Last updated on May 28, 2020
If you’ve ever seen the movie Gattaca, then you probably know something about the concept of genetic engineering. However fictionalized that movie may have seemed (my favorite part was a full genome sequence printed on one small tube of rolled up paper), it did bring up a lot of interesting ethical questions that are suddenly becoming more and more relevant. Widespread genetic engineering isn’t yet a reality, but it certainly could be within our lifetime. In the last decade, scientists have discovered a way to hijack the CRISPR-Cas9 system in bacteria to make efficient and targeted genetic editing possible. However, as we get closer and closer to widespread implementation of this technology, there is still a looming gap in the legal and ethical framework surrounding genetic engineering. Is nuanced and unbiased regulation of gene editing even possible?
Last week, I went over the mechanism of CRISPR and how it gives bacteria adaptive immunity to viruses. I gave a more generalized overview of how CRISPR operates, but actually there are three distinct classes of CRISPR that act in diverse ways to enact this immunity. In terms of genetic engineering, we are most concerned with Class II, known as CRISPR-Cas9. Each CRISPR class utilizes specific Cas proteins to perform the three main steps of bacterial immunity: adaptation, expression, and interference. The first step, adaptation, requires Cas1 and Cas2 to form a protein complex. This complex is responsible for chopping up viral DNA and integrating it as a spacer in the CRISPR DNA. This step is essential for establishing the molecular memory of a particular virus, and it is highly conserved across all three classes of CRISPR.
The second step, expression, is where the CRISPR DNA is transcribed into RNA and processed into CRISPR RNA (crRNA) by specific Cas proteins and other factors. The Cas proteins that turn this precursor crRNA (pre-crRNA) into mature crRNA differ between the three CRISPR systems. In Classes I and III, it is mostly Cas5 and Cas6 that perform this processing. But in CRISPR Class II, the pre-crRNA hybridizes with trans-activating CRISPR RNA (tracrRNA) transcribed from DNA upstream of the CRISPR region. This RNA duplex is trimmed by a RNase enzyme (a protein that can cut up RNA), and it quickly recruits the Cas9 protein, which performs the interference step.
The final step, interference, is perhaps the most important step in bacterial immunity. It is also the step that scientists hijack for genetic engineering. In Class II, the Cas9 protein carries the crRNA:tracrRNA duplex. The spacer in the crRNA directs Cas9 to the target viral DNA, where Cas9 uses molecular scissors to induce a double-strand break that neutralizes the DNA.
Proper targeting of the viral DNA is reliant on a specific sequence in the target DNA called protospacer adjacent motif (PAM), a short sequence of DNA next to the spacer’s target DNA that helps Cas9 distinguish target viral DNA from native bacterial DNA. The spacer is enough to recognize a specific target sequence, but that target sequence also exists in the bacteria’s own CRISPR DNA. To avoid destroying the bacteria’s immunity, Cas9 is programmed to only cut target DNA that contains the PAM sequence just downstream. But why does the viral DNA always contain a PAM next to the spacer target? Because, during the adaptation phase, the Cas1/Cas2 complex specifically selects a viral DNA “protospacer” that is next to a PAM sequence. The complex chops up the DNA around the protospacer and integrates it into the CRISPR DNA.
In terms of genetic engineering, the interference step of CRISPR-Cas9 presents a unique opportunity to induce double-strand breaks at very specific locations. There are only four elements needed: a Cas9 protein, a crRNA sequence that complements the target, a tracrRNA sequence that duplexes with the crRNA, and a target that contains a PAM upstream. Cas9 proteins can be isolated directly from bacterial cultures. Different strains of bacteria have different Cas9 proteins that recognize different PAM sequences, allowing scientists to choose a protein that recognizes a PAM near their desired target. For targets with no recognizable PAM sequences nearby, scientists have begun engineering modified Cas9 proteins to recognize new PAM sequences.
In 2012, Emmanuelle Charpentier and Jennifer Doudna engineered a synthetic fusion of tracrRNA:crRNA that was able to direct Cas9 targeted DNA cleavage. This discovery was made just one year after Charpentier discovered tracrRNAand its important role in the CRISPR-Cas9 mechanism. The tracrRNA:crRNA molecule, referred to as synthetic guide RNA (sgRNA), simplified the process of CRISPR-Cas9 targeted gene editing. Today, scientists can design and order customized sgRNA molecules for practically any gene target, as long as it contains a PAM that their Cas9 protein recognizes.
It may seem counterintuitive that a process used to destroy viruses can also be used to manipulate genes in a controlled way. In cells, when a double-strand break occurs in a section of DNA, the cell tries to repair the break. There are two main pathways for DNA repair: non-homologous end joining (NHEJ) and homology-directed repair (HDR).
In the absence of any template for the cell to model repair after, NHEJ is the dominant mechanism. This mechanism involves removing and adding base pairs from the ends of the break to make the pieces fit back together. But in that process, there is a two-thirds chance of a mutation occurring that completely disrupts the meaning of the gene (known as a frameshift mutation). In that case, the gene is completely inactivated and won’t be able to produce the protein it normally produces. Even though this only happens two-thirds of the time, the Cas9 protein will continue to cut target DNA that hasn’t mutated until it does mutate. This is a useful pathway for scientists looking to knock out a particular gene to either discover its function or prevent its undesirable effects.
Alternatively, if the scientist provides a template strand of DNA with the CRISPR-Cas9 system, then HDR is able to repair the double-strand break by copying the template into the gap between the ends. HDR repairs these breaks without introducing any dangerous frameshift mutations, and therefore does not deactivate the gene completely. Through this mechanism, scientists can insert new genes or make subtle changes in existing genes.
The field of CRISPR-Cas9 gene editing is still in its infancy. But as scientists continue to develop this technology, it is completely possible that human genetic engineering could be a reality within our lifetime. Next week, I’ll talk more extensively about what sorts of changes scientists are making with CRISPR-Cas9, and what changes they may be able to make in the future. I’ll also discuss some of the unique ethical challenges those changes may present.
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