8351 – Optimized CRISPR Base Editors

Technology Summary This technology includes optimized base editing tools that significantly improve DNA editing across multiple in vitro and in vivo model systems. Technology Overview The discovery and characterization of CRISPR DNA editing systems has revolutionized biotechnology, enabling unprecedented manipulation of host DNA for research and therapeutic applications. However, traditional CRISPR-Cas9 systems require introduction of a double-stranded DNA break, which has been demonstrated to carry risk of off-target effects and undesired recombination events. DNA base editors represent a promising alternative approach for targeting introduction of single-nucleotide variants (SNVs). By tethering DNA-modifying enzymes to nuclease-deficient Cas9 variants, base editors enable the direct conversion of C to other bases (T, A, or G) or A to inosine or G nucleic acids. However, current base editors are not optimized for expression in many in vivo model systems, reducing the applicability of the platform. This technology describes CRISPR base editors that have been reengineered to improve efficiency of DNA modification. The BE3 base editor was modified through codon-optimization of the Cas9n sequence that markedly increased protein expression and demonstrated 30-fold-higher target C-to-T conversion. These methods were also utilized to improve the expression of high-fidelity Cas9 (HF1), dramatically improving on-target DNA cleavage while maintaining little to no off-target activity. Further BE3 constructs were engineered that increased the range of editing (2X, through incorporation of an XTEN linker) or improved the efficiency of gene targeting (FNLS, through incorporation of an N-terminal FLAG epitope). Using the developed base editor toolkit, the inventors demonstrated target modification in human cancer cells, intestinal organoids, mouse embryonic stem cells, and mouse hepatocytes. As such, this technology promises to enable rapid generation of targeted SNVs in a variety of cell systems for both research and therapeutic applications. Potential Applications Genetic screens in a variety of in vitro and in vivo cell systems Generation of disease models for therapeutic development Correction of disease-causing mutations in genetic diseases and cancer Advantages Enhances DNA modification efficiency without increased off-targets effects Improves nuclear targeting through the inclusion of a nuclear localization sequence (NLS) Applicable to a variety of cell types and animal models Publications Zafra et al. “Optimized base editors enable efficient editing in cells, organoids and mice.” Nature Biotechnology. 2018. PCT Patent Application:WO/2020/033083. “Optimized base editors enable efficient editing in cells, organoids and mice.” Published Feb 13, 2020. Brian J. Kelly bjk44@cornell.edu 646-962-7045

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