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The discovery of forty novel adenine base editors with superior editing performance

In an exciting collaboration, Ranomics used its proprietary VariantFind platform to generate a series of site-saturation libraries for Beam Therapeutics’ Adenine Base Editor. The library was used for screening and identifying enhancing mutations in mammalian cells. Leveraging variant libraries synthesized by Ranomics , the research team at Beam Therapeutics identified forty novel eighth-generation adenine base editors with remarkable editing efficiencies, surpassing ABE7.10 (previous best version) in mammalian cells.

 

Ranomics' VariantFind platform played a pivotal role in this process, enabling the synthesis of a long range combinatorial library of TadA variants with all pairwise substitutions along the open reading frame. The resulting ABE8 variants proved to be a powerful suite of tools, showcasing superior editing properties and offering a game-changing solution for robust, programmable A•T to G•C DNA editing.

The detailed findings are available in the published article, emphasizing the potential of ABE8s in addressing critical challenges associated with gene therapy.

"Directed Evolution of Adenine Base Editors with Increased Activity and Therapeutic Application"

Key findings:

 Ranomics VariantFind Site-Saturation Mutagenesis Libraries:

  • Directed evolution of DNA-modifying TadA enzyme - with newly synthesized TadA*8.0 library

  • Library was designed to encode all 20 amino acids at each amino acid position in the previously published TadA*7.10 open reading frame.

  • Enabled access to a greater sequence space than is achievable with error-prone PCR techniques.

  • Identified new mutations that were unobserved with the previously published TadA error-prone PCR libraries. 

Activity of evolved TadA variants in mammalian cells:

  • ABE8s result in ∼1.5x higher editing at canonical positions (A5-A7) in the protospacer and ∼3.2x higher editing at non-canonical positions (A3-A4, A8-A10) compared with ABE7.10 

  • A sub-set of ABE8 constructs significantly outperform ABE7.10 at all genomic sites tested 

Editing efficiencies of Cas9 PAM-variant ABE8s and catalytically dead Cas9 ABE8 variants

  • 1.6 to 2.0 fold increase in A•T to G•C editing frequencies when comparing ABE8 variants to ABE7.10.

  • ABE8s retain high specificity for A-to-G conversion.

  • ABE8s causes no significant genome-wide, guide-independent adenine deamination.

Therapeutic application of ABE8s

  • Evaluated the clinical relevance of ABE8 variants in human hematopoietic stem cells (HSC) by reproducing Hereditary Persistence of Fetal Hemoglobin (HPFH) phenotype

  • Average ABE8 editing efficiencies at the −198 HBG1/2 promoter target site were 2-3x higher than either ABE7.10 construct at early time points (48h), and 1.3-2-fold higher than either ABE7.10 at the later time (144h)

  • 3.5-fold average increase in % γ-globin/α-globin expression in erythrocytes derived from the ABE8 treatment groups

  • ABE8s enabled efficient, multiplexed editing in T cells, opening new possibilities for cell therapies such as CAR-Ts.

  • Evaluation of off-target effects demonstrated minimal spurious editing of intracellular mRNA when using ABE8s delivered as mRNA constructs.

The collaboration between Ranomics and Beam Therapeutics has resulted in the development of ABE8s, a groundbreaking suite of tools for precise DNA editing. This advancement holds promise for therapeutic applications, marking a significant leap in the field of gene editing. ABE8s offer potential applications for correcting disease-associated SNPs, reactivating gene expression in CD34+ cells, and enabling multiplexed editing for adoptive T cell therapies.

Ranomics excels in synthesizing diverse mutagenesis libraries, ranging from degenerate NNK to codon-optimized versions, effectively addressing low diversity challenges. For a comprehensive understanding of VariantFind's capabilities, we invite you to download our informative brochure.

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