When it comes to gene editing technologies like CRISPR, the initial applications were focused around just that, the capability to edit and alter gene sequences. The main efforts after were to enhance the specificity of this activity and to improve further what could be changed. But in recent years we’ve begun to see an expansion beyond this base primary purpose of CRISPR and broad new functions have begun to be explored.
Jennifer Doudna, the arguably de facto “creator” of CRISPR or at least the first true publiciser and one of the most knowledgeable people on the topic, has been helping to lead the charge in finding what lies beyond our basic understanding of this innate bacterial immune system. While she started work on the ever popular Cas9, she chose to move on to the other discovered types of CRISPR to see what could be uncovered, even as most of the scientific world stayed behind.
CRISPR’s Other Half
Another form of CRISPR that has seen greater attention over the past two years is Cas12a, previously known as Cpf1, due to its unique features and production of sticky ends when cleaving genetic sequences. To describe more of what makes it special, it should first be pointed out that Cas9 has two catalytic domains, places of active enzyme activity within the complex usually related to cleavage. These domains are called RuvC and HNH and are the most important parts of CRISPR, along with the guide RNA packaged with the complex, guiding them to the target sequence to bind to.
Cas12 on the other hand features only a single domain in the form of RuvC, though has the same guide RNA and double stranded DNA (dsDNA) cleavage systems. Additionally, the protospacer adjacent motif (PAM) sequence for Cas12 is rich in T nucleotides, being a TTTA sequence. It can also make its own crRNA and create the aforementioned sticky ends when cleaving. These exclusive capabilities have drawn scientists to using the Type V form, but the actual mechanism of its cleavage and how specific the matching substrate sequence must be for cleavage activation has yet to be fully understood.
It was the latter (and in the same coin, the former) that Doudna was investigating along with University of California, Berkeley researchers. What they instead found and developed with their findings is something huge. Let’s go over their experiments from the beginning.
The Cas12 Massacre
Bacteria of the Lachnospiraceae family were used to test for single stranded DNA (ssDNA) cleavage capabilities. Their innate Cas12a system was obtained and pitted against Cas9 from S. pyogenes in order to see which could deal with the ssDNA phage called M13. Cas12a was found to rapidly and completely degrade the M13 genome using a cleavage mechanism that didn’t appear to be based on having a specific matching sequence to the guide RNA.
The shredding of ssDNA was not observed when using a catalytically inactive version of Cas12 and implied that it would be capable of degrading any ssDNA sequence when active, so long as it had a target activator. This was confirmed when the same results happened with a different guide RNA and with a ssDNA activator that had no matching homology to any part of the M13 genome. Therefore, so long as a complementary activator is bound to the guide RNA being used, Cas12 appears able to shred any ssDNA that binds with it.
The next test was to find out what specifically was capable of activating this activity and what the requirements are for activation. A test was done with complementary activators in the form of ssDNA, dsDNA, and ssRNA. Then an unrelated ssDNA or ssRNA sequence was added. They found that both DNA forms triggered cleavage, but the RNA did not and the unrelated RNA sequence that was added was also not degraded under any of the variants while the ssDNA was. This showcases that Cas12 has a DNA-targeted (DNase) and DNA-activated cleavage mechanism.
How Cas12 Cleaves
With that out of the way, they then had to figure out what amount of site-specific targeting is required for it to degrade a DNA sequence. Both target strand (TS) and non-target strand (NTS) activation was experimented with. They found that TS cleavage happened regardless of how long the complementary NTS strand was, but that NTS cleavage required at least 15 nucleotides of the TS to match the guide RNA. Thus, TS recognition in connection to the guide RNA is necessary in order to activate the manic ssDNA cleavage regardless of sequence.
