Universität zu Köln

Rheinbay, Sarah (2025). A hypercompact epigenetic editor based on a novel engineered Cas12f nuclease. PhD thesis, Universität zu Köln.

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Abstract

CRISPR-associated (Cas) nucleases have become the dominant genome editing tool. However, risks associated with double-strand breaks (DSBs) have triggered the development of next generation CRISPR technologies which circumvent DSBs. This includes epigenetic editing. The well-characterized epigenetic editor (EE) CRISPRoff induces persistent gene silencing by targeted gene methylation and histone modifications. Numerous disease targets require in vivo delivery for which adeno associated viruses (AAVs) are a promising vehicle. However, the large-sized Streptococcus pyogenes Cas9 (SpyCas9) and the additional epigenetic effector domains exceed the AAV packaging limit. Furthermore, existing hypercompact EEs such as OMEGAoff, CHARM, and EvoETR face constraints in accessing DNA target sites. Specifically, the IscB-based OMEGAoff suffers from a suboptimal PAM profile (5’ NTAAA-3’). In contrast, both EvoETR and CHARM are challenged through complications inherent to the development of ZFN technologies, which introduce additional layers of complexity in their design and implementation. Our objective was to integrate the advantages of hypercompact EEs with the efficacy of CRISPR nucleases by substituting SpyCas9 with hypercompact nucleases from the type V-F family identified through microbial genome mining. To evaluate the novel nucleases as DNA binding platforms, we first tested 15 candidates (~450 amino acids) for CRISPR activation (CRISPRa) in HEK293T cells. As a result, we discovered that nuclease Aeribacillus pallidus Cas12f (ApCas12f) led to substantial reporter gene activation. The nuclease activity was further improved through protein engineering efforts, resulting in the creation of the triple mutant enhanced-ApCas12f (en-ApCas12f). We developed assays for epigenetic editing read-out utilizing CRISPRoff based on existing literature and observed enduring silencing of the cell surface protein CD81 following a single hit-and-run treatment with the editor. Subsequent sequencing techniques, including amplicon sequencing and methylation sequencing, demonstrated that the promoter remained intact and exhibited high levels of CpG methylation. We assessed ApCas12f as EE by combining the enzyme with various epigenetic effector domains, achieving over >60% silencing efficiency of a reporter plasmid. Additionally, the system induced >40% promoter methylation of the locus Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9). These findings suggest that ApCas12f-EE holds promise as a versatile platform for precise gene regulation. Going forward, it could enable safer therapeutic approaches that are free from risks associated with DSBs. This will ultimately result in an increased number of treatment options for patients.

Item Type:	Thesis (PhD thesis)
Creators:	Creators Email ORCID ORCID Put Code Rheinbay, Sarah sarah.rheinbay@gmail.com UNSPECIFIED UNSPECIFIED
URN:	urn:nbn:de:hbz:38-812340
Date:	2025
Language:	English
Faculty:	Faculty of Mathematics and Natural Sciences
Divisions:	Faculty of Mathematics and Natural Sciences > Department of Biology > Institute for Genetics
Subjects:	Life sciences
Uncontrolled Keywords:	Keywords Language CRISPR; DNA methylation; epigenome editing; dCas; epigenetics; miniature Cas; protein engineering UNSPECIFIED
Date of oral exam:	17 October 2025
Referee:	Name Academic Title Gehring, Niels Prof. Dr. Baumann, Ulrich Prof. Dr.
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/81234