These days you can not talk about molecular biology or genetics without hearing the word CRISPR (Pronounced crisp-er, as in [spoiler alert] “The Lannister army was much crisper after its run in with Daenerys Targaryen.”) Anyway… The recent breakthrough in genetic engineering has the media and scientific community buzzing. Many are hopeful that CRISPR is a panacea of sorts, the first step towards a cure for HIV, an effective treatment for cancer, the eradication of malaria carrying mosquitos, the enhancement of human abilities, the production of reliable crops, and more. Others are weary of the unforeseen consequences of CRISPR’s unbridled potential. But what exactly is CRISPR? For something that makes such massive waves, there are surprisingly few coherent explanations of the genetic tool. We will frequently be discussing the uses and ethics of CRISPR here at the Science Distillery, so in this preliminary article we aim to provide an easily digestible breakdown of the technology for readers of varying scientific backgrounds. CRISPR has the potential to change the world for everyone, so let’s all take a second to understand the technology behind the genetic revolution.

 

CRISPR-Cas9 Overview

First of all, when people talk about CRISPR, they are almost always talking about CRISPR-Cas9 (the difference will be explained shortly). You can think of CRISPR-Cas9 as an extremely precise pair of molecular scissors that targets and cuts DNA at specific locations. If scientists so choose, this molecular tool can also be used to splice new segments of DNA into the cut thus editing the DNA. By cutting and/or changing segments DNA, scientists can study gene function and potential avenues for curing genetic diseases through gene editing. Genetic modification is by no means a new avenue of research. The field has just been revolutionized by CRISPR-Cas9’s incredible specificity (scientists can cut and insert new segments of DNA at extremely specific, pre-determined locations within large genomes), its ease of use, and relative cheapness compared to other DNA editing technologies.

In the following article we will first discuss the origins and mechanism of CRISPR-Cas9, and then explain how CRISPR-Cas9 has been modified for use in biotech research.

 

Bacterial Origins

Like many useful tools in biotechnology, CRISPR-Cas9 was first discovered in bacteria. In nature, variations of CRISPR-Cas9 act as an immune system for bacteria against bacteriophages (viruses that attack bacteria). When a bacteriophage attacks a bacterium, it injects its DNA into the bacterium. If the bacteriophage is successful, that DNA is incorporated into the bacterium’s genome. Remember that the genome, which is the sum total of DNA in an organism, is the instruction manual for making and maintaining that organism. When the bacteriophage DNA is inserted into the bacterial genome, the new DNA starts giving the bacterium new instructions. Instead of doing all the things a bacterial cell needs to do in order to be a healthy, happy bacterium, the bacterium starts building new bacteriophages. The bacterium is destroyed when the new bacteriophages burst forth into the world (sort of like how new aliens are born in the Alien movie) In other words, by inserting its DNA into a bacterium, the bacteriophage hijacks the bacterium’s machinery to carry out its own reproductive goals.  

viral attack

Variations of CRISPR-Cas9 are used by bacteria to defend themselves against such viral invasions. Once the bacteriophage DNA is injected into a bacterium, the bacterium produces CRISPR-Cas9. This molecular scissor identifies the foreign DNA and cuts it. Once cut, the bacteriophage DNA is inactive and can no longer hijack the bacterium.

Ok, thats cool. But HOW does CRISPR-Cas9 actually work, and what is CRISPR-Cas9 exactly?
What is CRISPR-Cas9?

Let’s start by breaking down the acronym. CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats.

Short Palindromic Repeats” refers to short segments of bacterial DNA (about 20-40 base pairs in length) that are palindromic. A palindrome is a sequence of letters that read the same in both directions. For example, “race car” still spells race car if you read it forwards or backwards. So, a palindromic segment of DNA is one whose nucleotides (adenine, guanine, thymine, cytosine) read the same in both directions.

Palindrome

In CRISPR, these identical palindromic sequences are repeated, hence the name “Short Palindromic Repeats”

CRISPR

The first half of the CRISPR acronym, “Clustered Regularly Interspaced” refers to the fact that the palindromic repeats are found clustered in one area of the bacterial genome and that the repeats are spaced apart at regular intervals. The scientists who were first studying CRISPR in the 1980’s and 1990’s were most fascinated with the DNA that filled the spaces between the palindromic repeats. These segments of DNA, called spacer DNA, were all unique and they matched perfectly to DNA found in bacteriophages (the viruses that attack bacteria).

CRISPR + spacer

Scientists also discovered other genes that are associated with the CRISPR DNA on the bacterial genome. These CRISPR associated genes, or Cas genes, provide the genetic instructions for building Cas proteins. Cas proteins are usually comprised of helicase enzymes (proteins that unwind DNA) and nuclease enzymes (proteins that cut DNA). Cas9 is just one type of Cas protein.

