CRISPR: the basics you need to know

CRISPR headlines make the news every day, served with a side order of alphabet soup that can make your head spin. To say the acronyms and initialisms are off-putting is an understatement. However, CRISPR technology is here to stay and it will change our lives forever, so a basic understanding is worth the effort.

At its core, CRISPR is a gene-editing tool. A gene is a string of genetic material, usually DNA, which resides at a specific spot on a chromosome and has a particular function. The instructions carried by the gene direct the formation of proteins or RNA molecules. Taken together, all the genes of an organism make up its genome. The genome of the honey bee, Apis mellifera, comprises roughly 10,000 genes, while the human genome sports approximately 20,000 to 25,000 genes.

In effect, editing a gene is not much different from editing an ABJ article. The editor reads through the strings of letters and changes the ones he doesn’t like. For example, I always write “further” and my editor (name withheld for privacy) always changes it to “farther,” a correction that improves the quality of my writing. Farthermore, it makes me sound smarter than I am.

Similarly, gene editing is a process in which the DNA or RNA can be modified to change its characteristics. Sequences can be altered, added, or deleted, depending on what the researcher is trying to do. Gene editing has been around since the 1970s, but the early techniques were time-consuming, costly, and often produced random and unpredictable results. But CRISPR technology, first developed about 15 years ago, revolutionized our ability to reliably edit the genes of nearly any organism.

Gene edits in the wild

CRISPR, pronounced like the plastic box in which you keep lettuce, is an acronym for Clustered Regularly Interspaced Short Palindromic Repeats. Crazy, right? Who would think of that? But oddly, the name helps explain how CRISPR was discovered back in 1987, and why it exists. Bear with me here because the story is cool in a nerdy sort of way.

CRISPR evolved naturally in some species of bacteria and archaea so they could protect themselves from invasion by viruses (called bacteriophages). Yes, you read that right. Bacteria and archaea get viruses just like we do, so they needed a way of coping. Since they don’t have complex immune systems, the bacteria and archaea devised a method of dealing with the intruders by editing their own genomes.

A short alphabet

As you know, the genes of living organisms are built with just four types of bases: adenine (A), cytosine (C), guanine (G), and thymine (T). The order in which the bases are strung together dictates the recipe for building a protein. But with only four “letters” in the alphabet, the words can be long and ungainly.

Early geneticists, especially those working with bacteria, sometimes noticed strange sequences of bases that formed palindromes. In case you forgot, a palindrome is a sequence of letters or numbers that read the same forward and backward. The ultimate palindrome appeared on your calendar earlier this year on 02/02/2020. If you ignore the punctuation, that date reads the same whether you start from the front or the back. Single words can be palindromes, too. Radar, kayak, racecar, and civic are perfect palindromes.

The discovery of palindromes in genetic code was odd, but even more perplexing was the code the scientists found sandwiched between the palindromes. For example, let’s say you found a sequence of the four bases that read a-c-c-t-a-g-t-a-g-c and then, a little farther along the chain, you found its palindrome: c-g-a-t-g-a-t-c-c-a. All well and weird, but between the two was more code, let’s say g-c-a-t-g-g-c-t.

All strung together, you would see c-g-a-t-g-a-t-c-c-a-g-c-a-t-g-g-c-t-a-c-c-t-a-g-t-a-g-c. Not knowing the meaning of any of it, some early researchers named the end pieces — wait for it — “clustered regularly interspaced short palindromic repeats (CRISPR),” while others called them bookends. The bookend analogy works well since they are mirror images bracketing some stuff in the middle.

The pieces in the middle didn’t seem to belong, so the researches called them spacers. The term “spacer” makes them seem insignificant, but it turns out that the most important part of the entire sequence — the “meat,” if you will — is actually the spacer. We’re getting to that.

Some enterprising researchers ran the middle pieces — the spacers — against enormous databases of genetic code. It took a while, but they found strings of similar code in the genomes of some viruses. But what was virus code doing inside bacterial DNA? And why was it offset by brackets of genetic palindromes? Even Hercule Poirot would be mystified.

Spoils of war

A battle between a bacterium and a virus is like a duel. First, a hapless bacterium gets infected with a virus. Not good, as we know. But by engaging its primitive immune system, the bacterium puts up a fight. During the fray, the bacterium excises a piece of the virus’s genetic code — Slash, slash. Take that, you vicious viral varmint! — which the bacterium copies and stores in its own DNA for future reference.

The stolen code is like a password. If the bacterium survives the viral attack, this stored password will allow the bacterium to recognize the same virus in the future and “remember” how to fight it. The one problem is how will the bacterium know which piece of code is relevant? Since code comes in strings, how can the bacterium know where the password starts and stops? Well, duh! It stores the code between bookends — two palindromic sequences that can be readily recognized.

Coded for life

<p>When you write a paragraph in HTML, the language of most websites, you start off with a code that means “the paragraph starts here.” Then, at the end of your paragraph, you use a similar code that includes a leading slash, meaning “the paragraph ends here.”</p>

CRISPR does the same thing. The palindromes mark the beginning and the end of the stolen password that resides in the middle, and the password identifies the particular virus that the bacteria may have to fight in the future. And since the bacteria — single-celled organisms — store the password within their genetic code, the information gets passed into future generations of baby bacteria.

