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Solution for Miniature Base Editors in Gene Therapy 
Miniature editors play a crucial role in genomic editing and gene therapy, particularly in addressing human genetic diseases caused by base mutations. Currently, approximately 60% of known human genetic diseases are attributed to base mutations. Traditional CRISPR technology poses risks such as double-stranded DNA breaks, whereas base editors can efficiently convert single bases at target sites without such breaks. Currently, the most widely used base editors include:
- Cytosine base editors (CBEs): used to mutate C-G to T-A.
- Adenine base editors (ABEs): used to mutate A-T to G-C.
Figure 1. CRISPR DNA Base-Editing Tools. (Kantor A, et al., 2020)
Importance of Delivery Efficiency and Tissue Specificity
The efficiency and tissue specificity of base editor delivery are critical for the success of gene therapy. Adeno-associated viruses (AAVs) have become the primary delivery tool for gene therapy due to their high efficiency and tissue specificity. However, base editors mediated by SpCas9 nickase (D10A) are too large to be packaged into AAVs, which have a packaging limit of approximately 4.7 kb. Thus, developing more compact and efficient base editors is essential.
Advances in Compact RNA-Guided Nucleases for Genomic Editing
Scientists have discovered and leveraged a series of compact RNA-guided nucleases for genomic editing, including Cas12f, Cas12m, Cas12n, IscB, Fanzor, among others.
Hu et al. developed the Un1Cas12f1 high-efficiency base editor. This nuclease comprises only 529 amino acids, which is just 39% the size of SpCas9. They designed editors for two types of base transitions: UminiABEs: For A-to-G transitions and UminiCBEs: For C-to-T transitions.
Han et al. engineered the OgeuIscB and its corresponding ωRNA to develop an efficient IscB system in mammalian systems, termed enIscB. By fusing cytidine or adenosine deaminase to the enIscB nickase, they generated miniature IscB-derived base editors (miBEs) that exhibit strong editing efficiency (up to 92%) for inducing DNA base conversions.
Chen et al. discovered the miniature V-U4 nuclease (termed Cas12n, 400-700 amino acids), which recognizes unique 5'-AAN PAM sequences, also required by TnpB. They designed an efficient CRISPR-Cas12n system, termed Cas12Pro, achieving insertion efficiencies of up to 80% in human cells. The engineered Cas12Pro is capable of performing base editing in human cells.
Miniature base editors not only address the size limitations of traditional base editors but also show great potential in gene therapy, especially for treating genetic diseases caused by single base mutations.
Directions for Further Development of Micro Base Editors
Enhancing Editing Efficiency: Optimize the catalytic domains and other enzymatic components to improve their activity and efficiency in editing targeted genetic sequences. Tailor sgRNAs to better guide the base editors to their targets, thereby increasing the overall efficiency of the editing process across various genetic targets.
Expanding Targeting Range: Create micro-base editors capable of recognizing and editing a broader range of DNA sequences, including those with non-standard PAM sites, to increase their applicability across different genomic contexts. Implement engineering strategies and novel molecular designs to enhance the precision of base editing, thereby minimizing undesirable off-target modifications.
Optimizing Delivery Mechanisms: Develop new delivery vectors or improve existing systems, such as AAV, to transport base editors more efficiently and specifically to target tissues or cells, accommodating the size constraints and ensuring effective delivery.
Combining with Other Technologies: Explore the potential of combining micro base editors with other gene-editing tools, such as prime editors, to create versatile and powerful therapeutic solutions, expanding the scope of possible genetic interventions.
Preclinical and Clinical Validation: Conduct comprehensive preclinical and clinical studies to ascertain the safety, efficacy, and therapeutic potential of micro-base editors in treating genetic diseases, ensuring their readiness for clinical applications.
Collaboration Opportunity with Creative Biogene
Creative Biogene, with its profound expertise in gene editing technologies, is dedicated to advancing and applying miniature CRISPR technologies. We offer a variety of pre-made viral particles for gene therapy and research, including AAVs and lentiviruses. Our Transfected Stable Cell Lines are highly efficient for long-term gene expression studies and drug screening.
Creative Biogene CRISPR PlatformCB is committed to providing comprehensive technical support, resource sharing, and professional services to help our partners achieve groundbreaking progress in this important field. We extend an earnest invitation to research institutions, pharmaceutical companies, and academic partners to collaborate with us in the further exploration and development of miniature CRISPR technologies. Together, we can make significant advancements in the treatment of genetic diseases.
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References:
- Han L, Mo Q, Du Z, Jiang W, Wu X, Zheng J, Xiao X, Sun Y, Ma H. Engineering miniature CRISPR-Cas Un1Cas12f1 for efficient base editing. Mol Ther Nucleic Acids. 2024 Apr 25;35(2):102201.
- Chen W, Ma J, Wu Z, Wang Z, Zhang H, Fu W, Pan D, Shi J, Ji Q. Cas12n nucleases, early evolutionary intermediates of type V CRISPR, comprise a distinct family of miniature genome editors. Mol Cell. 2023 Aug 3;83(15):2768-2780.e6.
- Han D, Xiao Q, Wang Y, Zhang H, Dong X, Li G, Kong X, Wang S, Song J, Zhang W, Zhou J, Bi L, Yuan Y, Shi L, Zhong N, Yang H, Zhou Y. Development of miniature base editors using engineered IscB nickase. Nat Methods. 2023 Jul;20(7):1029-1036.