AccuBase™ Base Editor Enzyme: Globally In Stock Now

AccuBase™ is a new generation base editing tool independently developed by Base Therapeutics with exclusive global intellectual property rights based on foundational technology patents. AccuBase™ base editor enzyme is characterized by high editing efficiency, and extremely low off-target activity. By leveraging its advanced enzyme design and expression platform, KACTUS has successfully established a large-scale production process and manufactured GMP-grade AccuBase™ enzyme and obtained authorization from Base Therapeutics for global manufacturing and sales as of November 2023. KACTUS and Base Therapeutics are providing bio-pharmaceutical customers with on-hand GMP-grade gene editing enzymes, jointly promoting the development of cell and gene therapy programs.

State of Gene Editing Technology

The future has arrived; the novel CRISPR technology has been implemented and approved as a drug in just a decade, driven by a well-defined gene editing mechanism. On November 16, 2023, the UK Medicines and Healthcare products Regulatory Agency (MHRA) officially announced the approval of the CRISPR gene editing therapy Casgevy, jointly developed by Vertex Pharmaceuticals and CRISPR Therapeutics, for the treatment of Sickle Cell Disease (SCD) and Transfusion-Dependent β-Thalassemia (TDT) patients. 

This milestone demonstrates how CRISPR-based editing, which relies on DNA double-strand breaks and subsequent repair, can modulate gene expression at specified target DNA sequences. At the same time, it underscores the need to control risks such as unwanted INDELs, chromosomal translocation and DNA horizontal insertions that can arise when double-strand breaks are introduced at one or more target site positions across multiple genes..

Multinational companies have also heavily invested in their early layouts for gene editing tools. Recently, Eli Lilly invested $50 million in Beam Therapeutics and acquired the option to purchase several base editing treatment projects of its partner Verve Therapeutics for a total amount of up to $600 million, thus strengthening its pipeline in cardiovascular diseases. One important reason for their significant investment is to acquire the cytosine base editors from Beam Therapeutics, known as "CRISPR 2.0", which theoretically does not need to damage the DNA double-strand breaks and can perform precise editing of a single point mutation or other specific point mutations at defined target DNA positions, helping reduce the probability of editing-related structural variants.

Expression & Manufacturing of AccuBase™

Many scientists and biotech companies are playing significant roles in developing new base editors and making breakthroughs and layouts in underlying patent technology via independent research and development. Among all of them, AccuBase™ is a new generation of base editing enzymes independently developed by Base Therapeutics with exclusive global IP. These types of enzymes are often difficult to express due to their fusion protein nature or low and unstable yields, making them extremely challenging to use as an off-the-shelf, ready-to-use enzyme, which is a well-recognized challenge in the industry. Impressively, KACTUS has addressed these constraints by optimizing upstream and downstream parameters within a scalable production process. Using a dedicated expression platform, KACTUS has produced GMP-grade AccuBase™ with high yield and activity, delivering both research-grade and GMP formats with various sample concentrations suitable for method development, positive control benchmarking, and scale-up. This work provides convenient, high-quality tools for gene editing workflows that require effectively editing bases with high fidelity.

Principles and Advantages of Base Editors

DNA base editing technology is a new genome editing technology developed based on the CRISPR/Cas system. It includes CBE (Cytosine Base Editors) and ABE (Adenine Base Editors), which use their fused deaminase to modify bases within the editing stage window, enabling the replacement of single bases through DNA replication or repair. Compared to traditional CRISPR/Cas9 technology gene editing mechanism implementations, CBE or ABE does not generate DNA double-strand breaks or require donor DNAs and is not dependent on the cell cycle for precise gene repair (Table 1. Comparison of AccuBase™ with other gene editing tools). Most human genetic diseases are caused by single-nucleotide mutations, and thus, DNA base editing technology can be broadly applied in the treatment of these genetic diseases. Base editing can also knockout genes by mutating start codons, introducing stop codons, introducing premature stop codons, or altering splice sites such as disrupting splice acceptor regions or inducing exon skipping, thereby shutting down downstream gene expression.

