
==== Front
Mol Ther Nucleic Acids
Mol Ther Nucleic Acids
Molecular Therapy. Nucleic Acids
2162-2531
American Society of Gene & Cell Therapy

S2162-2531(24)00189-6
10.1016/j.omtn.2024.102302
102302
Commentary
Advancing gene editing by engineering miniature CRISPR-Cas Un1Cas12f1
Tang Weimin Weimin.Tang@childrens.harvard.edu
1∗
1 Laboratory for Biomaterials and Drug Delivery and Department of Medical Critical Care, Boston Children’s Hospital, Harvard Medical School, Boston, MA 02115, USA
∗ Corresponding author: Weimin Tang, Laboratory for Biomaterials and Drug Delivery and Department of Medical Critical Care, Boston Children’s Hospital, Harvard Medical School, Boston, MA 02115, USA. Weimin.Tang@childrens.harvard.edu
29 8 2024
10 9 2024
29 8 2024
35 3 102302© 2024 The Author(s)
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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pmcMain text

CRISPR-mediated base editing allows precise single-base changes without double-stranded breaks, which holds great potential in gene therapy for diseases caused by single-base mutations.1 However, the clinical translation of these technologies has faced challenges, primarily due to the size limitations of delivery vectors like adeno-associated viruses (AAVs). The recent study by Hu et al. addresses these issues by developing highly efficient and compact base editors based on the Un1Cas12f1 protein.2 These engineered base editors (Figure 1), termed STUminiABEs (adenine base editors) and STUminiCBEs (cytosine base editors), demonstrate robust A-to-G and C-to-T conversion. Moreover, these editors can be packed into a single AAV vector for efficient gene editing, which could overcome the size constraints that hamper the clinical application of most CRISPR tools.3 However, challenges such as off-target effects remain, and addressing these issues will be crucial for the safe and effective use of these miniature base editors in therapeutic applications.Figure 1 Schematic of nuclease-dead Un1Cas12f1-derived miniature cytosine base editor (STUminiCBE) and adenine base editor (STUminiABE)

Images were created with BioRender.com.

The delivery of CRISPR components into target cells remains a significant challenge, especially for in vivo applications. The size of the Cas protein and associated components often exceeds the packaging capacity of delivery vectors like AAVs, whose packing sizes are limited to approximately 4.7 kb.3 Thus, a search for more compact alternatives that can be efficiently delivered in a clinical setting is urgently needed. Recent advances in the field have focused on miniaturizing the CRISPR-Cas system. One promising approach is the use of compact RNA-guided nuclease, which have been shown to retain gene editing capabilities while being more amenable to packaging within AAV vectors.1 The Un1Cas12f1 protein is a type V-F nuclease, which has a significantly smaller size compared to SpCas9, making it a promising candidate for developing compact base editors.2 Despite initial engineering efforts, the base editing efficiency of Un1Cas12f1 in mammalian cells remained below 10%, making it insufficient for therapeutic applications.4

The study by Hu et al. addressed this gap by introducing a series of innovations to enhance the base editing efficiency of Un1Cas12f1. By introducing specific mutations (D143R/T147R/T203R/E206R), the interactions between Un1Cas12f1 and the target DNA were enhanced, and the resulting variant with quadruple mutations (dUn1Cas12f1QM) showed an improved base editing effect. To further enhance the base editing efficiency, a non-specific DNA-binding protein, Sso7d, was fused to the N terminus of the deaminase in Un1Cas12f1. The resulting constructs, SUminiABEs and SUminiCBEs, showed enhanced binding affinity to target DNA and base editing efficiency when compared to unfused constructs. They also focused on optimizing the sgRNA scaffold to reduce the size and further improve the base editing efficiency. By truncating non-essential regions of the sgRNA, they developed a more compact version that retained high editing efficiency. The optimized editors, termed STUminiBEs, achieved remarkable A-to-G and C-to-T conversion efficiencies averaging 54% and 45%, respectively. This optimization not only improved the editing efficiency but also allowed the packaging of these components into a single AAV vector. Moreover, one of the engineered base editors, STUminiCBEs, was loaded onto AAVs and introduced a premature stop codon in the PCSK9 gene in HEK293FT cells. The C-to-T conversion efficiency at the target site reached up to 63.52%, highlighting the potential therapeutic applications of these base editors.

The ability to package these base editors into a single AAV vector not only simplifies the delivery process but also reduces the potential for immune responses and toxicity associated with high vector doses,5 underscoring their practicality for clinical gene therapy. However, the study also raises important questions and challenges that need to be addressed. The off-target effects observed with STUminiBEs highlight the efforts that are needed for further refinement and optimization. Additionally, the editing efficiency of these systems can vary across different genomic contexts; future study should focus on optimizing the sgRNA design and exploring alternative strategies to minimize off-target effects and enhance the specificity of these editors. Overall, the engineered STUminiBEs mark a pivotal step in the development of gene editing technologies. As research continues to refine and expand the capabilities of these miniature CRISPR-Cas systems, we may soon see the translation of these gene editing tools into clinical practice.

Declaration of interests

The author declares no competing interests.
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