Development of an Enhanced Class of Cytosine Base Editors Using Engineered TadA Variants
The study, conducted by researchers at Beam Therapeutics and utilizing TadA variant libraries synthesized by Ranomics, focuses on the enhancement of cytosine base editors (CBEs). These gene-editing enzymes are designed to introduce C·G-to-T·A transition mutations in the genomic DNA of living cells. Traditional CBEs had limitations, such as causing unguided, genome-wide cytosine deamination. The researchers developed a new class of CBEs referred to as CBE-Ts, which utilize engineered variants of TadA and demonstrated high on-target efficiency in converting C·G to T·A across diverse genomic loci, with minimal off-target effects.
The study also introduced cytosine and adenine base editors (CABEs), capable of catalyzing both A-to-I and C-to-U editing, thereby expanding the range of programmable transition mutations. The engineered TadA variants, especially in CABE-Ts and CBE-Ts, exhibited improved precision, reduced off-target effects, and compatibility with primary cell types like T cells and hepatocytes.
The comprehensive results of this study can be found in this publication, providing valuable insights into the potential therapeutic applications of these advanced base editors for precise gene editing in living cells.
Methodology & Key findings:
TadA Variant Libraries by Ranomics:
Synthetic libraries for directed evolution rounds one and two were obtained from Ranomics.
Round one involved the TadA*8.20 library, representing each amino acid position with all 20 amino acid substitutions at a frequency of 1–3 substitutions per library member (~10 million members).
Round two featured the TADAC1.02 synthetic library, representing each amino acid position with all 20 amino acid substitutions at a frequency of 2–3 substitutions per library member (~10 million members).
Directed Evolution Success:
Ten rounds of directed evolution were conducted, resulting in highly engineered TadA variants for CBEs (CBE-Ts).
Accumulation of over 29 substitutions in the most engineered CBE-Ts demonstrated the success of directed evolution in refining the gene-editing tools.
Improved Precision and Efficiency:
Crystal structures of TadA deaminase variants associated with CBE-Ts were determined, guiding structure-guided mutagenesis for improved precision.
CBE-Ts exhibited comparable on-target editing efficiencies to traditional CBEs (BE4) but with a more precise editing window and reduced guide-dependent off-target editing.
Cellular Compatibility and Therapeutic Potential:
CBE-Ts demonstrated compatibility with primary cell types such as T cells and hepatocytes, validating their potential therapeutic application.
Editing outcomes in therapeutically relevant cell types, including reduction of secreted PCSK9 protein, showcased the versatility and effectiveness of CBE-Ts.
The collaborative efforts of researchers at Beam Therapeutics and Ranomics resulted in the successful development of advanced CBEs, demonstrating improved precision and efficacy. The engineered TadA variants, especially in CBE-Ts and CABE-Ts, exhibited superior precision, reduced off-target effects, and compatibility with primary cell types. These findings have significant implications for the development of therapeutic tools for precise gene editing, offering a potential breakthrough in the treatment of genetic diseases and malignancies.
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