The principle of CRISPR System and CRISPR-CAS9 Technique (Part 1)

hello everyone in this video I'm going to speak about a very interesting topic which is CRISPR system so this time I'm uploading two videos part 1 and part 2 I'm going to think about the CRISPR system in general and the crystal castle on G engineer and technique I hope you enjoy this video let's first start a start talking about what is CRISPR system CRISPR system was first found in bacterial cells or in prokaryotic cells CRISPR system is like a type of immune system in these bacterial or prokaryotic cells so it's a region on the bacterial genome let's go Lister to see what is it exactly CRISPR so this is called the CRISPR and Marcus CRISPR locket from it's name is clustered regularly interspaced short palindromic repeats what does that mean so the CRISPR locus is a repeat of DNA so like this is this sequence is exact it's exactly like this is exactly like this these sweet Pete's are first of all shores and secondly or palindromic and what's palindromic means is that if you read them from left to right or from right to left you end up with the same sequence and they are received in regularly interspaced it means that they there are space your DNA between them and these spacer DNA's are regular so it between each two plus and crisper air repeats there is a DNA spacer so the CRISPR locus is DNA or a piece with space of DNA in between so this is what we call the CRISPR lockers CRISPR quick the lipids are ephemeral between it are between 28 and 37 base pairs in length and the space of DNA is between 32 and 38 days there now let's see how does the CRISPR system work now let's see let's say this is the bacterial cell there is something called B bacteria phages bacteria phages are viruses like this it's virus and this virus can infect the bacterial cells how does the bacteria phage infect the bacterial film the bacteria stage invade the bacterial cell if it attached itself on the bacterial surface and then it injects its genome inside the bacterial cell so this is the viral genome now this viral genome will force the cells to produce the viral proteins and the viral enzyme and then it will change the whole cell machinery in the bacterial cell and this is how the bacteria phage invade or infect the bacterial cell now the bacterial cell because a CRISPR system can like it can prevent this to happen a second time so the CRISPR system is somehow like the adaptive immune system in human so it's a kind of memory it's a kind of memory to prevent the same bacteria phage from infecting this gel and other times but how does it work now let's take closer the CRISPR system is a three the mechanism is three steps mechanisms the first is the space your acquisition I'm going to speak about each thing in detail so the first step is a spacer acquisition then we have the quick drying and processing and then we have the interference these are the three steps of the CRISPR system and we you should know that CRISPR system has three types so there's type 1 type 2 and type 3 depending on the bacterial cells ok so let's start talking about this spacer acquisition the this acquisition this step is the same in the three different types of CRISPR system I told you there are three types so this is a bacterial cell surface this is the bacteriophage and this is the bacterial genome sorry this is the bacteria phage this is the viral genome and this is the CRISPR locus so what's going to happen is that when the bacteria phage infected cell person for the first time the bacterial cells chop up the viral genome and take a piece of it and insert this piece into the G and a space of DNA so what you should know is that space your DNA's is nothing but pieces of different buyer viral genomes that have infected the cells previously so each time about a bacteriophage insects this cell to cell take a piece of it and insert it into the spatial DNA what you should also know is that near the CRISPR locus there are the gaps inside this cat's genes which is the cast enzyme the cap enzymes are many enzymes that are implicated in the clear in the crystal process the café's lines in general are most of them are nucleases or helicases to understand this so let's imagine this is a this is the DNA sequence nucleus can pop the DNA in this way it cuts the link between the nucleotides where is Henriques can can can cut the hydrogen bonds between the two strands and then it can separate the two strands of DNA from each other most of the cap enzymes are nucleus of any helicases in the fifth position we have two main players we have cash flow and cash - both of them are diamonds there - form that is form conflicts to get a Catholic a student conforming complex together in order to undergo spacer acquisition as I told you there's three types of CRISPR system type one two and three have the same spatial acquisition step gas form is a nuclei can be have nucleus and integrate activity so it can cut the viral genome and integrate the piece of genome in the spatial DNA whereas capture is in the ribonuclease so cast you can mainly cup RNA yeah some some bacterial pages have RNA genome so this here this bacterial page has DNA genome but other viruses have has RNA genomes so castor is an endo ribonuclease now let's go to the second step which is crispr RNA processing see rRNA is crispr RNA processing now we have this CRISPR largest we have different pieces of bacteriophages genomes in the spacer DNA what's going to happen now is that one of the two strands of DNA is going to be the transcribe transcribed into messenger RNA now this messenger RNA is exactly complementary to this trend and yeah it's complimentary to the lower strand and in this case we call the lower strand decoding turn because if this trend that was transcribed to a messenger RNA now this matthews RNA as i told you is exactly complementary to the lower sense which contains complementary sequences from the CRISPR repeats and the viral genome sequences now here we have three types of three different types of click for RNA processing in type one The Creeper repeat loops are are looked like this they form loops and then the messenger RNA will be cut using gas 6e or caste 6f enzyme the messenger RNA this sequence is going to be chopped up like this so we will end up with small pieces of RNA each piece contains the CRISPR the CRISPR sequence which forms a loop empty viral the piece of the viral genome as you see this is what we call crispr RNA these small pieces are the crispr RNA this is a platform in fact - so we have another player in this CRISPR processing it's called the tracer RNA there are these pieces of RNA which are bound to the to the CRISPR sequences on the messenger RNA so this star this is called the trace or RNA trace the TRA is from trans activating tra CR is from CRISPR and RNA these pieces of RNA as I told you bind to the