Degrading DNA with Cascade-Cas3

The versatility of CRISPR allows you to play with DNA in a number of ways, from small edits that change single base pairs, to chromosomal inversions and large deletions. Many of these methods rely on Cas9 or a derivative of Cas9, but the ever-expanding repertoire of CRISPR has brought many other Cas effectors to light.

One lesser-known Cas is waiting for its chance in the spotlight — Cas3. Cas3 can induce genomic alterations on a scale that other Cas effectors can’t, while maintaining relatively high efficiency. These alterations are achieved through DNA degradation, thanks to the unique capabilities of Cas3 and its partner in crime, Cascade.

Find Cascade-Cas3 plasmids in Addgene's collection!

A “complex” system

Cas3 is part of the Class 1 CRISPR family, which means it requires multiple Cas effectors to carry out genomic editing. Specifically, these types of CRISPR systems work using a multi-Cas complex called Cascade (CRISPR-associated complex for antiviral defense). Cascade is brought to the target location using a gRNA and can then recruit Cas3, which carries out the brunt of the mechanical work for editing.

Class 1 CRISPR systems make up the majority of CRISPR-based immune systems in bacteria and archaea. Class 1 is further broken down into different types, with Cas3 systems making up type I, the most abundant type in the Class 1 category. These type Is can then be further broken into subtypes of A–G, which come from different species and will have different compositions of Cascade. Although seemingly ubiquitous in nature, they are largely underutilized in research settings — mostly a consequence of Cascade's complexity.

Figure 1: Schematic of Class 1 CRISPR types. Class 1 includes type I with a Cas3 effector, type III with a Cas10 effector, and type IV with an unknown effector. Type I has subtypes A through G, with different compositions of the Cascade complex. Created with BioRender.com.

The Cascade complex

Cascade is an important component of the CRISPR Cas3 system. Most Cascade complexes are comprised of a combination of Cas5, Cas6, Cas7, Cas8, and Cas11. Type I-E Cascades contains all five of these Cas proteins, while type I-C Cascades do not contain Cas6. Regardless of the subtype, the mechanism of Cascade binding and subsequent DNA degradation appears to be conserved. This DNA degradation portion can be bypassed by leaving out Cas3 — a tactic some CRISPR transposase systems utilize with type I-F Cascades.

Figure 2: Cascade complex from CRISPR type I-E. Created with BioRender.com.

Thanks to this complex, developing Cas3 tools has been challenging. It can be difficult to ensure comparable expression of all components of Cascade for maximum efficiency of the process. Various labs have designed all-in-one Cascade plasmids — such as the Tomoji Mashimo lab and the Tilmann Weber lab — to increase efficiency of these systems, though consistent expression can still be a challenge.

A multitasking Cas

Most Cas effectors have one job — Cas see DNA, Cas chop DNA. Cas3, however, has two jobs, one as a nuclease and one as a helicase. Following recruitment by Cascade, Cas3 nicks the non-target strand. The helicase domain is then activated, and Cas3 begins unraveling the double-stranded DNA (dsDNA) in what is called a reeling mechanism (see next section for details). The newly formed single-stranded DNA (ssDNA) is then pulled through Cas3. After these initial steps, Cas3 can perform its helicase and nuclease functions simultaneously, by continuously unraveling dsDNA, pulling through the ssDNA, then cleaving the ssDNA strands. In this way, Cas3 eats away at both the non-target and target strands by making cuts every so often. Cutting both strands ensures that there isn’t a template strand left behind that the cell can use to repair.

Figure 3: General overview of the Cascade-Cas3 mechanism. Created with BioRender.com.

The reeling mechanism

The reeling mechanism is carried out much like the name implies — after nicking, Cas3 reels in the non-target strand of DNA much like a fisherman would reel in a big catch. It was originally suspected that Cas3 moved a couple of kilobases down the strand of DNA, Cascade caught up to it, and then DNA degradation continued. However, Cas3 cannot bind DNA on its own and needs to remain anchored to Cascade throughout the process. The reeling of ssDNA and unwinding of dsDNA occur simultaneously, which creates a loop in the target strand that is cut occasionally as ssDNA is pulled through the Cas3.

This mechanism is important for Cas3’s biggest trick. Cas3 likes to bluff, making you into think it’s more powerful than it is. The nuclease domain of Cas3 is considered weak, especially compared to the power of Cas9. However, by continually passing the ssDNA past the nuclease domain, Cas3 is able to repeatedly cut the DNA at an efficient rate. These cuts most often occur in thymine-rich regions on the non-target strand. Occasional cleaving of the target strand is not as well understood but likely occurs in similar regions. This combination of chewing up one stand and cutting the template strand causes large deletions of up to hundreds or even thousands of base pairs, stopping either at boundaries defined by anti-CRISPR proteins or running into other proteins naturally bound to the DNA.

