An Introduction to the Rapidly Expanding World of CRISPR

BY ELEANOR WANG | SQ ONLINE WRITER | SQ ONLINE (2018-19)

Many of us dream of having clearer skin, greater strength, increased height, or just having better genes. In the future, scientists may be able to develop ways to edit our genes and make all of this possible. Maybe this excites you. But maybe instead you’ve seen the movie GATTACA and find the concept of modifying our DNA appalling and unethical. Or perhaps you associate gene editing with Jurassic Park. With the power to manipulate DNA, could we bring back the dinosaurs? Can we turn unicorns from fantasy into reality? With the ever-expanding field of molecular biology and genome editing, these questions don’t appear as far-fetched as they might have seemed ten years ago. These hypotheticals are turning into real questions, thanks to a tool called “CRISPR.” You may even have heard a few months ago that the very first CRISPR babies have been born from the work of Chinese scientist He Jiankui. But to be honest, CRISPR is much more than dramatic headlines in the news, and He isn’t going to be the main focus of this article.

For good reason, gene editing is a major topic in bioethical debates. CRISPR has been widely introduced as a gene editing tool, but how exactly does it work? Its popularity can hardly be disputed—it has blown up all over the media—with CRISPR features on Radiolab and funny, informational videos on gene editing such as this one from John Oliver’s Last Week Tonight. Scientists and journalists are continually publishing more research on CRISPR biology and its development as a technology. In this article, I will introduce the basic mechanics of CRISPR and shed some light on the ethics behind its application as a tool for genome editing.

This is a general schematic illustrating how CRISPR-Cas9 operates. (source)
Transmission electron micrograph of multiple bacteriophages attached to a bacterial cell wall (source)

CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats. It’s a mouthful of an acronym, but simply put, CRISPR is an adaptive microbial antiviral defense mechanism that scientists have developed into a gene editing tool. What does this actually mean? Let’s break this down a little more:

Bacteria are in a competitive relationship with viruses called bacteriophages, which insert their genomes in the form of DNA or RNA into bacteria. To resist infection, bacteria have developed their own immune systems and defense mechanisms, including CRISPR. The basic components of CRISPR consist of an array encoded in the bacterial genome and an enzyme called Cas (CRISPR-associated). The array is a set of repeated DNA sequences with “spacers” in between that match up with viral DNA. The spacers are transcribed into a guide RNA (gRNA), and when a virus tries to infect a bacterium, the Cas enzyme forms a complex with the gRNA and uses it as a template to target the viral DNA before it can infect the bacterium. The Cas enzyme has catalytic domains that are analogous to “molecular scissors” that will cut up the viral DNA. Scientists have harnessed CRISPR-Cas as a programmable tool that they can retarget to desired DNA sequences. Additionally, genome-repair mechanisms can be manipulated to insert new sequences where the cuts are made. The most commonly known CRISPR system for genome editing is called “CRISPR-Cas9”.

Cas9 making a double-stranded break in DNA. (source)

Clearly the ability to cut and edit DNA is powerful, and although CRISPR-Cas9 isn’t the first gene editing technique, it is versatile and a lot easier to use than previous tools. CRISPR is now being applied to develop new and improved medical treatments. As a tool, CRISPR has also revolutionized basic scientific research, and scientists have developed many ways to apply it beyond cutting and pasting DNA sequences. For example, a technology called CRISPR inhibition and activation (CRISPRi and CRISPRa) allows scientists to turn genes on and off. This is done by inactivating the catalytic domains—or “repressing the scissors”—of Cas9, fusing transcription factors to Cas9, and targeting the new fusion protein to the locus of desire. Depending on the selected transcription factor, the targeted gene is either activated or inactivated. For further explanation, UC San Francisco has uploaded a video that explains how this process works in more depth.

Additionally, Cas9 is not the only CRISPR system. Microbes have evolved multiple types and classes of CRISPR systems to combat different types of bacteriophages. Simply put, Class 1 CRISPR-Cas systems operate with multiple proteins forming a complex, and Class 2 systems use one protein that operates as a single effector—Cas9 is Class 2 type II! Cas12, formerly named Cpf1, is another DNA-targeting CRISPR enzyme that has been shown in certain contexts to be more precise than Cas9. There are also CRISPR systems that target RNA instead of DNA. A family of CRISPR enzymes called Cas13 exclusively targets RNA sequences. The Cas13 family contains at least four known subtypes: Cas13a (formerly C2c2), Cas13b, Cas13c, and Cas13d. (I just want to give a little shout-out to Cas13d—I’ve been conducting research on it in the Hsu Lab at the Salk Institute for a full year as of May 2019!) Cas13 provides a more precise and efficient alternative to RNA interference. It also serves as a programmable RNA-binding module that is currently being developed for RNA editing, mRNA splice modulation, and live RNA imaging and tracking! If you’re curious about the details, here’s a recent review on applications of CRISPR in genetic engineering, and a “snapshot” of different Class 2 CRISPR systems!

