Due to its precise genome editing capabilities, the prokaryotic CRISPR–Cas system has revolutionized molecular biology – and has potential applications in gene therapy and biotechnology . Nonetheless, recent research has highlighted the need for more compact CRISPR-Cas complexes, which could be delivered to cellular systems more efficiently .
A candidate that meets the compact size criterion is the CRISPR-Cas12f1 complex, which has been studied in depth by researchers led by Ralf Seidel (Leipzig University) and Virginijus Siksnys (Vilnius University). These groups also first characterized the system, in 2021, in terms of its composition, DNA cleavage and target recognition .
A core tool in their research has been mass photometry, which helped them determine how the Cas12f1 proteins assemble into a complex with guide RNA and how they subsequently bind to target DNA.
We recently spoke to Selgar Henkel-Heinecke, a PhD student in Ralf Seidel’s group, about this exciting work and the role of mass photometry.
To find out more, read on and register for an upcoming webinar, in which Selgar explains his research.
“I am a PhD student at the Peter Debye Institute for Soft Matters Physics at University of Leipzig. I am part of the group of Ralf Seidel, who works with CRISPR proteins and DNA origami. I did my Master’s degree in Chemistry at the Martin Luther University Halle-Wittenberg.”
On LinkedIn: Selgar Henkel-Heinecke | LinkedIn
Why the miniature CRISPR-Cas12f1 complex would be ideal for gene therapy
The key benefit of the compact CRISPR-Cas12f1 system is that, due to its small size (400 – 700 amino acids), it can be packed into small vehicles for efficient delivery into cells . A recent study demonstrated that a miniature CRISPR-Cas12f1 complex could be delivered by an adeno-associated virus (AAV) into eukaryotic cells, where it then cleaved DNA with high specificity .
The experimental aim: Can theCRISPR-Cas12f1 system be used for genome editing in-vivo?
The aim of Selgar and his colleagues was to demonstrate experimentally that Cas12f1 nucleases can be used for genome engineering in eukaryotic cells. To do this, they had to first determine the biophysical and biochemical properties that underlie the assembly of the CRISPR-Cas12f1 complex and the mechanism it uses to recognize and cleave dsDNA. Mass photometry helped gain important insights.
Selgar explains that they used mass photometry, to explore “the composition and binding dynamics of Cas12f1-RNA-complexes.” They also used magnetic tweezers (a high-resolution technique that can determine the mechanistic properties of supercoiled DNA)for DNA untwisting measurements to better understand the target recognition process on the single-molecule level.
Using mass photometry to determine the composition and stability of CRISPR-Cas12f1 complexes
A critical step, according to Selgar, was “to determine how the Cas12f1 nuclease binds to guide RNA (gRNA), comprising the ribonucleoprotein (RNP) effector complex and how this complex subsequently interacts with double-stranded DNA.”
He and his colleagues used mass photometry to assess the assembly and oligomerization of CRISPR-Cas12f1 complexes from two different bacterial species – Syntrophomonas palmitatica (Sp) and Acidibacillus sulfuroxidans (As) –in solution. They also assessed the protein-RNA and protein-DNA interactions involved.
In both organisms, mass photometry showed that the Cas12f1 nuclease formed a binary complex with gRNA and, while bound, assembled into a ternary complex with the dsDNA target .
When they measured the mass of gRNA and Cas12f1 alone, they saw that both existed predominantly as monomers. They, therefore, expected an assembly with a 1:1 stoichiometry, coinciding with previous data for other class 2 effectors. However, Selgar reports that when they mixed gRNA and Cas12f1, “we were surprised to notice that the masses of the major species were 179kDa for SpCas12f1 and 168 kDa for AsCas12f1, which corresponded to a 2:1Cas12f1:gRNA stoichiometry”. This unique self-dimerization property of Cas12f1 enzymes could offer additional flexibility to the CRISPR-Cas toolbox, as the proteins could perform their nuclease and DNA binding functions simultaneously. This was confirmed from the composition of the Cas12f1:gRNA:dsDNA complex. In agreement with previous cryo-EM data , mass photometry showed that the ternary SpCas12f1 and AsCas12f1:gRNA:dsDNA complexes had a 2:1:1 stoichiometry, meaning that two nucleases bind one gRNA and one dsDNA molecule (Fig. 1).
