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Yarnall, Matthew T N; Ioannidi, Eleonora I; Schmitt-Ulms, Cian; Krajeski, Rohan N; Lim, Justin; Villiger, Lukas; Zhou, Wenyuan; Jiang, Kaiyi; Garushyants, Sofya K; Roberts, Nathaniel; Zhang, Liyang; Vakulskas, Christopher A; Walker, 2nd, John A; Kadina, Anastasia P; Zepeda, Adrianna E; Holden, Kevin; Ma, Hong; Xie, Jun; Gao, Guangping; Foquet, Lander; Bial, Greg; Donnelly, Sara K; Miyata, Yoshinari; Radiloff, Daniel R; Henderson, Jordana M; Ujita, Andrew; Abudayyeh, Omar O; Gootenberg, Jonathan S
Nature biotechnology, 04/2023, Volume: 41, Issue: 4Journal Article
Programmable genome integration of large, diverse DNA cargo without DNA repair of exposed DNA double-strand breaks remains an unsolved challenge in genome editing. We present programmable addition via site-specific targeting elements (PASTE), which uses a CRISPR-Cas9 nickase fused to both a reverse transcriptase and serine integrase for targeted genomic recruitment and integration of desired payloads. We demonstrate integration of sequences as large as ~36 kilobases at multiple genomic loci across three human cell lines, primary T cells and non-dividing primary human hepatocytes. To augment PASTE, we discovered 25,614 serine integrases and cognate attachment sites from metagenomes and engineered orthologs with higher activity and shorter recognition sequences for efficient programmable integration. PASTE has editing efficiencies similar to or exceeding those of homology-directed repair and non-homologous end joining-based methods, with activity in non-dividing cells and in vivo with fewer detectable off-target events. PASTE expands the capabilities of genome editing by allowing large, multiplexed gene insertion without reliance on DNA repair pathways.
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