A introduction to CRISPR
CRISPR Cas 9 is a gene editing system, and extensively used in neuroscience. Therefore, I felt it was appropriate to discuss how it works in detail, as we often see this technique used in various applications. As a broad overview, CRISPR-Cas9 works by cutting out a specific segment of the DNA, and then using the target cell's own repair mechanism to insert a new gene into the place of the recently excised gene. The CRISPR RNA is found in bacteria and archaea, and is used to excise bacteriophage DNA from the DNA of the virus.
More specifically, however, CRISPR-Cas9 works as such. First an appropriate CRISPR-Cas9 is constructed using the following components: crRNA, tracrRNA, and a Cas9. The macromolecules are then packaged into a plasmid that is later transfected into the target cell. Here crRNA is a CRISPR RNA. crRNA’s job is to cut a corresponding part of the target cell’s DNA. This break will be the new insertion point of the replacement DNA. The tracrRNA is a trans-activating CRISPR RNA. This particular piece of RNA acts as a ribozyme, or a RNA like enzyme, that is responsible for helping to cut the DNA strand that the crRNA has attached to. However, the tracrRNA also needs to be together in a complex with the Cas9 protein. This Enzyme is responsible for cutting the DNA in either a double stranded cut, or a single stranded nick. Either way, it is the Cas9+tracrRNA that cuts the targeted DNA. Another important part of the CRISPR-Cas9 system is that a combination of crRNA and traRNA, is called a single-guide sgRNA.
What makes CRISPR so special is that the crRNA is arbitrary, since the tracrRNA + Cas9 does the cut. Therefore it is very simple to synthesize your own crRNA strand. This allows one to choose exactly where in a DNA strand to cut. In order to create a crRNA strand all one needs is the RNA of the area one wants to cut. However, one constraint will need to be placed on the crRNA, as it must have a PAM or protospacer adjacent motif. This motif is often just 2 to 6 base pairs long. However, with base pair sequences that short, there are often many instances of this existing in the target DNA. All one needs to do is ensure that the target DNA contains that sequence at the end of the sequence.
From this, all of these components are constructed into a crRNA. This plus the RNA for the Cas9 protein are loaded into a plasmid. Then via Electroportation, the plasmid can be put into the target cell. From there the target cells' ribosomes will synthesize the Cas9 enzyme, which will then in turn form a complex with the sgRNA, and the target DNA. From here, the target gene will be cut.
The last step is to insert the gene that one wants to insert into the cut. This is just a matter of using Electroportation to insert the DNA of the gene you want to insert into the cut DNA. If you have constructed the inserted DNA correctly, such that either side of the gene matches the DNA on each side of the cut, the cell’s own repair mechanisms will insert this gene into where the cut occurred.
More specifically, however, CRISPR-Cas9 works as such. First an appropriate CRISPR-Cas9 is constructed using the following components: crRNA, tracrRNA, and a Cas9. The macromolecules are then packaged into a plasmid that is later transfected into the target cell. Here crRNA is a CRISPR RNA. crRNA’s job is to cut a corresponding part of the target cell’s DNA. This break will be the new insertion point of the replacement DNA. The tracrRNA is a trans-activating CRISPR RNA. This particular piece of RNA acts as a ribozyme, or a RNA like enzyme, that is responsible for helping to cut the DNA strand that the crRNA has attached to. However, the tracrRNA also needs to be together in a complex with the Cas9 protein. This Enzyme is responsible for cutting the DNA in either a double stranded cut, or a single stranded nick. Either way, it is the Cas9+tracrRNA that cuts the targeted DNA. Another important part of the CRISPR-Cas9 system is that a combination of crRNA and traRNA, is called a single-guide sgRNA.
What makes CRISPR so special is that the crRNA is arbitrary, since the tracrRNA + Cas9 does the cut. Therefore it is very simple to synthesize your own crRNA strand. This allows one to choose exactly where in a DNA strand to cut. In order to create a crRNA strand all one needs is the RNA of the area one wants to cut. However, one constraint will need to be placed on the crRNA, as it must have a PAM or protospacer adjacent motif. This motif is often just 2 to 6 base pairs long. However, with base pair sequences that short, there are often many instances of this existing in the target DNA. All one needs to do is ensure that the target DNA contains that sequence at the end of the sequence.
From this, all of these components are constructed into a crRNA. This plus the RNA for the Cas9 protein are loaded into a plasmid. Then via Electroportation, the plasmid can be put into the target cell. From there the target cells' ribosomes will synthesize the Cas9 enzyme, which will then in turn form a complex with the sgRNA, and the target DNA. From here, the target gene will be cut.
The last step is to insert the gene that one wants to insert into the cut. This is just a matter of using Electroportation to insert the DNA of the gene you want to insert into the cut DNA. If you have constructed the inserted DNA correctly, such that either side of the gene matches the DNA on each side of the cut, the cell’s own repair mechanisms will insert this gene into where the cut occurred.
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