very simply putting a template for recombination on a plasmid that is not going to be cut by the CRISPR. And the idea is that if our hypothesis is right, this should rescue the cells. And then when we transform our CRISPR system that targets this like the gene here, what we see is that without the repair template we do see a lot of deaths when we target the gene. But no way if we add the repair template we see that we rescue a lot of the cells. This confirms that the cells die because they don't have a template for repair. Then what we also noticed is that whenever we transform this CRISPR system to target in the position, whether it kills or not, we were wondering if this could trigger mutation at the target. And because when it kills it usually does not kill all the bacteria. You might have some that survive. And this is the case for this, like the two targets here where you have some bacteria surviving. But here when we played this on the exgal plate that allows us to see if the like the gene is intact or not, we see that we recover about half blue colonies and half white colonies. So that suggests that the CRISPR actually led to the introduction of mutation at this target position. And when we checked what kind of mutation we obtained, what we see is that we obtain a lot of very large deletions around the targets. Deletions up to about 40 kb. And in most cases, what was interesting is that these deletions involve these rep elements of E. coli. So I had no idea what these rep elements were before we studied these mutants. But those are basically repeat elements. So there are a lot of them in the genome of E. coli. I don't remember the exact number between 200 and 300. They are scattered throughout the genome. And basically the idea is that they have enough homology between them that if you introduce the Bostrand break, they can recombine together to repair the break and the cells might survive. If, of course, there were no essential genes in the deleted region. So then you can try to make the hypothesis. Why do eukaryotic cells survive CRISPR breaks so much better than bacteria? And one idea you could have is that, okay, bacteria, most bacteria, don't have non-homologous enjoining. But eukaryotic cells do. So maybe they are able to repair the CRISPR break with NHEJ, and that allows them to survive. So then we thought, okay, so let's try to introduce an NHEJ system in E. coli and see if that can rescue the cells. So that's what we did, and we took the NHEJ system from mycobacterium tuberculosis. So I told you that most bacteria don't have NHEJ. Actually there are a few bacteria species that do. And then we repeat the same experiment where we transform a CRISPR system that's going to cut in the chromosome. So in the control that doesn't target anything, we recover a lot of bacteria, but here, whether we have or we don't have the CRISPR system, the CRISPR system still kills most of the bacteria. So it does still kill most of the bacteria, but what we noticed is that when we played on this exgal plate that allows us to see there was a mutation introduced in like Z, we see that we start to recover more white colonies with the NHEJ system than without. And if we PCR the target position, we start to see small variation in size as a target position that are very typical of repairs made by NHEJ. And so we can map this small deletion and we see that when we have NHEJ present in the cell, we obtain a lot of deletion ranging from six bases to about 300 bases that are very variable in size but that are still very different from the big deletions we obtain without NHEJ in the cells. And something that was interesting is that these deletions were always very variable on one side but very... always within basically three bases on the other side. And that basically led to the idea that probably Cas9 might remain bound more strongly to this end of the break and might protect it from nucleases which might not be the case of the other side explaining the asymmetry in the repair. So, to sum up this part, basically, Cas9, when you introduce bacteria, if it's able to cut all copies of the chromosome at the same position at the same time as in the cell's cannodomalgoscommonation, it's going to die. But some bacteria might be able to survive by making, for instance, big deletions. And the other thing that we found out is that at least with the setup we had NHEJs not able to rescue the cells. It can make some repairs but very, very low efficiency. And efficiency is basically even too low to be able to use it really as a tool to introduce indels in bacteria. So now I want to talk about a slightly different thing you can do with this CRISPR system, which is to use the catalytic dead mutant of the Cas9 protein. And that's a very interesting mutant because it's still able to find its target position and bind to it very strongly, but it doesn't cut anymore. And the thing is that it buys strongly enough, for instance, to block transcription and so that you can use it to silence gene. So that's something that we demonstrated, the group of Sandley Chi, also published a very similar thing to this work. What you see here is just a GFP reporter gene and this is a relative fluorescence measure and then it's just a position we target with this guide RNAs, either within the gene or in the promoter of the gene and the fluorescent level we obtain. So you see that if you target in the promoter sequence you obtain very strong repression, in some cases really barely detectable, expression of GFP. If you target inside of the gene you can still get very strong repression up to 100-fold repression and it really depends on the orientation of the target in the gene. If you target the coding strand you can get good repression, if you target the template strand you can only have much weaker repression. And to understand what's happening here you can do a simple northern blot to see this is basically in the control of the full-length transcript of the GFP. If you target the promoter region you don't see any transcripts so basically you block the initiation of transcription. If you target within the gene you start to see a smaller transcript here appearing that the size of that transcript matches exactly what you would expect if the transcript is just stopped as a Cas9 binding position and if you target now the coding strand here you don't see any more the full-length transcript so you have very good repression and you produce this shorter transcript here. So basically this D-Cas9 protein is either to block initiation of