I'm not gonna lie, very late. This is also not it all. There will be more on CRISPR later.
CRISPR is an acronym for Clustered Regularly Interspaced Short Palindromic Repeat. CRISPR refers to somewhat palindromic sequences of DNA in microorganisms that are essential in their immune systems. How they work is by cutting up any intruding viruses. They can detect what to kill through stored memories of the genomes of viruses, stored in areas of CRISPRs known as spacers. If the virus genome doesn’t exist as a spacer already, it becomes one.
There are essentially three steps, then, to the CRISPR immune system:
- New virus adaptation: the creation of a new spacer to ‘adapt’ to a new virus or cellular invader.
- Production of CRISPR RNAs: the copying of the spacers from DNA into RNA, single chain versus the typical double chain DNA
- RNA guided virus fighting: CRISPR RNAs guide bacterial tools to seek and kill viruses.
That’s all fine and dandy for bacteria, but what’s in it for us? Why’s it become such a controversy lately?
Here are some applications of CRISPR:
Industrial: CRISPR was originally discovered in the bacteria Streptococcus thermophilus by the company Danisco. It was discovered that S. thermophilus was equipped with CRISPR sequences allowing it to be immune to certain viral/bacterial attacks. This could mean that cell cultures could be engineered with resistance.
Lab: CRISPR can be used to modify the genes of an organism by using cRNA, a process described in detail below.
Medical: CRISPR can be used as a medicine to target specific viruses or bacteria. It can also be a one use treatment for genetic or hereditary disorder.
WARNING: Technical portion coming up, some information may be inaccurate. If you wish to skip this, scroll down to the TL;DR for a summary.
CRISPR has already been used to revert a condition in mice, FAH deficiency, which also occurs in humans in the same mutation. This FAH is an enzyme, fullform fumarylacetoacetate hydrolase, that is the fifth in a series of five enzymes that breaks down the amino acid tyrosine. Tyrosine is common in many foods, and one of its byproducts known as fumarylacetoacetate is broken down in the FAH enzyme into acetoacetate and fumarate, through hydrolysis.
This behaviour is regulated through the FAH gene, and when one of 86 identified (and possibly even more, unknown ones) mutations occur, tyrosinemia type I can occur. The altered gene produces a faulty enzyme, either unstable or inactive, leading to minimal FAH enzyme activity. The most common one disrupts enzyme production instructions, leading to shorter enzymes.
Without enough FAH activity, a buildup of fumarylacetoacetate occurs in the liver and kidneys, of which high enough levels are thought to be toxic.
Meanwhile, tyrosinemia type 1 leads to generalized aminoaciduria in most cases (abnormal levels of amino acids in urine), hepatic failure in some, as well as other symptoms. But how does this relate to the mice in the experiment?
It doesn’t much, but you know, at least you learned something new. That is the disease the mice being experimented on had and was the one being treated.
In order for CRISPR Cas-9 nuclease enabled genome editing to work as intended, there has to be a guide RNA to tell the system what to target. The researchers did this by cloning three segments of mutated FAH genes that caused tyrosinemia type 1, labeled FAH 1, 2, and 3.
This, along with three other different injections, were prepared and injected into the mice:
- Saline
- A ssDNA oligo (single-stranded DNA oligonucleotide)
- ssDNA oligo plus pX330 that had an empty, unguided Cas9 (no sgRNA)
- The mentioned one, ssDNA oligo plus pX330 with Cas9 and a sgRNA(FAH1-3)
(pX330 is a plasmid that is also the root of a gene promoter, something that begins transcription of a particular gene. Couldn’t find much info on this matter, and I’m no biologist either. Oh, and saline is simply a mixture of sodium chloride in water. Kind of like a placebo, but with actual uses.)
SO, there were four sets of mice, and they all exhibited different effects. Mice with injection 1., 2., and 3. all lost 20% of their body weight and had to be culled (ah, the horror!). These were all kept without NTBC containing water, NTBC being 2-(2-nitro-4-trifluoromethylbenzoyl)- 1,3-cyclohexanedione. This is something that preserves and protects the liver from acute injury.
Mice with FAH-2 didn’t lose weight, and in mice with FAH-1 or -3 there was a weight loss of less than 20 after 30 days. This was also without NTBC water. They then were put on NTBC water for 7 days, and then on withdrawal for 28 days. Upon doing so, they regained the weight they initially lost.
Those with FAH-2 treatment had ‘substantially’ less liver damage than untreated mice, shown through liver histology.
TL;DR: CRISPR works, reduced liver damage in mice.
That technical bit should have given you a decent idea on how CRISPR Cas9 enabled genetic editing works. Here’s a better, concise description:
- sgRNA binds to the matching strand of DNA.
- Cas9 enzyme in turn binds to the sgRNA
- Cas9 cut both strands of DNA but not sgRNA
- Done. The cut is attempted to be repaired with a mutation. Then, you can insert desired genetic code.
That's basically the CRISPR immune system, and the gene editing it can result in explained in simple terms.
WORKS CITED
