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C4 - Targeted Gene Insertion (integrase mediated insertion -targeted or safe harbor)

193: Large Serine Integrase Off-Target Discovery and Validation for Therapeutic Human Genome Editing

Type: Oral Abstract Session

Presentation Details
Session Title: Targeted Gene Insertion

While numerous technologies for the characterization of potential off-target editing by CRISPR/Cas9 have been described, the development of new technologies and analytical methods for off-target recombination by Large Serine Integrases (LSIs) are required to advance the application of LSIs for therapeutic gene integration. Here we present the development of two complementary off-target recombination discovery technologies and a hybrid capture validation technology as a comprehensive framework for off-target characterization of LSIs. HIDE-Seq (High-throughput Integrase-mediated DNA Event Sequencing) is a PCR-free unbiased genome-wide biochemical assay capable of discovering sites with LSI-mediated DNA double-strand breaks and off-target recombination events. Cryptic-Seq is a PCR-based unbiased genome-wide biochemical or cellular-based assay that is more sensitive than HIDE-Seq but is limited to the discovery of sites with off-target recombination. Potential off-target loci discovered by HIDE-Seq and Cryptic-Seq can then be validated with our hybrid capture NGS sequencing of genomic DNA from cells after LSI-mediated gene insertion that has been qualified to 0.1% sensitivity. Hybrid capture sequencing of >450 discovered off-target sites led to the validation of 90 detectable off-target recombination sites with less than 1% frequency in cells. Furthermore, the lack of indels at these sites suggests that Bxb1 integrase does not generate significant double strand breaks, consistent with our HIDE-seq data. To demonstrate the sensitivity of our framework to discover bona fide sites of off-target recombination, we performed unbiased whole genome sequencing (WGS) on LSI-edited samples and found WGS detected only 2 of the 90 off-target sites discovered by our discovery and validation framework, yielding a 45-fold increase in sensitivity. We expect the dissemination of these sensitive technologies will advance the application of LSIs in therapeutic genome editing by establishing benchmarks for sensitivity of off-target detection.

Plain Language Summary
Large serine integrases are promising therapeutic enzymes to insert large pieces of DNA into cells to correct genetic diseases. Here we present new technologies to ensure accurate and specific gene correction in cells by these enzymes.

Dane Z. Hazelbaker, Japan B. Mehta, Didac Santesmasses, Connor McGinnis, Thomas Biondi, Anne M. Bara, Xiaoyu Liang, Brett Estes, Jenny Xie, Kaivalya Molugu, Ravindra Amunugama, Chong Luo, Jesse Cochrane, Sandeep Kumar, Matthew H. Bakalar, Jie Wang, Daniel J. O'Connell, Jonathan D. Finn

Tome Biosciences, Watertown, MA"

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