ᐊ P3-49
Open Readings 2025 • P3-50
P3-52 ᐅ

CHARACTERIZATION OF HYBRID CLASS 1 AND CLASS 2 CRISPR-CAS SYSTEMS

Brigita Duchovska1, Tomas Šinkūnas1, Tautvydas Karvelis1

1 Department of Protein-DNA Interactions, Institute of Biotechnology, Vilnius University, Lithuania

[email protected]

CRISPR-Cas systems are diverse microbial RNA-guided adaptive immune systems that protect bacteria from viruses and other mobile genetic elements by using guide RNAs to recognize and target them. These systems have also served as the foundation for several molecular technologies, most notably programmable genome editing. CRISPR-Cas systems are classified into two classes. In class 1 CRISPR-Cas, the effector complex consists of several different Cas proteins to recognize foreign DNA, while a separate component – Cas3 – is responsible for cutting the target DNA. Although Class 1 systems have been used to induce targeted deletions in genomic DNA, the number and size of their constituent components limit their wider application in the genome editing field [1]. In class 2 CRISPR-Cas systems, the effector complex comprises a single protein responsible for both target recognition and cleavage. Because of this feature, the class 2 RNA-programmed Cas9 and Cas12 effector proteins have been widely adapted to cut genomic DNA, which can be exploited to introduce precise changes in the genome [2].

A recent study has identified previously unknown associations with core CRISPR effector modules [3]. One of the associations included hybrid Cas12m-Cas3 systems, where Cas3 nuclease-helicase, characteristic of class 1 CRISPR-Cas systems, is located in the same genomic locus as class 2 Cas12m proteins. Interestingly, the compact Cas12m proteins of this type cannot cut DNA but can strongly bind it in a PAM (short motif adjacent to the DNA target recognized by the Cas effector complex) dependent manner [4]. The Cas12-Cas3 system is a putative class 1-class 2 hybrid system in which a Cas12m may have associated with a Cas3 helicase-nuclease that might enact an interference mechanism beyond DNA binding.

This project aims to biochemically characterize Cas12m-Cas3 hybrid systems to gain a better understanding of their molecular mechanisms and function. The successful implementation of this project could lead to novel tools with additional functionalities for biotechnological applications.

[1] Dolan, A. E., Hou, Z., Xiao, Y., Gramelspacher, M. J., Heo, J., Howden, S. E., Freddolino, P. L., Ke, A., & Zhang, Y. (2019). Introducing a spectrum of long-range genomic deletions in human embryonic stem cells using type i crispr-cas. Molecular Cell, 74(5), 936-950.e5. https://doi.org/10.1016/j.molcel.2019.03.014

[2] Wang, J. Y., & Doudna, J. A. (2023). CRISPR technology: A decade of genome editing is only the beginning. Science, 379(6629), eadd8643. https://doi.org/10.1126/science.add8643

[3] Altae-Tran, H., Kannan, S., Suberski, A. J., Mears, K. S., Demircioglu, F. E., Moeller, L., Kocalar, S., Oshiro, R., Makarova, K. S., Macrae, R. K., Koonin, E. V., & Zhang, F. (2023). Uncovering the functional diversity of rare CRISPR-Cas systems with deep terascale clustering. Science, 382(6673), eadi1910. https://doi.org/10.1126/science.adi1910

[4] Bigelyte, G., Duchovska, B., Zedaveinyte, R., Sasnauskas, G., Sinkunas, T., Dalgediene, I., Tamulaitiene, G., Silanskas, A., Kazlauskas, D., Valančauskas, L., Madariaga-Marcos, J., Seidel, R., Siksnys, V., & Karvelis, T. (2024). Innate programmable DNA binding by CRISPR-Cas12m effectors enable efficient base editing. Nucleic Acids Research, 52(6), 3234–3248. https://doi.org/10.1093/nar/gkae016