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C6 - Gene Targeting and Gene Correction New Technologies

17: Systematic Discovery, In Vivo Delivery, and DNA Repair Mechanism of Single-Strand Annealing Protein for Precision Integration of Large DNA Sequences

Type: Oral Abstract Session

Presentation Details
Session Title: New Technologies for Gene Targeting and Gene Correction

The challenge of accurately inserting large DNA sequences is a significant limitation in current genome editing tools, particularly for in vivo gene therapy applications. Our project explores the potential of microbial recombination systems, focusing on phage-encoded Exonuclease and Single-Strand Annealing Proteins (Exo-SSAPs), for efficient, cleavage-free gene editing in mammalian cells. Building on prior work demonstrating large DNA integration via SSAPs directed by catalytically inactive dCas9, we investigated a large range of microbial recombination systems, identifying over 26,000 recombination systems originating from both prokaryotic and eukaryotic sources. Via this systematic survey, we identified top candidates with three-fold efficiency compared to reported SSAP editors. This improved system is shown to be compatible with in vivo delivery using AAV vectors, successfully integrating knock-in sequences in primary mouse cells. Further, we explored Exo-SSAP mediated integration and DNA repair mechanisms in human cells, particularly with dCas9 genome targeting to avoid activation of double-stranded break (DSB) repair pathways. Our genetic and chemical perturbation results revealed that the Mismatch Repair pathway (MMR) proteins could play a pivotal role in the Exo-SSAP mediated DNA insertion. Specifically, by targeting MMR enzymes, we could further enhance the efficiency of Exo-SSAP integration in human cells, indicating room for future improvements. Overall, our study highlights the significant potential of microbial recombination systems for large DNA sequence integration in human cells. The mechanistic part indicates that mismatch repair proteins could be a previously under-appreciated mechanism that influences DNA integration when DSB is absent during the editing process. These results open new avenues for understanding and applying large DNA insertion in gene therapy, setting the stage for in vivo therapeutic applications.

Figure. Left: mining of over 26,000 Exo-SSAP systems reveal differential cellular activities distributed across evolutionary tree. Right, proof-of-concept demonstration of large insertion (800bp) in primary hepatocyte using improved Exo-SSAP system.

Plain Language Summary
Our research tackles a key challenge in gene therapy: inserting large DNA sequences into cells. Traditional methods often struggle with large-scale gene-editing processes, limiting options for basic and translational studies. We've turned to nature for inspiration, exploring how microbes naturally shuffle large DNA pieces. Our focus is on recombination proteins from viruses/phages, known as Exo-SSAPs (Exonuclease / Single-strand Annealing Protein). We searched ~26,000 microbial systems and found highly efficient recombination systems that can exchange DNA in human cells, without the usual cutting of DNA strands. We've successfully tested this method for in vivo use via viral delivery, and investigated how human cells repair DNA during the editing process. Our findings show that mismatch repair proteins play a key role, opening doors to further boost the efficiency and precision of our system. This new tool could serve as a promising option for more effective and safer treatment of genetic diseases.

Le Cong1, Di Yin2, Guangxue Xu3, Yuanhao Jerry Qu1, Chengkun Wang1, Xiaotong Wang2, William Arthur Johnson4, Gabriel Filsinger2, Tim Wannier5, George M. Church6, Lai Yee Phoon7, Boya Gao7, Li Lan7

1Pathology, Genetics, Stanford University, Stanford, CA,2Stanford University, Stanford, CA,3Stanford University, Palo Alto, CA,4Stanford University Laboratory for Cell & Gene Medicine - Palo Alto, CA, Stanford, CA,5WildMicrobes, NA, CA,6Harvard Medical School, Boston, MA,7Duke University, Durham, NC"

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