Genome Editing with CRISPR-Cas9

crisper cast 9 based genome editing uses the cast 9 nucleus for targeted DNA cleavage the cast 9 is an endonuclease technically cine nucleus is a ribonuclear protein it is made up of three components the cine protein itself the Tracer RNA and a crisper RNA crisper RNA is partially complimentary to the Tracer RNA the 20 nucleotides at the five Prime end of the crisper RNA make up the spacer or protospacer region of the crisper RNA all three of these components form this active cast 9 endonuclease typically in practice Tracer RNA and crisper RNA are combined through

a stem Loop structure to get a single RNA called single guide RNA when this was created researchers made a handful of variations and the plus 85 Tracer RNA tail structure worked the best so in literature you may see sgrna being referred to as the plus 85 regardless of technicality when speaking about about sgna the 20 nucleotides at the five Prime end of the sgna the spacer part is quite liberally also sometimes called the guide RNA that is the portion which guides C9 nucleas to make Cuts therefore C9 nucleus is an RNA guided DNA endonuclease the

guidance of C9 actually starts with the Pam interacting domain of the C9 protein which scans the target DNA for an NG sequence the NG sequence is a Pam short for protospacer adjacent Motif recall that the 20 nucleotide region is called the protospacer The Motif adjacent to it is therefore protospacer adjacent Motif an important point to remember here is that the protospacer RNA is part of the C9 r&p the Pam sequence on the other hand is only found in the Target DNA I'll come back to this in a moment the Pam scanning step is called Pam

verification which leads to the spacer or guide RNA sequence pairing with the target DNA and when it base pairs the nucleus domain of cast 9 cuts that paired DNA this is essentially the interference step of cast 9 mechanism that I discussed in crisper adaptive immunity video which is linked Below in case you want details all right let's get into the specifics of crisper cast 9 suppose this is the single guide RNA that is going to guide the cast 9 nucleus this implies that the matching Target DNA is expected to base pair with the guide RNA

something like this but before the matching can happen the NG sequence needs to be verified by the Pam interacting domain only after the Pam is verified the 20 nucleotide spacer is allowed to pair the pairing starts at the three prime end of the spacer and around 10 to 12 nucleotides in this region or even as little as six nucleotides called the seed region is where the cast 9 protein expects to find a perfect match the leftover portion is more flexible and a minor mismatch in their base pairing is tolerated this hybrid structure of RNA and

DNA is called the RNA Loop the DNA strand that pairs with the guide RNA is referred to as the target strand with this Logic the unpaired Strand is called The non-target Strand I want you to take away a very important point from this the Pam sequence the NG is always in the non-target Strand and is not part of the guide RNA so it does not base pair or engage in the r Loop formation okay so once the guide RNA pairs with the target strand C9 makes a cut on the target DNA three nucleotides Upstream of

the Pam the H&H nucleus domain strictly Cuts three nucleotides Upstream of NG keep in mind that guide RNA is never cleaved only the DNA is cleaved The non-target Strand is cut by the roofy domain of cast 9 also three nucleotides Upstream of NG but roof C is often times flexible in its cut position sometimes it can cut four bases Upstream sometimes five or even six in some instances this means that the double stranded DNA is cut on both strands and if both domains cut three bases Upstream of the Pam you get a blunt doubl stranded

brake in the Target DNA alternatively you can get a staggered cut if the roof seed decides to cut a little further Upstream one side note the NGC sequence is what cast 9 prefers as a Pam over all three base combinations instead of this canonical Pam sequence sometimes cast 9 can also use sequences like n or NGA and a couple more although with low efficiency but NG is always preferred if you understood all that here's a little exercise for you we discussed this target DNA and guide RNA configuration and where the Pam sequence is and the

location of the cut site there are other possible configuration for Target DNA and guide RNA just like this example can you identify the location of Pam sequence and cut sites in these configurations pause the video and try it out you will find the answer to this exercise later in the video hopefully by now you're convinced that C9 nucleas is essentially making a double stranded DNA break in the Target DNA in UK carots generally there are two major Pathways to repair double stranded brakes one of them is called non-homologous enjoining simply put through this mechanism a

cell can just paste the broken DNA back together this works if you have a blunt DNA cut and by this repair mechanism no error or mutation is generated this is called the classic nonhomologous enjoining mechanism alternatively you have a variation of NH EJ mechanism called microhomology mediated and joining where the ends of the broken DNA are reected this can convert blunt into a staggered end or a staggered ends can be filled in to make a blunt end depending on the choice the blunting and reection can delete some of the bases from the broken DNA this

will result in a small dedion in contrast if the cell decides to extend the broken staggered cut you may add extra bases to the broken ends generating a small insertion the insertion deletion resulting from this repair is therefore error prone nhj is simply an joining mechanism the other repair pathway called homology directed repair is not as simple in this pathway the homologous chromosome participates in the DNA repair it starts with a long range resection of the broken DNA end causing the non-resected Strand to recombine with the homologous chromosome through complementarity once the base pairing is

established with the homologous chromosome DNA polymerase starts DNA synthesis where the homologous chromosome is used as a template for the synthesis this results in the copying of information from the homologous chromosomes if the information that is copied and replaced is the same as the original information present then technically this repair mechanism is error free it is not just how the repair Pathways work but even the timing of their use is different the classic enjoining mechanism is active at all times in the cell cycle the rest of them are only active during the S and G2

phase no notice that the error-free repair using a template chromosome is statistically outcompeted by other repair Pathways throughout the cell cycle crisper cast 9 based genome editing relies on these dsb repair Pathways and depending on your application you are interested in outcomes from these specific Pathways here's a common list of applications when editing a genome using crisper cast 9 when trying to disrupt a specific location in a genome this could be an Exon a per motor or something else the expectation may be to create some random indels for this you're using a single cast 9

complex to make the cut and rely on nhj mechanism to randomly make indels alternatively your aim may be to delete a sizable part of the genome to achieve this you use two different cast n complexes each targeted at a desired location to cut the genome an nhj mechanism again kicks in to liate the broken ends together and hopefully the cutout portion of the genome is not religated back a fairly common application of crisper cast 9 is to perform a targeted insertion for targeted insertion you have to provide a repair template the repair template has regions

that are identical to the flanking DNA where the cut is made these are called homology regions in between the homology region is the piece of DNA that you're trying to insert HRS are typically designed to be in 500 to th000 B Bas pair range this synthetic template now has to compete with the homologous chromosome and get used as a template in the HDR pathway when the HDR pathway is complete the broken genome ends up containing the new information copied from the synthetic repair template conceptually this may seem simple but there is a practical tedium to

crisper cast 9 generally a crisper Castine experiment is done on a culture of cells for deployed ukar cells Maman cells for instance it is possible that one cell gets a cut in only one chromosome giving you a heterozygous cast 9 editing if it goes through the other cell may be cut on the other homologue and you obtain a hat for a different chromosome in some rare cases you cleave both the chromosomes and your expected DSP repair goes through and you obtain a homozygously edited cell population given these three application and many possible outcomes your job

may get complicated if you're trying to obtain a specific clonal population for a specific alal or maybe both to make a permanent cell line out of your crisper experiment