transcription or to block even the elongation of transcription and it can be very easily reprogrammed to bind any place you want in the genome so you can use that to silence genes in a very convenient way. Then you can start to do even more things with this D-Cas9. You can fuse protein domains to it to introduce functions at a specific position in the genome. And here for instance what we did is to fuse it with the omega subunit of the RNA polymerase in order to turn it into a transcription activator. And then we target it in upstream of weak promoter sequence and we should see activation of the downstream gene. And so we targeted it to a different position upstream of the weak promoter, either on one strand or on the other strand, and we see that at a very specific distance from the promoter and in the right orientation we can get up to a 23-fold activation of the GFP. So I also just want to mention work by other groups related to these very interesting applications of D-Cas9, for instance including in imaging if you fuse a GFP to D-Cas9 and here for instance we targeted it to bind to telomere sequences. You can see here in this picture every little dot is a different telomere in the cells and here you don't have to fix the cell or anything, the cells are live so you can follow the dynamics of different loci in the cell and it's a very powerful tool for people studying the architecture chromosome organization and dynamics. So the last application I want to talk about is something that we're really focusing at the moment is to develop screens, functional screens based on CRISPR. And this comes from the fact that you can so easily reprogram this CRISPR system that you can construct libraries of CRISPR or of guide RNAs that are going for instance to target up to 10 to the 5 position here in the genome of E. coli and then we can introduce this library of guide RNAs in our cells and then you can perform a functional assay of interest. You might be interested in studying sub-MIC antibiotic concentrations, some different stresses you might want to combine it with some query gene knockout, etc. And the readout of this experiment is going to be done through deep sequencing. What you do is you sequence the CRISPR library both before you perform the experiment and after you perform the experiment and by comparing the proportion of each guide RNA changes during the course of the evolution you can basically compute the thickness of each mutant in the population. In series there are two ways you can try to perform these CRISPR screens. You can either think of doing them using CAS9 to introduce knockouts in combination with NHEJ and that's something that people have already published and are doing in eukaryotic systems where you have this endogenous NHEJ in the cells and these screens work very well. And you can also think about doing screens based on D-CAS9 where here you're not going to introduce knockouts but to knock down and silence the target position. And I explained to you that NHEJ repair at least in E. coli is very inefficient so we cannot do this but we can do this type of screens. But ideally we might be interested in both and both screens have actually different properties that might be complementary. So for D-CAS9 screens you can think that you would only have partial silence and polar effect but you actually know what the polar effect are. For instance in an operon if you knock down target a gene early in the operon it will also block expression of older downstream genes. A big advantage of D-CAS9 knockdown is that it's actually both inducible and reversible. So knockouts with CAS9 and NHEJ here the good thing is that you can have complete deletion but the problem is that the edits made are going to be quite unpredictable and you don't really understand what the polar effects will be. I just want to give you a flavor of what kind of data we can get with this type of screen and this is just a very raw data showing you the number of reads we get for each guide in the library both before the experiment and after the experiment. So this is a control where we don't induce the expression of D-CAS9 so everything falls more or less nicely along the diagonal meaning that the number of guides in the library didn't change between before and after the experiment. But now if you start to induce the expression of D-CAS9 you see a lot of points either going down or points going up and points going down here means that basically those guides provide a fitness defect to the cells in the population and when guides going up we provide a fitness advantage to guides in the population. And so then you can of course focus on some specific points and try to see what's going on and here for instance it's the exact same figure except that I just kept the targets in this mirror A gene that's an essential gene in E. coli involved in cell wall synthesis and what you see is that the control experiment everything is still nicely along the diagonal but now when you induce you see that here's all these pink points basically go down and the green one, blue one stays there and what these points highlight is these blue points actually target the template strain and if you remember from a few slides before I explained that targeting the template strain gets you only very weak repression but if you target the coding strain to that those are these red points here you have very good repression and you see depletion of this guide in the library which is expected because it's an essential gene. So like this you can very easily figure out what are all the essential genes in the cells and of course if you do that in a specific condition where you want to do functional screens you can also get more interesting information something that we think is also very interesting and that's linked to the fact that this is something that you can induce at a precise moment you can actually follow the evolution of each guide over time and here we sequence for instance this library after 20 generations of culture or after 40 generations of culture and this is targets in the mercy or secd gene and here I kept the same color code the blue ones target the coding strain so good repression, the red one target template strain, weak repression so you see that all the red lines are basically straight horizontal lines and the blue lines here both these genes are essential genes but it turns out that if you target mercy the guides are depleted much much faster in the population than if you target secd so both genes are essential but maybe you