1. Introduction: The Brave New World of Human Genetic Modification is Here

On November 25, 2018, Dr. He Jiankui, a scientist working in China, announced that he had created the first genetically edited human babies.

Dr. He used CRISPR-Cas9, the technology we’ll be discussing in this tutorial, to disable a gene called CCR₅ in two twin girls, increasing their resistance to infection by HIV.

Figure 0: **H.I.V. uses the CCR₅ protein to infect Helper T cells**

The CCR₅ gene codes for a receptor protein (also called CCR₅) that HIV (the virus that causes AIDS) uses to infect cells.

In the diagram at left, Phase 1 shows HIV bumping into a Helper T Cell. The Helper T cell is like the general in our immune system’s army. HIV has a surface protein, gp120, shown at “b,” that binds with a protein called CD4 (letter “e”) on the Helper T Cell’s membrane (c).

In Phase 2, the bound CD4 protein forms a complex with the CCR₅protein (at “d”). The formation of this complex induces receptor-mediated endocytosis (shown in Phase 3). In subsequent phases (not shown) HIV integrates itself into the Helper T cell’s nucleus, transforming the cell into an HIV factory. Ultimately, this kills the T-cell and destroys an infected person’s immune system. (If you want to learn more, click here for my tutorials about the immune system, and here for a tutorial about the life cycle of H.I.V. All links on this page will open in a separate tab.)

Many humans (including about 10% of Europe’s population) have a natural resistance to HIV infection because they carry a mutation in the CCR₅ gene. That mutation results in a much smaller version of the CCR₅ protein that doesn’t sit on the outside of the membrane, blocking the entry of HIV into helper T cells (use this link to read about CCR₅ on Wikipedia).

The twins were born to a Chinese couple in which the father was HIV positive. Dr. He explains in this youtube video that his motive was to protect the girls from HIV infection and subsequent discrimination and that the birth of these girls had given the twins’ father “a reason to live.”

The response to Dr. He’s work was worldwide condemnation (see this report from the Associated Press, this article in the NY Times, and this article on Wikipedia. While the Chinese press initially lauded Dr. He’s accomplishments, he was subsequently investigated for ethical misconduct by the Chinese government, fired from a professorship at his university, fined, and imprisoned.

Why? Human gene editing of any type opens up many ethical issues, and Dr. He had taken it upon himself to cross a line that humanity hasn’t come to any clear agreement about. Additionally, the type of edit that Dr. He carried out was the one that people worry most about. It wasn’t merely a change in the cells of body tissues to cure a disease (a proposed therapy that I’ll describe below). Rather, Dr. He had changed the genome of every cell in the body of these twins, including their germ cells. If these girls reproduce, they can pass on the edited gene to their offspring. That kind of change brings up questions for which there are no clear answers:

What are the conditions (if any) that justify human genetic modification? For example, is it okay to do gene-editing to prevent a couple from passing on a genetic disease like hemophilia or cystic fibrosis to their children? Is it okay for a couple to ensure that their child will have curly hair? Brown eyes?
What are the risks that genetic editing brings to the offspring?
What will the socio-economic consequences of genetic editing be? Are we paving a path to a world where there will be those who can afford to genetically modify their children and those who can’t? Are there ways to ensure that the benefits of this technology are used wisely, and distributed in a way that makes our world more just and equitable?

Those are some of the big issues. But even on a much more superficial and technical level, Dr. He’s work was medically unjustifiable. First, the girls were not at risk for HIV infection, and editing their genomes to prevent the expression of the CCR₅ gene was unnecessary. Many well-established measures could have been used to protect them from HIV infection in utero and during birth. Second, genetic modification entails unknown risks: other genes in the girls’ genomes might have been affected (though Dr. He has made assurances that this is not the case). Third, in one of the twins, the gene edit was only partially successful: one allele still contains the working CCR₅ gene, meaning that she’ll express the protein, and be just as much at risk of contracting HIV as an adult as anyone else.

With CRISPR-Cas9, modifying the genome of any organism — humans included — has become easier than ever before. To get an overview of how CRISPR works, watch the video below and then answer the questions that follow.

2. Overview Video of CRISPR-Cas9

3. CRISPR-Cas9 Video Quiz

[qwiz qrecord_id=”sciencemusicvideosMeister1961-CRISPR 1 (Video) M17″]

[h]Genome editing with CRISPR-Cas9: Video Quiz

[i]

[q] CRISPR first evolved as a system that bacteria used to protect themselves from attack from [hangman]. x

[c]IHZpcnVzZXM=[Qq]

[q] When CRISPR-containing bacteria are attacked by a virus, they produce two types of [hangman]. These molecules form a complex with a protein that’s called [hangman].