A developing embryo, credit to Pascal Goetgheluck/Science Photo Library (source)

Beyond this, CRISPR is being applied to execute a genetic engineering technology called gene drive, in which a specific gene is selectively propagated through a population. Ethan Bier, a professor in Cell and Developmental Biology at UC San Diego, is leading efforts to apply gene drive in mosquitoes to prevent the spread of malaria. Over the past decade, development of CRISPR-based technologies has greatly expanded our toolbox for conducting research and for developing new treatments for disease.

Recently, however, a Chinese scientist named He Jiankui used CRISPR to alter the embryonic genomes of two twin girls: Lulu and Nana, who were born in November 2018. He Jiankui is a genome-editing researcher at the Southern University of Science and Technology of China in Shenzhen. In this YouTube video, he announces that the girls are healthy and that DNA sequencing showed that the editing worked, although these claims have not been verified by independent genome testing, nor have results been published in a peer-reviewed journal. He targeted Lulu and Nana’s CCR5 (C-C chemokine receptor type 5) gene to make them resistant to HIV. He states in the video:

“For a few children, early gene surgery may be the only viable way to heal an inheritable disease and prevent a lifetime of suffering…Their parents don’t want a designer baby. Just a child who won’t suffer from a disease which medicine can now prevent. Gene surgery is and should remain a technology for healing…I understand my work will be controversial, but I believe families need this technology and I’m willing to take the criticism for them.”

Editing human embryos, especially when it is unknown whether this process was safe for the twin girls or not, was highly unethical. Prior research studies with CRISPR-Cas9 have reported off-target effects. Additionally, edited genes will continue to be passed onto future generations, meaning that editing the genomes of children has consequences that extend far beyond their wellbeing, but to their own offspring. But the fact that He Jiankui edited human embryos was not the only dicey part of this story.

He’s selection of HIV and CCR5 itself is questionable. CCR5 is a protein on the surface of white blood cells and is involved in signaling pathways in the immune system. (It’s a G protein-coupled receptor!) CCR5 provides a method of entry for HIV, and populations that have inherited a genetic deletion of CCR5 have previously been shown to be resistant to specific strains of HIV. Genome-editing scientist Fyodor Urnov at the Altius Institute for Biomedical Sciences in Seattle, Washington says “that there are ‘safe and effective ways’ to use genetics to protect people from HIV that do not involve editing an embryo’s genes. ‘There is, at present, no unmet medical need that embryo editing addresses.’” Furthermore, some strains of HIV don’t actually use CCR5 to enter cells, and instead use a protein called CXCR4, meaning Lulu and Nana are not completely resistant to HIV despite being CCR5-negative. “This experiment exposes healthy normal children to risks of gene editing for no real necessary benefit,” says Julian Savulescu, director of the Oxford Uehiro Centre for Practical Ethics at the University of Oxford, UK.

He Jiankui’s research methodology was also flawed—he chose a family with an HIV-positive father and HIV-negative mother. There are already methods to prevent transmission of HIV from parent to child, such as semen washing prior to in vitro fertilization (IVF) when the carrier is the father, and delivering the child via caesarean section to avoid infection during childbirth when the mother is HIV-positive. CCR5 also “plays a vital role in the inflammatory response by directing cells to sites of inflammation” and knocking out CCR5 may leave Lulu and Nana more susceptible to other infectious diseases. A strongly worded online statement by more than 100 Chinese biomedical researchers says:

“This is a huge blow to the international reputation and the development of Chinese science, especially in the field of biomedical research. It is extremely unfair to the large majority of diligent and conscientious scientists in China who are pursuing research and innovation while strictly adhering to ethical limits.”

Incidents like these make it much more difficult for scientists conducting responsible and ethical research to carry out their work. The work was also done in secrecy. Southern University of Science and Technology reported in a statement on November 26, 2018 that they were “unaware of He’s experiments, that the work was not performed at the university and that He has been on leave since February.”