can start to see some degrees that's what I want to say so where we're going with this basically is that this single knockdown or knockout screens are actually very cool and in some ways are actually also pretty similar to what people have been doing already in the past for instance with transposon, tag metagenesis TN-seq kind of approaches but what we think we can do that's very exciting with this is now go after genetic interaction by doing multiple knockdown simultaneously in the cells and so that's something that we're really excited in doing right now and we're just starting to be able to construct these double knockdown screens and study them in the same way so with this I'd like to thank people in my group so we have a very nice lab space at the institute Pasteur, we just started a year and a half ago and some of my collaborators and funding, thank you very much we have time for a couple questions about the curiosity what's known about CRISPR and denocoxide denocoxide constitute a film bacteria that are able to sustain thousands of DNA double grades and repair them they typically have no less than four copies of the genome per cell so I was wondering whether some studies have been done on these lines it would be super easy to check whether denocoxide tend to have or not CRISPR system it turns out that's something like 50% of bacteria have CRISPR systems so there is this very nice database CRISPR database on lines that we could look into I never checked for denocoxide any questions? a few slides back in your mercy data for the different generation times some of the lines on the graph went up and 40 generations is that just an artifact of the way you're measuring or is there some recovery? so we're not quite sure yet we're just starting to look into this type of things so the true answer is I don't know I suspect that it might actually be some mutant guide RNAs that might survive and then have a regular fitness or recover a bit or something like that but I honestly I'm not quite sure so you showed that the CRISPR is killing these cells unless you provide a template for repair that is which means it would be an even better mutagenesis to a targeted insertion so you're putting your finger on a very good point and the answer is that we first believed so as well and it actually turns out not to be the case we were lucky to have this experiment where actually the template uses cells efficiently but it turns out that this works only if what you introduce is a point mutation and only for a specific homology the length of your homology arms needs to be very specific and as soon as you want to for instance introduce a small deletion efficiency drops dramatically and etc so we don't understand really why yet and this is something that we're investigating as well David, I was really nice but I think I'm just not quite understanding the CRISPR screen so it's similar to TM-seq or transposon-seq in some ways so one I was wondering if you had a comparing contrast in methods and then the second is how are you preventing death in this case so here it's using the D-Cas9 so we're not cutting the genome it's just going to silence expression of the target positions but you're perfectly right that it's very interesting to compare this type of method with TM-seq and we're actually doing that at the moment so we're conducting in parallel a D-Cas9 and a TM-seq screen and we're going to sequence them and analyze the results very soon following up on that how do you construct the library do you design your 20 base pair RNAs and then or is that random so you can design them so now you have these companies including probably the ones that is most commonly used is oligo arrays so I mean I don't have any shares in them whatsoever just think the ones that we're using they provide, you can order a pool of oligo and you can precisely determine the sequence of up to 10 to 5 different oligos you want to be in this pool and they just generate it for you so the technologies that they're using are very similar to the technology used to generate micro arrays except that then they just cleave it off the chip and recover it in a pool and you get that and then you have this pool of oligo and then you just use it in a standard cloning procedure so what's the role of the PAM sequence you showed this one promoter where you had nicely spaced target RNAs but then what is so you absolutely need a PAM motif to be able to Cas9 to bind so Cas9 is really first looking for the PAM motif there is no PAM motif it's not going to bind and so in the experiment where I showed that for instance we had tested many different positions along the promoter we specifically engineered a promoter to contain many PAM motifs so that we can test precisely different positions would that be a problem if you want to target a gene and then you would want to target the Cas1 or the minus 10 or something but there is no PAM so the answer to that is that you actually have quite a broad range of position you can target even if what you want to block is the initiation of transcription you see here it's a 100 base pair range where you are still very good blocking the initiation so GG motif is very frequent it's randomly every 8 bases one last question you were talking about immunizing with that CRISPR when you are treating the population to kill the bad guys how long does that immunization last so it should last as long as the cells keep the CRISPR system then how long will they keep it will depend on the stability of the plasmids that you inject and many other things so that we haven't really investigated the cells themselves how long do they persist the cells which keep active your resistance the cells were I'm not sure I understand which cells you are talking about well previous question was about the period of the existence of the resistance and you refer this period to the cell survival so cells how long do they survive in your system I mean if they are not killed by the CRISPR systems they are going to survive indefinitely there is apparently one question over here just a question about the natural CRISPR system how many repeats does the natural bacterium maintain it how big is it so that's very very very variable it's basically from 1 to 600 and it depends on different bacterial species and this is really not understood why some species of bacteria tend to have shorter CRISPR while some have larger CRISPR arrays I cannot really speak to that it's really still a mystery we need to ask more questions during the discussion thank you David thanks