The CRISPR-Cas9 Trilogy, CRISPR 101 Part I: What… is it… even?

If you’re a biology nerd, like, the kind who attends the Friday afternoon seminar series for no extra credit and schedules your classes around PCR runs, you have heard of the relatively new CRISPR-Cas9 system. You know it is very important and powerful, and that it has the potential to save mankind from our own genome. That’s obvious, especially for biology nerds. Further, as a biology nerd, you know that because of its importance, it will go down as one of the few developments in basic science that makes the leap from the knowledge quorum of biology nerds to public consciousness.

But even most biology nerds don’t know how it works, or to what extent it can be a cure-all for genetic maladies. From my experience, the CRISPR-Cas9 system was introduced, and continues to be discussed, in hushed, reverent tones. This further drives home to me that it is something very important and powerful. It is presented as something I should understand, as a proper biology nerd.

Well, a few things don’t add up: 1) If I am one thing, I am a biology nerd. But I don’t know how it works. Pretending I do is easy though, because when discussing CRISPR-Cas9 with my gaggle of biology nerd friends, upon deeper inquiry, it becomes apparent that no one else has really done their homework on this either. Often, we’ll just describe and revere it in very vague, idealistic terms and get excited. 2) Even though people tell me it has the potential for incredible things, that’s all I know that it can do. Incredible things… what are these incredible things? Beats me. 3) Nobody aside from biology nerds is talking about it. And when these nerds do talk about it, the consensus is that it is huge and will change everything, like, tomorrow. But this CRISPR system has been up and running for a few years now, and judging by the gravitas surrounding it, one would predict that the system should have rocked everyone’s world by now. Where is it? Who is using it?

Well, I have had enough with these discrepancies. Recently, I tugged at the shroud of mystery surrounding CRISPR (googled) and the subject of an epic Alma Matter post took shape before me. Nay, multiple Alma Matter posts. This will be a CRISPR saga, or at least a two-parter. This first post will be technical, the next, controversial, and the final, ethical. I give you: CRISPR 101. And by “you” I mean everyone, not just biology nerds.

1. This is how CRISPR-Cas9 works, but to understand it entirely we’ll start with a crash course on how genomes work, generally:

This CRISPR-Cas9  system operates on our genome, the blueprints for life. This design is encoded in a language of four letters, which all stand for the different, interchangeable pieces that make up our DNA. These letters are A, T, G, and C. These letters are strung together in multiple long, long, long run-on sentences. These sentences are coiled into chromosomes, and humans have 23 of them. All of these chromosomes hang out in the middle of each cell, or the basic unit of all of our body parts (blood, guts, skin, brain). Stuff in our cells read the genome, and make more body parts, simple as that.

But there can be typos in those sentences. This makes sense because they are ridiculously long so the chances of one being “misread” or “mis-transcribed” aren’t high, per-se, but high enough. So when the stuff in our cells read the wrong instructions, they don’t make our body parts quite right. These typos are specific, and often the same typos occur in different individuals, and you’re familiar with them, they include: cancer, Huntington’s, multiple sclerosis, sickle-cell anemia, cystic fibrosis… you get the idea.

So, with this understanding of basic genomics, and to exhaust the aforementioned analogy, CRISPR-Cas9 is exciting all of the biology nerds because it has the potential to autocorrect these typos. “Seek and destroy” is the typical way to describe its method of action when handling typos in our genome

2. So how does it autocorrect?

• CRISPR-Cas9. Let’s break down this weird moniker (usually biology nerds are so good at naming things, i.e. the sonic hedgehog gene, the tdTomato or mCherry fluorescent proteins… This is a lapse in creativity.). This name has two parts, because it is a two-part system. There is a part that is made of genomic material (the letters, A, T, C, and G), and a part that snips out the typo. CRISPR refers to the genomic part. Cas9 is the snipper. CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeat, but that really isn’t helpful at all to understanding the mechanism, so I’ll make a footnote for it if you’re still curious.

• CRISPR: This genomic portion of the system refers to the way in which genes of interest are “flagged” for editing. Again, going back to the typo analogy: when one misspells a word in a word processor, a little red line usually occurs underneath it. The acronym CRISPR describes the “little red lines” made of genomic material (A, T, G,and C) that appears with the genes that need to be edited. [A little more genome 101 is necessary: when genes are expressed, only specific pieces of the genome are copied to express specific genes. Picture them as words lifted from the huge run-on sentence that is our genome. These pieces (the “words” figuratively, but known as mRNA, literally) interact with other parts of the cell to become expressed (or put another way: used to make our body parts)]. When genes are marked with CRISPR’s “little red lines,” those “little red lines” are copied with the gene when it is transcribed into mRNA before it is expressed.

1. Cas9: This is a molecular machine, called an enzyme, that exists independently of the genome, and essentially “scans” its area for the “lifted words” (transcribed DNA aka mRNA) that have the “little red lines” (CRISPRs) associated with it.

   Search and destroy: much like how police sniffer dogs are given a scent to track down, the Cas9 enzyme grabs the CRISPR’d (little red lined) mRNA (aka crRNA), hangs onto it, and whenever it encounters another string of genomic material that matches its crRNA sequence, Cas9 snips it out and destroys it, and as a result, that particular gene is rendered totally ineffective.

3. Hijacking:

Scientists observed that the CRISPR system occurs naturally in bacteria as a weapon against repeated viral infection. The Cas9 enzyme in this case is “assigned” viral DNA so that upon viral entry, Cas9 chomps up any invading viral DNA so the bacteria does not get infected. The invader’s DNA is then inserted into the bacteria’s genome, flagged with the “little red lines” (CRISPRs) for future reference. The scientists who identified this found it to be a pretty sweet system, especially if you could use it to CRISPR out the faulty genes that cause cancer or Huntington’s or Tay-Sach’s disease by programming a Cas9 enzyme to “seek and destroy” the genomic culprit that causes each disease. So, the team who discovered this system, and outlined the possible implementation in humans, led by Jennifer Doudna of UC Berkeley and Emmanuelle Charpentier of Umea University in Sweden, published their findings in June 2012. As a result, labs all over the world set out to meet the challenge of successfully executing CRISPR-Cas9 in plants and animals.

Patent wars (of course): The original discoverer, Duodna, accomplished this in January 2013, but these results were published a mere four weeks after two other papers were published simultaneously, reporting the same feat. These two other teams include George Church of Harvard and Feng Zhang of MIT.

The ensuing battle for ownership of this idea is the topic of part II of the Alma Matter’s CRISPR saga.