Over time, strains of bacteria acquire defenses against many types of virus. Within the DNA of these bacteria, long strings of alternating bookends and passcodes are called CRISPR arrays. As soon as a virus strikes, the CRISPR section of the DNA unwinds so the bacteria can copy the array into RNA segments. The new RNA segment is much like a big keyring. The RNA floats around inside the cell, trying key after key, looking for the one that matches the virus. If the bacteria is lucky and recognizes the attacker, it can then destroy it. But how does that work?

Crispr’s little helpers

You will often see CRISPR written with an additional notation, such as CRISPR/Cas9. In this example, the add-on is shorthand for CRISPR-associated protein 9, although there are many others. Cas9 is an enzyme that attaches to both ends of the CRISPR segment. Once the bacterium recognizes the virus, it uses the Cas9 like a pair of tin snips — snip snip — clipping that segment of DNA right out of the virus, both strings of it. With luck, the virus is toast.

Adapting the system

This miraculously efficient system of gene editing is at work every single day in the natural world. While humans once believed gene-editing resulted from higher-level thinking, things we can’t even see have been doing it for eons without thinking at all, a humbling thought. But as soon as scientists realized what was happening inside a lowly bacterium, the question arose: “How can we use the CRISPR system of passwords and tin snips to do our own editing?” It turns out to be a piece of cake, at least compared to prior methods of gene editing, which is both good news and bad.

After we inject a stretch of CRISPR array into a cell along with a pair of snips such as Cas9, the RNA floats around until it finds the part that matches and then cuts it away. Finding the right part seems impossible considering how much code is in the DNA, but like Google, it finds the string it’s hunting without delay.

Incredibly, if we provide some alternative code and inject it along with the CRISPR strings, the cell may use it as a patch to fix the broken DNA string: “Oh! Look what I found!” says the cell. “Let’s mend the DNA with this.” And sure enough, it often works, allowing unwanted strings of code to be replaced with ones we select. In fact, researchers have refined the system to the point where they can delete, edit, or replace strings of code, or even single letters, with amazing speed and accuracy.

Since a bacterium is a single-celled organism, any DNA changes made in the nucleus will be passed along when the cell divides. But in complex multi-celled organisms, you would have to change every cell separately — a job more boring than scraping mountains of brood frames. The workaround is editing the organism in the fertilized-egg stage so that every subsequent cell division contains the edited DNA. Alternatively, if we edit stem cells, the changes will affect only parts of an organism.

Examples from the field

Most likely, CRISPR technology will revolutionize the world of agriculture — including beekeeping. Whether you agree or disagree with the ethics surrounding gene editing, the science is evolving at a startling rate.

One of the early successes occurred with the development of non-browning button mushrooms (Agaricus bisporus) at Penn State University. After discovering that browning was regulated by several genes, the scientists used CRISPR/Cas9 to remove just a few base pairs and produce the mushrooms. The snow-white fungus is valuable because of its appearance and its unusually long shelf life.

However, the mushrooms became controversial when the USDA ruled in April 2016,that they would not be regulated — and therefore not labeled — as a genetically-modified organism (GMO). The department based the decision on the fact that the mushrooms do not contain DNA from other organisms, so they did not fall under regulatory authority.

Before CRISPR, genetic modification was typically achieved by incorporating gene sequences from one living thing into another, which is why you saw photos of glow-in-the-dark cats. Green iridescent felines resulted from incorporating a gene produced naturally in some jellyfish. The researchers used the glowing biomarker to track changes within a cat’s body. Another example of a trans-specific organism is “golden rice,” which received genes from a daffodil (Narcissus pseudonarcissus) and a bacterium (Erwinia uredovora) that enabled the rice to produce high levels of beta-carotene.

Regulators are considering updating the rules as technology progresses. At this writing, however, simple edits that do not incorporate foreign genetic material, and knockouts, which completely delete a gene, do not fit the GMO definition.

Excessive snipping

One of the problems with CRISPR can be overzealous snipping by Cas enzymes. Sometimes, the enzymes snip a little more than they should, which may or may not be important in the life of the organism. However, it is one of the big fears when considering human-genome edits. What if something important is snipped away? What if the damage isn’t discovered until adulthood or until the next generation? Or what if we change a human in a not-so-human way? Until we can answer such questions, CRISPR gene editing will most likely be limited to organisms that can be destroyed if things go awry.

Crispr and honey bees

Some honey bee researchers dream of the day when we can simply delete the genes that make honey bees susceptible to foulbrood, or insert genes that neutralize deformed wing virus, or make fat bodies poisonous to varroa mites. If genetic strings could be found — or manufactured — to do this, CRISPR could most likely deliver them to the right place in the honey bee genome. But for a number of reasons, honey bees are particularly difficult to work with.

Probably the biggest problem is that honey bees don’t do well in confinement. They are designed to inhabit open spaces with acres of forage and plenty of room to roam. In confinement, colonies languish and perform poorly, making them hard to study.

The problem, of course, is you can’t edit the genome of a line of bees and set them loose in the environment. If something went wrong, if the editing process induced a harmful genetic sequence, you wouldn’t be able to backtrack and call them all home. Whatever you unleash into the population would stay there, free to replicate in the wild, and the results could be catastrophic. The discovery of how to simultaneously confine and field test honey bees is lagging far behind the science of how to change them.

The future of crispr

At the moment, CRISPR technology is evolving at breathtaking speed. New and more accurate clipping enzymes are being tested and unique methods of delivering designer pieces of code are under development. Enhanced techniques for assuring more precise cuts that reduce over-clipping and other uncertainties will soon be standard in labs everywhere. In the meantime, deep thinking about what we should or should not do with such power should be a worldwide priority.

Rusty
Honey Bee Suite