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Nucleic Acid Enrichment And Sequencing Method For Detecting Off-Target Base Editing Events

Published:
Lead Inventor: Weixin Tang
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SUMMARY

Inrich-seq is a new method that selectively enriches and sequences inosine-containing DNA or RNA, enabling highly sensitive, genome-wide detection of off-target effects from adenine base editing in human cells

The Unmet Need: Techniques need to rigorously evaluate and profile any unintended genetic modifications

  • Adenine base editors (ABEs) represent a powerful class of genome editing tools designed to convert adenine to guanine in DNA. This precise genetic manipulation holds immense therapeutic potential for treating a wide array of genetic diseases. However, as with any genetic intervention, ensuring patient safety is paramount before these therapies can transition into clinical settings. Thoroughly assessing potential off-target effects is an essential prerequisite to guarantee the safety and efficacy of ABE-based treatments in human patients.

  • Despite the urgent need for rigorous safety assessments, current methods for detecting off-target editing events are fundamentally inadequate. Existing detection techniques suffer from a severe lack of sensitivity and specificity, resulting in high false-positive rates. Furthermore, previous chemical approaches used to identify editing intermediates exhibit broad reactivity, failing to distinguish target molecules from other naturally occurring nucleic acid bases like thymidine. These conventional methods also rely on harsh chemical conditions that degrade nucleic acids and destroy vital sequence information. Ultimately, these limitations render current approaches entirely incompatible with modern next-generation sequencing workflows, leaving a significant gap in clinical safety evaluation.

The Proposed Solution: Inrich-seq is a genome-wide sequencing technology that detects off-target adenine base editing by utilizing a novel inosine-specific chemical reaction to selectively enrich inosine-containing nucleic acid fragments

  • The faculty inventor developed inosine-containing nucleic acid enrichment and sequencing (Inrich-seq) designed for the unbiased, genome-wide detection of off-target editing events induced by adenine base editors. The solution functions by identifying inosine, an intermediate formed during adenine-to-guanine editing. It utilizes a novel inosine-specific chemical reaction, enzymatic processing, and biotin-streptavidin pulldown to selectively isolate inosine-containing nucleic acid fragments. These enriched fragments are then processed through next-generation sequencing and analyzed using a dedicated data pipeline. This end-to-end workflow enables the efficient, scarless enrichment of target fragments across DNA and RNA, providing a streamlined approach to profiling base editor activity.

  • This technology is highly differentiated from existing detection methods due to its unprecedented sensitivity and specificity. Previous chemical approaches exhibited broad reactivity that failed to distinguish inosine from natural bases like thymidine, utilizing harsh conditions that degraded nucleic acid sequence information. In contrast, Inrich-seq introduces optimized reagents that are strictly selective for inosine and operate under gentle conditions, preserving structural integrity. By seamlessly integrating these chemical advances with standard sequencing workflows, the solution achieves a remarkably low false-positive rate. This provides the high fidelity required to rigorously assess the clinical safety of therapeutic genome editing applications.

ADVANTAGES

ADVANTAGES

  • Provides highly sensitive, unbiased, and genome-wide detection of off-target adenine base editing (ABE) events

  • Delivers exceptional specificity with a low false-positive rate, significantly outperforming existing off-target detection methods

  • Utilizes a gentle, highly selective chemistry that enables "scarless" enrichment of inosine without degrading nucleic acid sequences

  • Offers a streamlined, end-to-end workflow that seamlessly integrates with standard next-generation sequencing (NGS) and purification protocols

  • Demonstrates versatility by being applicable for inosine detection in both DNA and RNA

  • Facilitates the clinical translation of ABE therapies by providing the rigorous safety and fidelity assessments required for therapeutic genome editing

APPLICATIONS

  • Off-target base editing detection

  • RNA inosine modification mapping

  • Gene therapy safety screening

  • Base editor tool development