CRISPR Crash Course

Final sentence of Charles Darwin’s Origin of Species (forceps image credit: Mortech Manufacturing, Inc).

My informal CRISPR education began in early 2016 with a forwarded email from my dad titled “Crisp lady.” Attached to the email was a Nature report on Emmanuelle Charpentier, a French molecular biologist whose work with Jennifer Doudna, a biochemist at UC Berkeley, resulted in a revolutionary tool: CRISPR-Cas9.

Emmanuelle Charpentier (left, image credit: Nature) and Jennifer Doudna (right, image credit: The Mercury News).

In the article, Charpentier unspooled the incredible potential of this system to edit the genome of virtually any living organism. I felt surprised, intrigued, and an insatiable curiosity. What’s the science behind CRISPR? What are its implications? And, more importantly, how do we responsibly move forward with this new technology?

To answer these questions, we need to unpack four dimensions of CRISPR: its science, limitations, applications, and ethics. A lot has happened in the world of CRISPR research since 2016. Let’s get started.


Science

CRISPR stands for “clustered, regularly-interspaced, short palindromic repeats.” Although it’s a bit of a mouthful without context, CRISPR describes the placement and regularity of a stretch of nucleotides in the genome of a bacterium.

To a bacterium, CRISPR is the key weapon in preventing viral infections from getting out of hand. When a virus attacks a bacterium, it squirts its genetic material into the cell. The cell’s machinery then reads this genetic material and is tricked into mass-producing all the components needed to synthesize new viruses and continue the spread of the infection. This process tends to kill the host cell, making it vital that bacteria have a way to intervene before viral genetic material can be read and used as template.

Schematic credit: Khan Academy.

The CRISPR array enables the bacterial cell to fight back before this critical step. Two components make up the array: redundant repeat regions (shown as black diamonds above) and variable spacer regions (shown as colored rectangles). Each spacer region is a short segment of DNA taken from a viral invader; it’s the equivalent of a molecular mugshot. If the same virus dares return to a bacterium that was previously infected by it, then the bacterium will deploy the CRISPR-Cas9 system to eliminate the threat. By providing bacteria with an immune system memory, the CRISPR array ensures that the host cell recognizes an attacking virus as a threat rather than ignoring it.

Colored rectangles are spacer regions and black diamonds are repeat regions. As always, the CRISPR array is king in determining how CRISPR-Cas9 fights back against viruses (schematic credit: Lander Cell 164, 18 (2016)).
But how does the bacterium actually attack the invading virus?

Although the CRISPR array is key for recognizing threats, it can’t neutralize these threats alone. Instead, bacteria retaliate against viruses by pairing CRISPR with Cas9, a nuclease that can cut viral DNA and induce a double-stranded break. When you zoom out from the CRISPR array, you can see that it is chaperoned by several other key players along what is called the CRISPR locus. A well-studied model from S. thermophilus, a bacterium widely used in yogurt, is pictured below:

Annotated CRISPR locus of S. thermophilus, containing the CRISPR array and Cas gene cassette (schematic credit: Lander Cell 164, 18 (2016)).

When the CRISPR array is transcribed to RNA, it has three primary products of interest for eliminating viruses: tracrRNA transcript from the tracrRNA gene, pre-crRNA transcript from the CRISPR array, and a Cas9 nuclease from cas9 gene. After some editing, the two transcripts link together to form a gRNA (guide RNA), which forms a complex with Cas9. The gRNA acts as a GPS and contains the RNA complement of an invading virus’ DNA. When Cas9 and the gRNA come together, you have a tracrRNA:crRNA:Cas9 complex, or CRISPR-Cas9.

In bacteria, the main goal when using CRISPR-Cas9 is to absolutely destroy the vDNA (viral DNA). To do this, the complex must first be able to distinguish between self and vDNA. Here, the crRNA sequence is key. It consists of 20 RNA bases and controls the specificity of CRISPR-Cas9, ensuring that Cas9 only cuts vDNA and does not randomly slice and dice the bacterial genome. CRISPR-Cas9 finds its viral victims by zipping along available DNA and checking for PAMs.

Schematic credit: Jinek et al. Science 337, 816 (2012).