Table 1. Comparison of AccuBase™ with other gene editing tools

	CRISPR Nucleases	BE4max	AccuBaseTM
DNA double-strand break	Yes	No	No
Target Insertion/Deletion	Yes	No	No
Single base editing	No	Yes	Yes
Editing efficiency	80-100%	80-100%	80-100%
Off target; Unwanted INDEL; chromosomal rearrangements	1%-5% Chromosomal translocation, Large insertion/deletion, abnormalities (1 to 3 gene editing)	Off target mutations	Close to zero
Size	4Kb	around 5.5Kb	around 5.5Kb

 

Due to the exposure of the deaminase, the traditional base editors are prone to random deamination, leading to off-target effects. This limits the application of base editors. To overcome the off-target problem, Base Therapeutics designed and engineered a new type of base editor, AccuBase™, which maintains high editing activity while significantly reducing the occurrence of off-target effects.

 

Principle of Gene Knockout with AccuBase™

The commercialized AccuBase™ by KACTUS is a cytosine base editor. This enzyme, when combined with sgRNA (including sgRNA for SpCas9), forms an RNP complex. Once introduced into the cells and bound to the target site, the RNP complex converts the cytosine at the respective position (the editing window is at positions 3-12, with the first position being far from the PAM site) into uracil without generating DNA double-strand breaks, eventually creating uracil to thymine mutation via DNA repair. The C->T mutation can also create gene knockout by generating stop codons or disrupting splicing sites on the target sequence (Figure 1).

In practice, this mechanism supports efficient base editing of a single point mutation or selected codons across multiple genes, without the need to produce DNA horizontal insertions or rely on donor templates.

Figure 1: Application of AccuBase™ in generating point mutation and gene knockout

 

High Base Editing Efficiency of AccuBase™

AccuBase™ is widely used in gene knockout in different cell types, with the highest knockout efficiency in T cells nearing 100%. AccuBase™ RNP by electroporation into T cells, followed by flow cytometry detection (Figure 2), shows that at the protein level, the AccuBase™ base editing system can efficiently knock out PD1, B2M, and TRAC proteins in T cells. Among these, the knockout efficiency of PD1 and B2M is above 80%, and the knockout efficiency of TRAC reaches 96%.

Figure 2: Knockout efficiency of AccuBase™ in T cells evaluated by flow cytometry.

 

Near-to-Zero Off-Target Occurrence with AccuBase™

The ingenious structural design and the inherent characteristics of single-base editing make AccuBase™ an editor with near-to-zero off-target effects compared to other editing tools, revolutionarily solving the issue of safety concern, the biggest pain point in the world gene editing industry.

 

By utilizing GOTI (genome-wide off-target analysis by two-cell embryo injection) to analyze the off-target levels of AccuBase™ across the genome (Figure 3), it was found that compared to the group using the other editor, (where over 700 SNVs were detected), the SNVs detected within the AccuBase™ group were close to the level of the GFP (negative) control group. These results indicate that the AccuBase™ is of higher safety compared to other gene editing tools.

Figure 3: Comparison of off-target effects by GOTI

 

By leveraging its Structure Aided Design and Multiplex Screening SAMS™ platform, KACTUS underwent process development for AccuBase™, a series of steps including screening of protein expression system, purification process optimization, and formulation development, and successfully produced DNA base editing proteins with high stability, high purity, and high activity. The manufacturing and filing for AccuBase™ is conducted in strict compliance with GMP standards, meeting the requirements in both the United States and China for clinical project filing. Currently, KACTUS and Base Therapeutics have reached an agreement on the production and sales of the GMP-grade base editor AccuBase™ around the world.

 

Product Information for AccuBase™

KACTUS provides both Research-Grade and GMP-grade AccuBase™ products on the market, which are currently in stock. Feel free to reach out and request a testing sample today!

Product Name	Catalog	Size
BS-EP1	KD-0001	200μg/500μg/1mg
BS-EP1 (GMP grade)	GMP-KD-0001	1mg

NOTE: BS-EP1 is product name, AccuBase™ is the commercial name.