Fig. 1: Mass photometry determined the composition of CRISPR-Cas12f1 binary and ternary complexes For both a) SpCas12f1 and b) AsCas12f1, gRNA and Cas nuclease existed predominantly as monomers. The binary SpCas12f1 and AsCas12f1:gRNA complexes (green line) had a 2:1 stoichiometry. The ternary SpCas12f1 and As:gRNA:dsDNA complexes (blue line) had a 2:1:1 stoichiometry. Figure source: 
In addition, Selgar used mass photometry further – to assess the stability of the complex in different temperatures and salt concentrations. This gave him enough information to progress with experiments using single-molecule magnetic tweezers. “The nice thing about mass photometry,” Selgar says,“ is that it happens in solution, without requiring labels or sample fixation. Therefore, I am sure of what to expect when I use magnetic tweezers since I know how long my complex can remain stable at the desired temperature and conditions.”
“The nice thing about mass photometry is that it happens in solution, without requiring labels or sample fixation. Therefore, I am sure of what to expect when I use magnetic tweezers, since I know how long my complex can remain stable at the desired temperature and conditions.”
The untwisting measurements with magnetic tweezers showed that the RNP complex adopts a stable R-loop formation that is required for DNA cleavage and is driven by a conformational change within the complex.
Due to their compact size and self-dimerization, Cas12f1 enzymes are promising candidates for efficient cellular delivery and genome editing. Recently, the system successfully modified genomic DNA, after being delivered by an AAV into eukaryotic cells. As the authors of that study hypothesized, CRISPR-Cas12f1 complexes could be used to efficiently delete pathogenic exons or introns, and potentially treat diseases such as Usher’s syndrome or Duchenne muscular dystrophy .
Using mass photometry, Selgar and his colleagues gathered further insights about how these complexes operate, an important step towards wider therapeutic applications. They elucidated the composition and binding dynamics of the CRISPR-Cas12f1 effector complexes and determined how they interact with guide RNA and target DNA.
This was certainly not the first time the group had tried mass photometry, Selgar reports. Due to its simplicity and fast data generation, mass photometry is used routinely in Seidel’s lab – to check the purity of protein and DNA samples in solution, and analyze the binding and oligomerization states of complexes. Selgar says he is looking forward to how the technology could develop further, for example when combined with fluorescence, for even more applications.
This note describes in detail how to use mass photometry to measure the mass, purity and relative abundance of DNA, in the range of 100 to 5000 base pairs.
Here you can learn how mass photometry can be used to measure protein oligomerization in different experimental conditions – and even detect rare oligomeric species.
In this webinar, Dr. Margaret Stratton (UMass Amherst) talks about her work on the role of CaMKII in different tissues. Mass photometry has been used to quantitatively determine the stoichiometry of different variants of this crucial oligomeric enzyme.
In this webinar, mass photometry is used to determine how the compact Cas12f1 nucleases interact with guide RNA (forming a binary complex)and double-stranded DNA (forming a ternary complex). This mechanistic understanding can help improve genome editing applications in the future.
 S. Nidhi et al., “Novel CRISPR–Cas Systems: An Updated Review of the Current Achievements, Applications, and Future Research Perspectives,” International Journal of Molecular Sciences, vol. 22, no. 7, p. 3327, Mar. 2021, doi: 10.3390/ijms22073327.
 D. Y. Kim et al., “Efficient CRISPR editing with a hypercompact Cas12f1 and engineered guide RNAs delivered by adeno-associated virus,” Nature Biotechnology, vol. 40, no. 1, Art. no. 1, Jan. 2022, doi: 10.1038/s41587-021-01009-z.
 G. Bigelyte et al., “Miniature type V-F CRISPR-Cas nucleases enable targeted DNA modification in cells,” Nature Communications, vol. 12, no. 1, Art. no. 1, Oct. 2021, doi: 10.1038/s41467-021-26469-4.
 S. N. Takeda et al., “Structure of the miniature type V-F CRISPR-Cas effector enzyme,” Molecular Cell, vol. 81, no. 3, pp. 558-570.e3, Feb. 2021, doi: 10.1016/j.molcel.2020.11.035.