Cas3 or Cas9?

Large genomic deletions can be achieved using Cas9, but Cas3 may be a better option. To get large deletions using Cas9, two or more gRNAs must be used along the same chromosome. During DNA repair, there is a chance that the two breakpoints will repair without the section between the two gRNAs, creating a deletion. However, there is the chance of a chromosomal inversion, or just random mutations if the chromosome is repaired with all the original DNA. With Cas3, you don’t have to play these odds — Cas3 will definitively degrade the targeted portion of DNA using only a single gRNA. Cas3 appears to have fewer off-target alterations, likely due to the multi-Cas mechanism. These benefits of Cas3 do come at a cost — the previously mentioned issues with expressing all Cascade components can make Cas9 easier to work with than Cas3.

Future targets

Cas3 has the potential to be incredibly useful in therapeutics. Imagine deleting a problematic copy of a gene that causes a debilitating disease! This is, of course, only hypothetical at this point. More time and energy must be devoted to studying Cas3 and its Cascade partners to understand it and use it to its full potential.

Hopefully more people will want to utilize Cas3’s degradative power in the future!

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References and Resources

References

Loeff, L., Brouns, S. J., & Joo, C. (2018). Repetitive DNA reeling by the Cascade-Cas3 complex in nucleotide unwinding steps. Molecular Cell, 70(3), 385-394.e3. https://doi.org/10.1016/j.molcel.2018.03.031

Xiao, Y., Luo, M., Dolan, A. E., Liao, M., & Ke, A. (2018). Structure basis for RNA-guided DNA degradation by Cascade and Cas3. Science, 361(6397). https://doi.org/10.1126/science.aat0839

Yoshimi, K., & Mashimo, T. (2022). Genome editing technology and applications with the type I CRISPR system. Gene and Genome Editing, 3–4, 100013. https://doi.org/10.1016/j.ggedit.2022.100013

O’Brien, R. E., Bravo, J. P., Ramos, D., Hibshman, G. N., Wright, J. T., & Taylor, D. W. (2023). Structural snapshots of R-loop formation by a type I-C CRISPR Cascade. Molecular Cell, 83(5), 746-758.e5. https://doi.org/10.1016/j.molcel.2023.01.024

Morisaka, H., Yoshimi, K., Okuzaki, Y., Gee, P., Kunihiro, Y., Sonpho, E., Xu, H., Sasakawa, N., Naito, Y., Nakada, S., Yamamoto, T., Sano, S., Hotta, A., Takeda, J., & Mashimo, T. (2019). CRISPR-Cas3 induces broad and unidirectional genome editing in human cells. Nature Communications, 10(1). https://doi.org/10.1038/s41467-019-13226-x

Cameron, P., Coons, M. M., Klompe, S. E., Lied, A. M., Smith, S. C., Vidal, B., Donohoue, P. D., Rotstein, T., Kohrs, B. W., Nyer, D. B., Kennedy, R., Banh, L. M., Williams, C., Toh, M. S., Irby, M. J., Edwards, L. S., Lin, C., Owen, A. L. G., Künne, T., . . . Sternberg, S. H. (2019). Harnessing type I CRISPR–Cas systems for genome engineering in human cells. Nature Biotechnology, 37(12), 1471–1477. https://doi.org/10.1038/s41587-019-0310-0

Makarova, K. S., Wolf, Y. I., Alkhnbashi, O. S., Costa, F., Shah, S. A., Saunders, S. J., Barrangou, R., Brouns, S. J. J., Charpentier, E., Haft, D. H., Horvath, P., Moineau, S., Mojica, F. J. M., Terns, R. M., Terns, M. P., White, M. F., Yakunin, A. F., Garrett, R. A., Van Der Oost, J., . . . Koonin, E. V. (2015). An updated evolutionary classification of CRISPR–Cas systems. Nature Reviews Microbiology, 13(11), 722–736. https://doi.org/10.1038/nrmicro3569

Additional resources on the blog

• CRISPR 101: Cas9 vs. The Other Cas(s)
• CRISPR 101: Anti-CRISPR Proteins Switch Off CRISPR-Cas Systems
• INTEGRATE: Bacterial Genome Engineering Using CRISPR-Transposons

Additional resources on Addgene.org

• CRISPR guide
• Cascade-Cas3 Plasmid collection