Rational Mutants of Bxb1 Derived from Structural Insights

22225 – A method for site-specific integration of exogenous DNA into a cell genome using an editing polypeptide composed of a DNA nickase and a reverse transcriptase to insert an integration sequence, followed by an integrase enzyme that inserts the desired DNA payload at that site. The system uses a guide RNA with primer binding and integration sequences, enabling precise and programmable genomic modifications across diverse cell types and therapeutic applications.

23554 – Engineered serine integrases with improved efficiency and specificity for precise, programmable gene insertion in mammalian cells—non-DSB platform for gene and cell therapy. The sequences for thousands of novel serine integrases are covered in the filing. These integrases exhibit robust activity at endogenous or synthetic attB/attP sites, tolerate sequence truncations, and function efficiently when linked to DNA-binding domains (e.g., Cas9 nickases or zinc fingers) and reverse transcriptase domains to increase the efficiency of targeted integrations, which has previously not been shown for integrase-based genome editing.

23685 –  The gene editing complex comprises a CRISPR-based nickase, a fusion protein containing a reverse transcriptase domain linked to an RNA-binding protein, and a guide RNA engineered with a primer binding site, reverse transcription template, and protein-recruiting stem-loop. Together, these components enable programmable, site-specific integration of genetic material without double-strand breaks, using reverse transcription and integrase-mediated recombination at defined genomic loci.

24104 –  A programmable integration system that uses a pair of guide RNAs to encode integration sites via reverse transcription, enabling precise and flexible DNA insertion by splitting the integration sequence across two templates. Each gRNA contributes a portion of the integration site and includes a reverse transcription template and primer binding site, allowing for efficient, strand-specific integration while reducing sequence constraints and expanding targeting options.

24105 – Compact system using trans-template RNA (ttRNA) with aptamer and integration sequence for reverse transcription at nicked sites, followed by integrase-mediated DNA insertion. The ttRNA acts as a modular, programmable template separate from the guide RNA, enabling flexible delivery of integration sequences and recruitment of editing proteins via aptamer-binding domains—allowing precise, RNA-encoded site-specific DNA insertion without requiring extensive modification of the guide RNA itself.

25675 – Novel mutations in the Bxb1 integrase which increases its activity.  The variants are capable of binding attB/attP, engineered recognition site pairs, or endogenous pseudosites within the human genome and offer increased integration efficiencies over the WT enzyme for potential to increase levels for editing systems . 

25676 – Novel mutations in the Bxb1 integrase which increases its activity.  The variants are capable of binding attB/attP, engineered recognition site pairs, or endogenous pseudosites within the human genome and offer increased integration efficiencies over the WT enzyme for potential to increase levels for editing systems . 

26511 - Tangible property: Includes all available plasmids that can be transferred to a company to jumpstart its R&D program for PASTE genomic editing—available for licensing. 

**These technologies are available for exclusive licenses for use in human therapeutics, and non-exclusive licenses for applications in research tools/kits, agricultural biotechnology and animal genome editing.

Technology

The PASTE system operates through a precise, multi-step process that enables safe and efficient gene insertion. First, a guide RNA (gRNA) directs a Cas enzyme—such as a Cas9 nickase or Cas12—to a specific location in the genome. Instead of creating a double-strand break, the enzyme introduces a single-strand nick at the target site, minimizing the risk of genomic instability. A reverse transcriptase then uses the gRNA, which includes a primer binding site and an integration sequence, to transcribe the desired genetic payload into DNA directly at the nicked site. This payload can include large and complex sequences, such as full-length therapeutic genes, reporter constructs, or regulatory elements. A serine integrase subsequently recognizes specific attachment sequences on both the genome and the payload and catalyzes their site-specific integration. This approach allows for stable and predictable insertion of sizeable genetic sequences without relying on error-prone repair mechanisms.

Problem Addressed

This technology addresses several major challenges in genetic engineering: the inefficiency and unpredictability of inserting large DNA sequences, the risk of off-target effects, and the potential damage caused by DSBs. Traditional genome editing methods, such as CRISPR-Cas9, depend on creating DSBs that are then repaired by the cell’s natural repair machinery—processes that are often error-prone and can lead to insertions, deletions, or chromosomal rearrangements. These issues are especially problematic when trying to introduce large or complex genetic payloads, which are typically inserted with low efficiency and little control over the integration site. Such limitations pose serious safety concerns and reduce the reliability of gene therapies, making it difficult to develop robust and precise treatments for genetic diseases. The present technology overcomes these limitations by enabling programmable, site-specific insertion without DSBs, using a Cas-guided nickase, reverse transcriptase, and integrase to safely and efficiently install large genetic payloads at defined genomic locations.

Advantages

• Precise, site-specific integration of single bp small DNA sequences, and large DNA sequences (up to >30 kb) without relying on double-strand breaks
• Reduced genomic instability by using single-strand nicks instead of double-strand cuts
• High efficiency and fidelity in inserting complex or therapeutic genetic payloads
• Potential for all RNA-mediated genomic editing system
• Minimized off-target effects, increasing safety for clinical applications
• Versatility and scalability for use in gene therapy, synthetic biology, and functional genomics

Publications

• Yarnall, M.T.N., Ioannidi, E.I., Schmitt-Ulms, C. et al. Drag-and-drop genome insertion of large sequences without double-strand DNA cleavage using CRISPR-directed integrases. Nat Biotechnol 41, 500–512 (2023). https://doi.org/10.1038/s41587-022-01527-4.
• Rose, Joshua, et al. Engineered Bxb1 Variants Improve Integrase Activity and Fidelity. bioRxiv, October 21, 2024. https://doi.org/10.1101/2024.10.21.619419.
• Hazelbaker, Dane Z., et al. Large Serine Integrase Off-Target Discovery and Validation for Therapeutic Genome Editing. bioRxiv, August 23, 2024. https://doi.org/10.1101/2024.08.23.609471.
• Nan, Angela Xinyi, et al. Ligase-Mediated Programmable Genomic Integration (L-PGI): An Efficient Site-Specific Gene Editing System That Overcomes the Limitations of Reverse Transcriptase-Based Editing Systems. bioRxiv, September 27, 2024. https://doi.org/10.1101/2024.09.27.615478.
• Bakalar, Matthew H., et al. Large Serine Integrase Off-Target Discovery with Deep Learning for Genome Wide Prediction. bioRxiv, October 10, 2024. https://doi.org/10.1101/2024.10.10.617699.
• Estes, Brett J.G., et al. Breaking Free: Development of Circular AAV Cargos for Targeted Seamless Integration in the Liver. bioRxiv, October 26, 2024. https://doi.org/10.1101/2024.10.26.620313.
• Xie, Jenny, et al. Curative Levels of Endogenous Gene Replacement Achieved in Non-Human Primate Liver Using Programmable Genomic Integration. bioRxiv, October 12, 2024. https://doi.org/10.1101/2024.10.12.617700.