Drag-and-drop genome insertion of large sequences without double-strand DNA cleavage using CRISPR-directed integrases.

Autor: Yarnall MTN; McGovern Institute for Brain Research at MIT, Massachusetts Institute of Technology, Cambridge, MA, USA., Ioannidi EI; McGovern Institute for Brain Research at MIT, Massachusetts Institute of Technology, Cambridge, MA, USA.; ETH Zürich, Zürich, Switzerland., Schmitt-Ulms C; McGovern Institute for Brain Research at MIT, Massachusetts Institute of Technology, Cambridge, MA, USA., Krajeski RN; McGovern Institute for Brain Research at MIT, Massachusetts Institute of Technology, Cambridge, MA, USA., Lim J; McGovern Institute for Brain Research at MIT, Massachusetts Institute of Technology, Cambridge, MA, USA., Villiger L; McGovern Institute for Brain Research at MIT, Massachusetts Institute of Technology, Cambridge, MA, USA., Zhou W; McGovern Institute for Brain Research at MIT, Massachusetts Institute of Technology, Cambridge, MA, USA., Jiang K; McGovern Institute for Brain Research at MIT, Massachusetts Institute of Technology, Cambridge, MA, USA.; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA., Garushyants SK; National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD, USA., Roberts N; Integrated DNA Technologies, Coralville, IA, USA., Zhang L; Integrated DNA Technologies, Coralville, IA, USA., Vakulskas CA; Integrated DNA Technologies, Coralville, IA, USA., Walker JA 2nd; Synthego Corporation, Redwood City, CA, USA., Kadina AP; Synthego Corporation, Redwood City, CA, USA., Zepeda AE; Synthego Corporation, Redwood City, CA, USA., Holden K; Synthego Corporation, Redwood City, CA, USA., Ma H; University of Massachusetts Chan Medical School, Worcester, MA, USA., Xie J; University of Massachusetts Chan Medical School, Worcester, MA, USA., Gao G; University of Massachusetts Chan Medical School, Worcester, MA, USA., Foquet L; Yecuris Corporation, Tualatin, OR, USA., Bial G; Yecuris Corporation, Tualatin, OR, USA., Donnelly SK; PhoenixBio USA Corporation, New York, NY, USA., Miyata Y; PhoenixBio USA Corporation, New York, NY, USA., Radiloff DR; PhoenixBio USA Corporation, New York, NY, USA., Henderson JM; TriLink Biotechnologies LLC, San Diego, CA, USA., Ujita A; TriLink Biotechnologies LLC, San Diego, CA, USA., Abudayyeh OO; McGovern Institute for Brain Research at MIT, Massachusetts Institute of Technology, Cambridge, MA, USA. omarabu@mit.edu., Gootenberg JS; McGovern Institute for Brain Research at MIT, Massachusetts Institute of Technology, Cambridge, MA, USA. jgoot@mit.edu.
Jazyk: angličtina
Zdroj: Nature biotechnology [Nat Biotechnol] 2023 Apr; Vol. 41 (4), pp. 500-512. Date of Electronic Publication: 2022 Nov 24.
DOI: 10.1038/s41587-022-01527-4
Abstrakt: 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.
(© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)
Databáze: MEDLINE
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