CRISPR repeats on the messenger RNA and then the messenger RNA will be chopped out by cast 9 and another enzyme called RNA s 3 and we'll end up with these pieces of RNA so in this case you have the CRISPR repeat we have the viral the piece of the virus genome and the tracer RNA together and these are the whisper RNAs in type 2 type 3 is very simple so type 3 cast 9 homologue is going to chop up them and message or indirectly and we will end up with the quiz for RNA containing the CRISPR repeat and the piece of viral genome now let's move to the third step to Thursday the interference which is also different between the three types of CRISPR system but in general so there is like a general thing in general the crispr RNA will be integrated with the cap protein to end up with the conflicts containing the caps proteins with a piece of RNA inside which is decreased for RNA this is in general this is the interference the interference is between the cal specific tasks protein and RNA sequence they are going to Mir to be merged together to form this complex now the differences between the three types let's go to task one in fact one as I told you as you remember if you remember we have the CRISPR the CRISPR sequence is looped then then what's going to happen is that when this when the stage when the bacteriophage infects the cell and all the time so so as I told you in the bacteriophage infected this the cell the first time this beach was taken from from this state and now when this page comes another time to infect this bill this piece of RNA can recognize this piece of DNA because it's complimentary now you should know that there is a very very important important player in type 1 and type 2 which is this yellow sequence here it's called with Pam and Families protospace their adjacent motive what is the tablet let me tell you a little bit about the time because it's important scientists have found that when - like when the bacterial cells choose a piece of of the viral genome to take it it doesn't take just any piece of the viral genome it takes the piece of the viral genome which is adjacent to the Pam sequence so the the bacterial cells can recognize the Pam sequence and then it takes the adjacent seeker in order to add it into the space your DNA and then in order to form the crispr RNA and the CRISPR complex from it why why the time is important because the cast enzyme can recognize the time sequence so here we increase the specificity because not only the the RNA sequence can recognize the DNA sequence here but also the cast enzyme can recognize the time sequence here and in this case we are increasing the specificity of recognition tom is very important in type 1 and type 2 but not in type 3 so what's going to happen here is that the casts enzyme will recognize the pam sequence and then the RNA sequencer is going to recognize the adjacent sequence here which is exactly complementary to this sequence so it's going to happen if that be the viral genome will bind to the R and H like this - the complementary strand because this is complementary to this and then the viral genome will bind the lower strand was copied so the lower strand is complementary to this and then the lower strand will bind to the RNA sequence now when this happens now I'm speaking about type 1 when this binding happens this will activate a cascade of of cat enzyme so many gas enzymes will be activated and it's very complicated actually no one exactly knows the process until now but at the end this gas cascade will recruit we recruit Castries to chop up the viral genome like this so in type 1 gas 3 will chop up the or on will cut the viral genome to end up with a liquid degraded viral genome so the virus cannot invade the cell anymore this virus in us cannot infect the cell anymore this is platform in type 2 as I told you type 2 isn't the most important in fact to the main player is cast 9 so as I told you before the time sequence is also important in type 2 because cast 9 enzymes can recognize the time sequence and then this RNA sequence can recognize this DNA sequence and then they will bind together to form like as I told you the RNA is complementary to the lower strand and then they will bind together and then this cast enzyme itself will undergo something called the double strand break in the viral genome and what double strand break means is that the cast enzyme will break the two strands exactly at the same place it's called a double strand break the cast enzyme has two domains so this is what we end up with the viral genome will be chopped up the cap enzyme has two domains called the air HMC and the roof see they are like RNase H like in the nucleus domains and the cache enzyme can use these two domains in order to do this double strand break this is type 2 now going to type 3 type 3 is very simple also there is a CAF enzyme there is no pam here in fact 3 so the RNA sequence we recognize it's complimentary here on the viral genome and then they will bind together and there will be also a cascade of cache enzymes like type 1 and then the viral genome will be chopped up like this now if you want to summarize everything with Joe per system is a three-step system the first step is special acquisition in which the bacterial cells took a piece of the viral genome which invades the bacterial cell for the first time and if it is too great this sequence into the spacer DNA so we will have several pieces of different viruses into the spacer DNA's then in the crystal RNA processing this strand will be copied to the messenger RNA and then the messenger RNA will be chopped up to give the twist paralanguage contains a complementary to the piece of crystal speed and the piece of viral genome and in the third step the interference this crispr RNA will be merged with the cast enzyme to form this coke like and then when the viral when the same virus will invade the cell or will try to infect this cell another time this cast enzyme and the piece of RNA will recognize the complementary piece here and this will cause the chopped-up in the viral genome this is everything I wanted to tell you about Chris particular about CRISPR system just swept the end of the part one video and the part to begin I'm going to speak about the CRISPR caste 9 technique used used in in gene silencing gene engineering and so if you like you can go and watch part two if you liked this video please don't don't forget to Like share subscribe the channel if you have any questions you can write in the comments if you have any suggestions for for other topics you can also write in the comics comments and see you in the other bit in the next video bye

Channel: Biomedical and Biological Sciences

Views: 138,355

Rating: 4.9354224 out of 5

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Id: VtOZdThI6dM

Length: 19min 41sec (1181 seconds)

Published: Wed Mar 01 2017