He Jiankui was fired from his university for his gene-editing experiments. (source)

If you’re interested in looking at some of the data, here is a Google Drive folder of He’s slides from the Second International Summit on Human Genome Editing in Hong Kong. This was a three-day summit organized by the Academy of Sciences of Hong Kong, the Royal Society of London, the U.S. National Academy of Sciences and the U.S. National Academy of Medicine, where the “goal was to reach a global scientific consensus on how scientists might some day ethically use powerful new gene-editing techniques such as CRISPR to edit the human genetic blueprint.”

Many prominent CRISPR scientists have called for a moratorium on implantation of edited embryos in response to He Jiankui’s actions. The following statement is by Dr. Feng Zhang, whose lab pioneered the development of CRISPR-Cas9 as a genome editing tool for use in eukaryotic cells. Zhang is also an investigator at the McGovern Institute for Brain Research at MIT, the James and Patricia Poitras Professor of Neuroscience at MIT, and an associate professor at MIT, with joint appointments in the departments of Brain and Cognitive Sciences and Biological Engineering, and an investigator at the Howard Hughes Medical Institute. (He was the thesis advisor for my current principal investigator’s Ph.D.):

“Given the current early state of genome editing technology, I’m in favor of a moratorium on implantation of edited embryos, which seems to be the intention of the CCR5 trial, until we have come up with a thoughtful set of safety requirements first.

“Not only do I see this as risky, but I am also deeply concerned about the lack of transparency surrounding this trial. All medical advances, gene editing or otherwise and particularly those that impact vulnerable populations, should be cautiously and thoughtfully tested, discussed openly with patients, physicians, scientists, and other community members, and implemented in an equitable way.”

The moratorium, however, was not implemented at the time. (There are new updates to the moratorium since March 2019 that I will discuss shortly.) At Harvard University’s Stem Cell Institute, IVF doctor and scientist Werner Neuhausser says he plans on using CRISPR to edit sperm and “create IVF babies with a greatly reduced risk of Alzheimer’s disease later in life.” Again, this is highly unethical for several reasons. Alzheimer’s disease pathology is nowhere near well-understood, meaning it is virtually impossible to quantify whether the edits made would be beneficial in the long run. This is particularly important because Alzheimer’s disease develops late in life and is influenced by a multitude of different genetic and environmental factors. Neuhausser wants to apply a technology called DNA base editing to reduce risk of Alzheimer’s by modifying a gene called ApoE that has been associated with it. Neuhausser says, “It’s one letter, G to A. You take it from risk to non-risk,” but this is a vast oversimplification. CRISPR DNA base editors have been demonstrated to cause an enormous number of off-target mutations, putting the potential child at a high risk for unpredictable mutations across their entire genome.

How do we approach CRISPR and ensure that it is being applied safely and ethically? Clearly, guidelines need to be set. The solution cannot be to completely ban the use of gene editing for an indefinite period of time, nor is it to say that anything goes. On March 13, 2019, a call to adopt a global moratorium on heritable genome editing was released by 18 signatories including scientists and ethicists from 7 different countries, such as Feng Zhang, Eric Lander, Emmanuelle Charpentier, and other major figures in CRISPR biotechnology and biomedical ethics. Their thorough statement details several important considerations for heritable genome editing.

Rather than a permanent ban, they “call for the establishment of an international framework in which nations, while retaining the right to make their own decisions, voluntarily commit to not approve any use of clinical germline editing unless certain conditions are met.” The statement begins by stating the need for a period of time in which germline editing (heritable genome editing) is completely banned while “discussions about the technical, scientific, medical, societal, ethical and moral issues” can take place and an international framework can be established. The National Institutes of Health (NIH) has supported the statement, and currently, the “US National Academy of Sciences, the US National Academy of Medicine and the UK Royal Society are leading an international commission to detail the scientific and ethical issues that must be considered, and to define specific criteria and standards for evaluating whether proposed clinical trials or applications that involve germline editing should be permitted.”

Although this is the first time the world has seen the birth of genetically modified children, it is likely that this is not the last time. As developments in biomedical engineering continue to proceed and biotechnology continues to advance, regulation of ethics in biomedical research continues to be a vital aspect of science. As for my personal opinion, I hope that global standards for bioethics can continue to be established and revised, that scientists can effectively communicate their exciting work to the public, that people across the globe have the opportunity to access educational opportunities for participation in these discussions, and that they can benefit from exciting improvements in healthcare.

in vitro fertilization (source)