CRISPR in the wild as immuno-defense

In the wild, bacteria need to protect themselves from bacteriophages, viruses that target bacteria. In order to due this, bacteria evolved a defense mechanism called CRISPR. CRISPR stands for clustered regularly interspaced short palindromic repeats. I will explain the significance of this name shortly. However, here, I will explain how it is used.

When a bacteria realizes it is being attacked by a virus, it sends out an enzyme complex. An enzyme complex is two or more enzymes (think of an enzyme as something does a chemical reaction). That enzyme complex is Cas1-Cas2. What it does, is look in the DNA that the virus is injecting into the bacteria, for any nitrogenous base followed by a sequence of Guanine Guanine (at least for S. Pyogenes). That nitrogenous base and Guanine Guanine, is called the protospacer adjacent motif (PAM). The protospacer is what the Cas1-Cas2 care about, and I will talk about it in a second. Adjacent means next to, and motif basically means repeating. The protospacer adjacent motif is a section of virus DNA, that is repeated in a lot of viruses, and is next to the protospacer. The protospacer is a section of virus DNA, typically ~20-26 nitrogenous bases long, which Cas1-Cas2 will cut out, and insert into the CRISPR array. The CRISPR array is a portion of bacterial DNA, which contains spacers, or the cut out portion of virus DNA, encapsulated by repeating sections called repeats. In the CRISPR array, we can see why it is called CRISPR. Cluster comes from all the different parts of the CRISPR array – the repeats and the spacers – are all together in one cluster. The regularly interspaced refers to the fact that each spacer is interspaced at an interval of one repeat per spacer. The palindromic repeat refers to the repeats containing palindromes, or stuff read the same in either direction. These palindromic portions of the DNA are used for bonding with a certain type of RNA later on.

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Every so often, RNA-polymerase will transcribe the entire CRISPR array into an RNA molecule, called pre-crRNA, or pre-CRISPRRNA. There is something called pre-tracrRNA, or pre-tracerRNA, which is transcribed from a different portion of the DNA. The pre-tracrRNA has portions which are complementary to the pre-crRNA, and will proceed to bond to the pre-crRNA, specifically to the repeats. Then, another enzyme, called RNAase (sick name), will cut in the repeat regions, resulting in a piece of RNA, that has a spacer, a portion of the repeat that is bonded to a tracer RNA via hydrogen bonds. This new RNA molecule is called cr:tracrRNA.

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This new cr:tracrRNA then bonds, and becomes part of a Cas9 enzyme. The Cas9 enzyme grabs and takes the cr:tractRNA. This new molecule, is called gRNA, or guide RNA. The Cas9 enzyme, while it is one long polypeptide, or protein, contains several different regions, each with different functions. The first region I want to cover, is the HNC/RuvC portions. They have an important role in the cell – actually cutting the DNA. They are what are called “nucleotide domains”, where domain just means region. The other main region, is the PI region, or PAM-interacting region. Its job is to identify, and bond to, a PAM, (protospacer adjacent motif). The majority of the rest of the gRNA is simply there in order to keep the guide RNA inside the Cas9 enzyme.

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When the Cas9 identifies any DNA, it looks for a PAM region. Upon finding the PAM region, it unzips the DNA upstream of that region. DNA is double stranded, and the two sides are “zipped” together, so the Cas9 unzips them, and goes and looks in the opposite strand of the PAM, for a region complementary to the gRNA. When Cas9 finds complementary bases, it cuts the DNA ~4 nitrogenous bases upstream of the PAM. The Cas9 then leaves. While this may not seem helpful for, say, gene-editing, which is why CRISPR-Cas9 is so famous, it is helpful for the bacteria’s use of CRISPR-Cas9, which is as an immune defense, in order to neutralize the virus.

sgRNA

There is another type of gRNA, which I will shortly cover. sgRNA stands for single guide RNA. It is synthetic, and is created by geneticists in a lab. It is designed as artificial gRNA, which does what the scientists want it to do. It is used in place of now gRNA.

Gene Editing – non homologous end joining (NHEJ)

Now that we have covered how CRISPR-Cas9 works in the wild in order to destroy DNA and to protect from viruses, we are going to cover how geneticists/scientists use CRISPR-Cas9 to modify genes. The first, is non homologous end joining. Non homologous end joining works by using CRISPR-Cas9 to break part of the cell. This break, will attract the enzyme Ku 70/80. Ku 70/80 in turn attracts DNA Protein Kinase Catalytic subunit (what a name), which in turn attracts a string of proteins XRCC4 and XLF, who IN TURN attract DNA ligase, which acts as a hot glue gun, and glues together the two ends. This is how knock-out genes are made. DNA ligase is very error prone, and as such, it tends to create genes which aren’t properly working, and as such, stop doing what they’re supposed to be doing. The gene is effectively knocked out liked a wrestler!

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Gene Editing – Homology Directed Repair (HDR)

Homology Directed Repair is another way that the cell can fix the DNA breakage caused by CRISPR-Cas9. When done using Homology Directed Repair, the fix tends to be of higher fidelity, with less errors, as the cell uses other DNA to help with that, hence the homology. The first part of Homology Directed Repair, is called Resection to Chi. In this part, the 5` end of both strands of the DNA is cut (aka resectioned) by the enzyme RecBCD. This cut goes on, until the RecBCD reaches something called a Chi Site. A Chi site is a portion of DNA containing nitrogenous bases in the format GCTGGTGG. This creates a sort of overhang. Then, some homologous DNA ends up near the 3` part of the DNA, and an enzyme called RecA will essentially drag the 3` down into that homologous DNA, finding a complementary part of it. DNA polymerase then extends the cut DNA until it reaches a chi site, at which point the attraction between both parts of the cut DNA is strong, and so the chi site part of the cut DNA attracts the other part, and they rebind. The other overhang is then fixed, by having DNA polymerase simply extend it using the already fixed part, until DNA ligase comes in and pastes it together, more accurately this time due to only one strand, not both, being broken. Now, you may already see how this repair mechanism can be hijacked by scientists in order to insert a foreign gene. What the scientists will do, is create some synthetic DNA, which is “capped” essentially by homologous DNA, so that the DNA is repaired using the synthetic DNA strand, thereby inserting scientist selected nitrogenous bases. [5]

_{[1] https://explorebiology.org/collections/genetics/crispr-cas9-a-new-tool-for-genome-editing\n[2] https://sites.tufts.edu/crispr/crispr-mechanism/\n[3] https://blog.addgene.org/components-of-crispr/cas9-our-new-crispr-101-ebook\n[4] https://en.wikipedia.org/wiki/Non-homologous_end_joining\n[5] https://sites.tufts.edu/crispr/genome-editing/homology-directed-repair/}