A PAM (protospacer adjacent motif) is a short sequence of bases directly before the protospacer, or the region on the vDNA where Cas9 will cut. In the above schematic, the protospacer consists of the DNA bases on the complementary strand that match with the yellow-highlighted portion of the crRNA-sp2. A PAM is analogous to a handle that the complex grabs onto and then uses to shimmy apart the bases. Once the bases are slightly unzipped, the gRNA sniffs around for complementary sequences. If there is not a match, the complex continues its journey. If there is a match, then the gRNA binds to the protospacer and cutting commences.

The heavy-duty cutting action is done with the help of two parts of the Cas9 protein, HNH and RuvC. When the target vDNA is bound, they change conformation and cleave the offending virus so it can no longer hijack the cell, as shown below:

Video credit: UC Berkeley.

With the vDNA destroyed beyond recognition, the bacterial cell can rest easy — for now.

So what? Who cares?

From the perspective of a researcher, CRISPR is a wonderfully adaptive, affordable, and precise tool. It’s more than just a defense mechanism. Instead, it’s a scalpel for editing genomes with surgical precision to perform a diverse number of functions.

Some of the main research targets addressed using CRISPR-Cas9 are:

  1. Gene knockouts.
  2. Editing to introduce an outside DNA sequence into a DSB site or repair a mutation.
  3. Gene activation and inactivation.

Additionally, the gRNA is an incredibly modular and flexible component. By swapping out different crRNAs, you can target radically different parts of the genome. This generates some contentious questions on where the boundaries lie for genome editing, especially in its early developmental stages where much of the underlying biology remains to be known.


Limitations

The technical limitations of CRISPR come in three main categories: targetability, specificity, and availability.

In terms of targetability, gRNA design is limited by whether or not the sequence of interest is preceded by a PAM. The primary PAM used is NGG (where N is any nucleotide), but some alternative PAMs do exist. This dependence on PAMs, however, leaves the researcher dependent on the sequence to see whether or not s/he can successfully apply CRISPR.

With regards to specificity, concerns exist that CRISPR may have off-target effects in the periphery of the genome. Additionally, recent research has shown that human cells edited using CRISPR-Cas9 activate similar defense strategies as those used against cancer. Once the double-stranded break is induced and repair mechanisms begin, the gene p53 is activated. p53 detects when DNA damage occurs and may intervene so that the DNA containing the repair is no longer replicated. This leads edited human cells to either cease replication or to die off; when researchers inhibited p53 in healthy human cells, the edits were successful. This has concerning implications and suggests that CRISPR may be inefficient in humans and, by disabling p53, we are introducing the potential for dangerous, cancerous mutations to accumulate in the human genome alongside intended edits.

Availability is also an issue in two senses: (1) in the mechanical in vivo delivery of CRISPR to the organism and (2) in whether or not the target DNA is physically accessible. Theoretically, you could inject the organism with CRISPR-Cas9 or have it delivered via a virus. Viral delivery varies based on whether you want long-term Cas9 activity, which can accumulate off-target lesions, or if you want short-term editing. Lipid nanoparticle delivery also presents an additional alternative.

However, even if CRISPR-Cas9 successfully arrives at the site in question, chromatin may be obscuring its access to the PAM and preventing recognition of a protospacer that is complementary to the gRNA. At the moment, the biology of how exactly the complex enters the nucleus is unclear — does it couple with specialized proteins so it can be trafficked through the nuclear pore? Does it only edit cells that are in prophase, when the nuclear membrane is dissolved? Further research is needed to clarify the biological basis of these fundamental processes.


Applications

Although there are some limitations to CRISPR’s widespread efficacy, the technology holds incredible promise as it is further refined and developed. In terms of cost, CRISPR is also highly affordable; a graduate student with lab access could start a project with only $75. CRISPR is also highly customizable and, at the moment, has yet to meet an organism that it can’t edit. Its universality, flexibility, and cheapness are strong drivers of its promising future to revolutionize many domains ranging from healthcare to agriculture.

However, due to ethical concerns and the duration of clinical trials, CRISPR-Cas9 experimentation with mammalian cells proceeds at a much slower pace than in plants. As a result, some of the more interesting applications of CRISPR-Cas9 in the field originate from genome editing in agriculturally-significant crops such as rice and tomatoes.