 

CRISPR-Cas9

CRISPR-Cas9 at work

When a bacteriophage attacks a bacterium and injects its DNA, the bacterium builds the CRISPR-Cas9 complex to fight back. The Cas gene is translated into a Cas protein with the ability to unwind and cut the invader DNA, and the CRISPR DNA is transcripted into segments of guide RNA. Each segment of guide RNA (gRNA) is the exact complement to one segment of spacer DNA plus one palindromic repeat. The Cas protein and gRNA combine to form the CRISPR-Cas9 complex.

making CRISPR-Cas9

Because each segment of gRNA is the exact complement to a segment spacer DNA plus a palindromic repeat, and, as you recall, segments of spacer DNA match exactly to segments of DNA found in bacteriophages, it follows that gRNA also contains exact complements for segments of DNA found in bacteriophages. When the bacteriophage injects its DNA into a bacterium, the CRISPR-Cas9 complex recognizes the invader DNA because the gRNA matches perfectly a segment of the viral DNA. The Cas9 protein unwinds the viral DNA (helicase function), and the gRNA binds to its complementary segment of viral DNA. The Cas9 protein then cuts the viral DNA (nuclease function), deactivating it.

virus attack

New bacteriophage and no matching guide RNA

Sometimes a bacteriophage will attack a bacterium, and the bacterium will not have a segment of spacer DNA that matches that exact virus’s genome. In this case, the bacterium will produce a different class of Cas protein that will break apart the viral DNA at random locations and then copy  a segment of the new bacteriophage DNA into the bacterial genome as a new CRISPR spacer. If that same bacteriophage ever attacks again, the bacterium will produce a CRISPR-Cas9 complex using the new spacer DNA to specifically identify and destroy the viral DNA. When the bacterium divides (reproduces), all of its daughter cells will also carry a copy of the new bacteriophage DNA spacer. These daughter cells will be immune to the bacteriophage without ever having been exposed to it before.

new virus

Harnessing CRISPR-Cas9 as a revolutionary biotechnology

CRISPR-Cas9 is cool enough just as a mechanism for a bacterial immune system, but then scientists found a way to harness it as a tool for genetic manipulation.

Recall that the guide RNA that identifies and binds to a specific segment of bacteriophage DNA is derived from a spacer DNA segment (which matches a segment of bacteriophage DNA) and a palindromic repeat. Scientists realized that they could engineer/ build/ write their own spacer DNA segments that could match any sequence of DNA they want. For example, let’s say a researcher wants to determine if a certain sequence of DNA found in mice is part of a gene that plays a role in pigment production. The researcher could construct a segment of spacer DNA that matches exactly to the segment of mouse DNA in question. The CRISPR-Cas9 DNA (containing the engineered spacer segment) can be introduced into the cells of a mouse embryo. The CRISPR-Cas9 complex with the engineered gRNA will be constructed inside the cells and then precisely locate and cut the segment of mouse DNA in question. The mouse’s internal DNA repair mechanisms will try to fix the cut, but will introduce errors in the process. The gene is deactivated. If the targeted gene did indeed play a central role in pigment production, then the resulting mouse will be born without pigment. This is an example of researchers using CRISPR-Cas9 to study gene function.

Scientists can also use CRISPR-Cas9 to introduce changes to specific locations in a genome by hijacking the internal DNA repair mechanism. Template repair DNA can be introduced along with the engineered CRISPR-Cas9 DNA. When the CRISPR-Cas9 complex cuts the mouse DNA, and the cell’s internal repair mechanisms attempts to repair the damaged DNA, the repair mechanism will copy the template repair DNA into the location chosen by the researchers. The genome has been edited. CRISPR-Cas9 may one day soon be used to effectively treat diseases with strong genetic components such as cystic fibrosis, Duchenne Muscular Dystrophy, Huntington’s disease, or sickle cell anemia by cutting out and replacing the problematic segments of DNA.

DNA Repair

CRISPR has wonderful potential as a tool for genetic manipulation. The ability to precisely edit DNA may soon pave the way to new treatments for many genetic diseases, cancer, or HIV.  Perhaps it will be used to eradicate malaria carrying mosquitoes or to grow more nutritious foods. CRISPR is an incredibly powerful technology with diverse applications, but as such it also has the potential to be used irresponsibly, causing significant damage. Should humans have the ability to eradicate or revive entire species? What types of genetic conditions should or should not be genetically modified? Many members of the Disabled community or the Deaf community would argue that their quality of life, or level of happiness, is no less than that of others. Should their “conditions” be “fixed” before birth, before they have the option to decide? What unforeseen consequences may arise from our new found abilities? We will discuss these questions and more in future articles at the Science Distillery. CRISPR will no doubt affect massive change, but in order to assure an overwhelmingly positive outcome, we must proceed with forethought and great caution.

 

Sources:

Doudna, Jennifer A., and Emmanuelle Charpentier. “The New Frontier of Genome Engineering with CRISPR-Cas9.” Science, vol. 346, no. 6213, 28 Nov. 2014, doi:10.1126/science.1258096.

Horvath, Philippe, and Rodolphe Barrangou. “CRISPR/Cas, the Immune System of Bacteria and Archaea.” Science, vol. 327, no. 5962, 8 Jan. 2010, pp. 167–170., doi:10.1126/science.1179555.