[c]IFJOQQ==[Qq]

[c]IGNhczk=[Qq]

[q multiple_choice=”true”] Cas9 is a nuclease. That means that it’s an enzyme that’s capable of cutting up

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[f]IE5vLiBUaGluayBhYm91dCB3aGF0IHZpcnVzZXMgaW5qZWN0IGludG8gY2VsbHMgd2hlbiB0aGV5IGF0dGFjayB0aGVtLg==[Qq]

[c]IHByb3RlaW5z[Qq]

[f]IE5vLiBUaGluayBhYm91dCB3aGF0IHZpcnVzZXMgaW5qZWN0IGludG8gY2VsbHMgd2hlbiB0aGV5IGF0dGFjayB0aGVtLg==[Qq]

[c]IA ==bnVjbGVpYyBhY2lkcw==[Qq]

[f]RXhjZWxsZW50ISBDYXM5IGlzIGEgbnVjbGVhc2UsIGFuIGVuenltZSBjYXBhYmxlIG9mIGN1dHRpbmcgdXAgRE5BLg==[Qq]

[q] CRISPR-Cas9 uses guide [hangman] to find matching DNA from a [hangman]. When it does, it makes a double-stranded cut in the viral [hangman], which disables the virus and keeps it from destroying the cell.

[c]IFJOQQ==[Qq]

[c]IHZpcnVz[Qq]

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[q]In recent years, scientists have engineered the CRISPR-Cas9 system so that instead of only cutting DNA from a [hangman], it can cut any targeted sequence of [hangman] in any organism. All that was required was changing the [hangman] RNA.

[c]dmlydXM=[Qq]

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[q multiple_choice=”true”] After the Cas9 enzyme cuts up targeted DNA in a cell, the cell

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[f]IE5vLiBBcG9wdG9zaXMgaXMgYSByZWFsIHBoZW5vbWVub24sIGJ1dCBjZWxscyB1c3VhbGx5IHNhdmUgdGhhdCBtb3ZlIGFzIGEgbGFzdCByZXNvcnQuIE5leHQgdGltZSwgY2hvb3NlIGFub3RoZXIgYW5zd2VyLg==[Qq]

[c]IHJlY29ubmVjdHMgdGhlIGJyb2tlbiBETkEsIGJ1dCBvZnRlbiBpbiBhIHdheSB0 aGF0IHJlc3VsdHMgaW4gbXV0YXRpb25zIHRoYXQgZGlzYWJsZSB0aGUgZ2VuZS4=[Qq]

[f]IFRoYXQmIzgyMTc7cyBleGFjdGx5IHJpZ2h0LiBDZWxscyByZXNwb25kIHRvIEROQSBicmVha3MgdGhyb3VnaCBETkEgcmVwYWlyIG1lY2hhbmlzbXMuIE9uZSBtZWNoYW5pc20gcmVjb25uZWN0cyB0aGUgRE5BLCBidXQgb2Z0ZW4gaW4gYSB3YXkgdGhhdCBkaXNhYmxlcyB0aGUgZ2VuZS4=[Qq]

[c]IHN0YXJ0cyBkaXZpZGluZyBvdXQgb2YgY29udHJvbCwgb2Z0ZW4gYmVjb21pbmcgYSB0dW1vciB0aGF0IGNvdWxkIGRldmVsb3AgaW50byBjYW5jZXIu[Qq]

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[q]If scientists add working DNA to a cell where CRISPR-Cas9 is at work, the cell might cut out a defective gene and [hangman] in a working copy of that gene (hint: think of how you cut and ______ words in a word processor).

[c]cGFzdGU=[Qq]

[q]If CRISPR-Cas9 and foreign [hangman] are introduced to a fertilized egg, the result can be a transgenic organism.

[c]RE5B[Qq]

[/qwiz]

4. Understanding CRISPR

As you just saw, CRISPR-Cas9 evolved as a molecular defense used by bacteria to fight off attacks from bacteriophages (viruses that attack bacteria).

Viruses are constantly attacking bacteria. That’s one part of an unending war that has been going on between bacteria and viruses for billions of years. The viral attack plan is captured in the diagram at left, which shows a process known as the lytic cycle. Note that if you want to know more about viruses, you can do so at this tutorial.

When a virus (“a” and “b”) attacks a bacterial cell (d), the virus injects its DNA (e) into its bacterial victim. The viral DNA converts the bacterial cell into a virus factory (steps 3 and 4). This process begins as the virus shuts down the bacteria’s chromosome (at “c”). Next, the virus uses the bacterial cell’s molecular machinery (such as polymerases and ribosomes) to assemble new viruses (step 5). At some point, the bacterial cell becomes so full of viruses that the newly assembled viruses (h) burst out of the cell (step 6). This last step is called lysis, from which the lytic cycle gets its name.