The actual kinetics of this was shown to be that Cas12 binds to a targeted and site-specific dsDNA sequence at the PAM sequence with the help of guide RNA. Cleavage then occurs, but leaves behind a dsDNA segment bound to the PAM as an activator. The rest of the CRISPR complex comes unbound from the already cleaved end of the sequence, releasing it from the RuvC catalytic site. This opens up this site to access by ssDNA, which binds and is immediately cleaved, regardless of the ssDNA’s actual sequence, as the prior dsDNA activator is still bound to the PAM sequence.
The efficiency and rate of cleavage of this turned on system is incredibly high. Interestingly, whether a ssDNA or dsDNA sequence is bound to the PAM matters. The former cleaves 250 sequences per second, while the latter cleaves 1250. The efficiency of the dsDNA bound system approaches the rate of diffusion, meaning the physical speed at which ssDNA sequences can even reach the complex. This difference suggests that the NTS of the dsDNA sequence bound to the PAM helps to stabilize the overall complex into the most optimal configuration for cleavage. In short, it works better when bound to a double-stranded sequence than a single stranded one in regards to activation.
The specificity of the bound PAM/DNA complex is also incredibly high, requiring a precise PAM sequence and precise dsDNA sequence in order to function properly. Any amount of mismatches severely reduces the overall speed of cleavage, Though this requirement seems to only be so high when binding to a dsDNA and not a ssDNA, meaning that the PAM sequence in Cas12 is directly targeted toward dsDNA, which was already pointed out in the previous paragraph anyways.
Detecting Viruses With DETECTR
Once the entirety of how Cas12’s cleavage mechanisms and functions were found, Doudna et al moved onto the final and most important part of their study. Could this CRISPR variety be used to detect specific DNA in a swift and accurate manner? The researchers wanted to test whether it could distinguish between two types of human papillomaviruses (HPV), both dsDNA organisms. The target sequence next to the PAM was chosen to vary by six nucleotides between the sequences of both HPV16 and HPV18.
Plasmids containing the HPV genomes were incubated with the Cas12 targeting one or the other, along with a ssDNA reporter that had an attached fluorophore quencher. If the Cas12 successfully matched with one of the HPV’s, its ssDNA cleaving abilities would be stimulated and the FQ-ssDNA reporter would be cleaved, allowing the fluorophore attached to the CRISPR complex to begin fluorescing. This would indicate that identification of the HPV has been accomplished. To improve the sensitivity, the Cas12 would have an added Recombinase Polymerase Amplification system included to amplify and duplicate any DNA sequences in the sample, improving the chances of matching with the target if it existed in the sample.
All together, this system was named by the team as the DNA Endonuclease Targeted CRISPR Trans Reporter (DETECTR). So long as it is programmed with the correct target sequence, its specificity is on the attomolar level, meaning that a very low concentration of the target is needed to find it.
The last dual field test used samples of cultured human cells infected with one of the HPVs and a control sample without them. Cas12 by itself was unable to locate the HPV, but the combined DETECTR system had no issues. Finally, patient samples were taken and applied, with DETECTR being able to correctly identify 25 out of 25 of the HPV16 samples and 23 out of 25 of the HPV18 samples. The two misses did give off weak signals, but Doudna expected that better guide RNA design would fix that issue.
Beyond Gene Editing
For a bacteria, it can be presumed that Cas12 works quite adequately. It is targeted to work best against dsDNA phages, but can also deal with ssDNA phages regardless of them matching its PAM sequence. It can even deal with both kinds simultaneously, along with a mobilized Cas12 being able to randomly target the ssDNA that forms from phage replication and transcription.
But for science, Cas12 has now proven itself to be far more than just an arguably more useful alternative gene editor to Cas9. With its activated capability to target ssDNA at transcription bubbles, forks, and loops, it is far more versatile than any single other CRISPR system. Add on this precise and speedy DNA detection system that Doudna et al have made in DETECTR and we can guess to be only seeing the tip of the iceberg of new science and medical innovations that this will lead to.
The sky is truly the limit or, perhaps in this case, we can go beyond even that.
Photo CCs: Influenza virus particle color from Wikimedia Commons