A contributing factor to the booming applications of CRISPR in agriculture is the USDA’s ruling that it would not regulate the development of CRISPR-edited crops. This stands in stark contrast to its continued oversight of GMOs, and indicates US agencies’ enthusiasm to develop and apply this technology to domestic and global problems of hunger and nutritional insecurity. The rationale behind this decision is that transgenic crops, more commonly known as GMOs, have foreign DNA integrated into their genomes (for example, a tomato with a cold-hardiness gene sourced from a cold-water fish). CRISPR, on the other hand, neatly edits the DNA already within the organism and doesn’t involve crossing the species barrier.

Non-browning mushrooms edited using CRISPR-Cas9 (image credit: PSU News).

A famous example of a crop edited using CRISPR includes non-browning mushrooms. Pioneered by Dr. Yinong Yang at Penn State University, this genetically engineered mushroom variant keeps its color when sliced and has a longer shelf-life. Currently, Dr. Yang is contemplating the commercialization of his innovation.

“This technology holds promise for precision breeding of crops with many desirable traits, such as low levels of food allergens or toxins, disease resistance, drought tolerance, and efficient nitrogen and phosphorus utilization. These agronomic traits not only help reduce pesticide, fertilizer, and water usage, but also improve food quality and safety.” — Dr. Yinong Yang on the potential of CRISPR-Cas9

However, Dr. Yang is not alone. Several companies were founded to support the application of CRISPR to agriculture, including PLANTeDIT, Pairwise Plants, and Caribou Biosciences.


Bioethics

There’s no question that the science behind CRISPR is breathtakingly beautiful, elegant, and customizable. The ethics of CRISPR, however, are much less pretty. A variety of debates arise from the question of how best to develop and deploy CRISPR, or even if we should. However, taking into account the explosive increase in patents, publications, and funding for CRISPR research, there is no way to halt its spread. Pandora’s box has been opened. But to exercise control over the biochemical elements that make you, you throws the concept of self into question. Once CRISPR-Cas9 is optimized, the universal language of life is at its mercy; no organism or gene is completely off-limits.

NAS and NAM’s report on how we should proceed in applying CRISPR-Cas9 to human genomes (image credit: The National Academies Press). The CRISPR patent, funding, and publications landscape as of 2014 (infographic credit: Ledford Nature, 2015).

To address these concerns and set guidelines for the application of CRISPR-Cas9 in human genome editing, the National Academies of Sciences and Medicine released a report in 2017. In the report, they demanded that all edits involving humans proceed only under certain considerations like transparency, respect for the person, and fairness.

However, even these principles become contentious with regards to the issue of somatic versus germline cell editing in humans. Somatic cells are any cell that is not passed on by an organism to its offspring. Your skin cells, for example, are somatic cells. Germline cells are egg or sperm cells that are passed down to all of an organism’s offspring. If an edit would be performed on a germline cell, it would have repercussions — good, bad, or all of the above — that stretch across future generations.

This past week, that boundary of somatic versus germline cell editing was crossed when Chinese scientist He Jiankui reported that twin baby girls had been born with an edited CCR5 gene. Many international organizations condemned his work as dangerous, lacking transparency, and largely — from a scientific perspective — indefensible. His research breaches an important ethical dilemma in CRISPR-Cas9 that requires further public discussion.

Gif credit: It’s OK To Be Smart, PBS.

Now what?

I got a shock this Monday when an email popped up in my inbox titled “First CRISPR-edited baby was given anti-HIV gene, claims Chinese researcher.” This email was a far cry from the casual messages I exchange with my dad. I’m used to emails with cavalier titles like “Crispy HooHa” or “Cool CRISPR Agriculture Article.” I found it easier to avoid the tangled, non-binary bioethics of CRISPR when I could lose myself in the science and pretend the two were separate.

But this week presents an example where the science is so deeply interwoven with the societal consequences that they can’t be disentangled. The immediacy and proximity of CRISPR-Cas9 to our lives and collective future demands that we take an active role not only in understanding CRISPR, but also in joining the debate. The NAS and NAM’s report on human genome editing was deliberated among scientists, but for the future, science conversations among scientists alone just won’t cut it. Through the resources linked in this article, I hope you can take the first steps to join the conversation.

Sophia Swartz

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