Bacteria have survived this onslaught because of various countermeasures. These include restriction enzymes and the CRISPR-Cas9 system. Both systems are capable of detecting injected viral DNA and destroying it by breaking it apart. The CRISPR-Cas9 system, however, doesn’t only destroy viral DNA: it’s also a part of a system that remembers viral invaders, making the bacterium (and its descendants) more efficient at fending off future attacks from the same virus. CRISPR-Cas9, in other words, is like our immune system. It remembers previous attacks and becomes more efficient at fighting off future ones.

CRISPR is an acronym that stands for clustered regularly interspersed short palindromic repeats. Along a bacterial chromosome (“c,” above, “A” below) are clusters of repeated sequences (B), interspersed with what are called “spacers” (C). The spacers are non-repetitive DNA, and they consist of fragments of invading viral DNA.

Figure 2: The CRISPR Array. Adapted from an image presented by Jennifer Doudna, credited to Jillian Banfield, UC Berkeley. Also credited to Bolotin, Mojica, and Pourcel, 2005.

How does this viral DNA become integrated into a bacterial chromosome?

Figure 3: CRISPR Immunization: Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4051438/. Permission Pending. Click the image to view a larger version.

The image at left shows a bacteriophage (a) injecting its DNA (c) into a bacterial cell (b). Nuclease enzymes that are part of a CRISPR-associated complex (also known as Cas Complex enzymes, shown at “d”) attack the viral DNA, breaking it into fragments (e). One of these fragments (f) becomes incorporated into the CRISPR array (j) as a new spacer (g). The letter “i” shows spacers that were incorporated into the CRISPR array after previous infections. “H” shows repeats.

The Cas nuclease complex shown above at “d” is a kind of innate, or non-specific, immunity. It’s part of a bacterium’s generalized arsenal for fighting off bacteriophages. Once the new spacer (g) has been integrated into the CRISPR array, however, the cell has a kind of acquired immunity against further attack from this virus. Here’s how this works. Note that you have to scroll the text below the diagram to read the entire explanation.

**Figure 4: CRISPR-Cas9 Immunity:** Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4051438/. Permission Pending. Click the image to view a larger version in a new tab.

STEP 1: A phage (at “a”) attacks the cell by injecting its DNA. Note that an identical phage had previously attacked this cell (or one of its ancestors), and a spacer with a fragment of this phage’s DNA is in the cell’s CRISPR DNA (at bottom right).
STEP 2: Because a clone of this phage had previously attached the cell, the cell now has a new weapon with which to counterattack. The cell transcribes its CRISPR DNA into pre-CRISPR RNA. Note that CRISPR is an operon: a series of genes that get transcribed together into a single RNA transcript, labeled in this diagram as “pre-CRISPR RNA.”
STEP 3: The cell’s counterattack requires transcription of additional genes. One of these is the Cas9 gene (shown a little bit to the right of and below number 3). As we learned above, Cas9 is a nuclease. With proper guidance from CRISPR RNA, it will be able to attack and destroy the viral DNA that’s been injected into the cell. An additional gene is the tracrRNA gene, which is transcribed into tracrRNA. As you can see below, tracrRNA bonds with mature CRISPR RNA to form a functional CRISPR-Cas9 complex. In some sources, it’s described as a “handle” that Cas9 holds on to as it carries out viral target surveillance and destruction. Study the image below to see all of the components of CRISPR-Cas9.
STEP 4: Multiple CRISPR-Cas9 complexes form as the Cas9 protein, the RNA transcribed from the spacer DNA, and the tracrRNA all come together. Once associated with Cas9, the spacer RNA now graduates to become crRNA. The crRNA is the guide that the complex uses to find injected phage DNA. RNAase III is yet another enzyme that edits the various RNAs into their mature forms.
STEP 5: Of the various CRISPR-Cas9 complexes built, one has the crRNA that complements a 20 nucleotide sequence in the DNA from the invading phage. That stretch of recognizable DNA is called a protospacer.
STEP 6: When the CRISPR-Cas9 encounters phage DNA, the viral DNA is split open and lined up against the crRNA. If the crRNA complements the protospacer in the viral DNA …
STEP 7: … then the viral DNA is broken apart, preventing the virus from taking over the cell.

5. Understanding CRISPR-Cas9: Interactive Diagrams

[qwiz style=”width: 700px !important;” qrecord_id=”sciencemusicvideosMeister1961-CRISPR 2, Interactive Diagrams (M17)”]

[q labels = “top”]

[l]Cas Nucleases