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Tiny Protein Has Huge Potential in CRISPR

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An array of CRISPR proteins now exist, each presenting with their own unique properties. Last year the smallest CRISPR protein, Cas14 was discovered. This CRISPR protein’s tiny size means that it is hyper specific in the delivery of gene editing. Now, Mammoth Biosciences have received exclusive licencing to Cas14 from UC Berkeley for use in all fields – completing their CRISPR protein toolbox. We spoke with the scientist that discovered Cas14, Lucas Harrington, to learn more about the protein, and what it's discovery means for the future and application of CRISPR research. 

Molly Campbell (MC): CRISPR technology looks set to revolutionize the medical field as we currently know it. What applications may it have, and how important is it to continue developing the CRISPR method?  

Lucas Harrington (LH):
There's definitely still a long way to go for CRISPR to deliver on the promise of revolutionizing genetic medicine. To do so, we need to push the protein-discovery front, find new types of CRISPR proteins and develop their applications. Each kind of CRISPR protein has its own strengths and this needs to be explored thoroughly. Additionally, there is an ethical discussion surrounding CRISPR that needs to take place not only within the scientific community but also in the general public so we can collectively decide on how to use this tool going forward.

MC: Can you tell us about how the novel CRISPR protein, Cas14, was discovered? What technologies allowed this discovery to be made?

Cas14 is the most recent protein we discovered using meta genomics. Rather than sequencing organisms that we can grow in the lab, our collaborators went out in the field to collect samples from a variety of places, including hot springs, sewage, and animal waste products. Using this approach, they are able to sequence a variety of life forms rather than simply cultivate organisms. The Cas14 protein is derived from Archaea, a more ancient form of life that was not grown in a lab. However, through metagenomic sequencing, we were able to discover the valuable proteins encoded in their genome.

MC: Why is the size of the CRISPR protein used in research important?

LH: Most CRISPR proteins are typically between 1000 to 1400 amino acids long, which is particularly large. Cas14 is less than half the size, approximately 400 amino acids, which is amazing considering that the protein is a complex molecular machine that is responsible for the precise DNA cutting that its larger relatives are capable of. Its small size is also useful for delivering the protein to an organism, whether it be a human or an animal. Viral packaging systems, one of the most effective CRISPR delivery system available, have a limited capacity to what they can hold, which poses as an issue with larger proteins. With a smaller protein such as Cas14, you are increasing the payload of what you can deliver. Rather than simply delivering the protein, you can incorporate other items, adding new functionality to the CRISPR system.

MC: Mammoth Biosciences has received exclusive licencing for three CRISPR proteins. What are your next steps in CRISPR research and utilizing these proteins? 

Mammoth Biosciences is a diagnostic company, so we are focused on using CRISPR systems to help diagnose disease, whether it’s infectious disease or cancer. However, we are also looking at non-health related applications, such as agriculture. For diagnostics, each of the CRISPR proteins has a unique property that makes it appropriate for certain applications, and so it’s great that we have this complete arsenal of proteins available. We have been able to detect double stranded DNA and RNA very accurately; however single stranded DNA was difficult to detect with precision. Cas14 overcomes this issue and completes the set of proteins that we need to cover all areas of molecular diagnostics.

MC: What are some of the technical challenges that researchers face in complex CRISPR research?

LH: One challenge we face is dependent on where the proteins come from -- for example Cas14 comes from Archaea, which are hard to work with in the laboratory. Transferring these proteins over to a more common system for experiments can be a shot in the dark; sometimes it works and sometimes it doesn’t. Sometimes the systems do not work because the experimental techniques used to thoroughly analyze them are not available. Another challenge is knowing what’s a real CRISPR system and what’s not. As of now, this process is time consuming and erroneous because we have to go through a long list of CRISPR systems to try and deduce from bioinformatics whether they are real.

MC: Are there things that we still do not know about CRISPR? How can we continue to further CRISPR research? 

LH: There are a few big areas that researchers are working tirelessly to figure out. The first is the safety of the systems; for example, when we discuss using CRISPR for therapeutics, we need to be confident that the proteins are only cutting the areas that we require them to. If the system is introduced into medicine before it has been refined, there are dangers that CRISPR could cut the DNA in the wrong location and cause disease, which would prevent the field’s progress. The second challenge is the delivery of CRISPR. In a large organism, it becomes much more difficult to conduct targeted delivery; so, in the future, there is going to be an ongoing evolution as to what vehicles we use to deliver the proteins. 

Lucas Harrington was speaking to Molly Campbell, Science Writer, Technology Networks.