Single-stranded DNA with internal base modifications mediates highly efficient knock-in in primary cells using CRISPR-Cas9.

Autor:	Kanke KL; Center for Gene Therapy, Abigail Wexner Research Institute, Nationwide Children's Hospital, 700 Children's Drive, Columbus, OH 43215, USA., Rayner RE; Department of Veterinary Biosciences, The Ohio State University, Columbus, OH 43210, USA., Bozik J; Center for Gene Therapy, Abigail Wexner Research Institute, Nationwide Children's Hospital, 700 Children's Drive, Columbus, OH 43215, USA., Abel E; Center for Gene Therapy, Abigail Wexner Research Institute, Nationwide Children's Hospital, 700 Children's Drive, Columbus, OH 43215, USA., Venugopalan A; Center for Gene Therapy, Abigail Wexner Research Institute, Nationwide Children's Hospital, 700 Children's Drive, Columbus, OH 43215, USA., Suu M; Center for Gene Therapy, Abigail Wexner Research Institute, Nationwide Children's Hospital, 700 Children's Drive, Columbus, OH 43215, USA., Nouri R; Center for Gene Therapy, Abigail Wexner Research Institute, Nationwide Children's Hospital, 700 Children's Drive, Columbus, OH 43215, USA., Stack JT; Center for Gene Therapy, Abigail Wexner Research Institute, Nationwide Children's Hospital, 700 Children's Drive, Columbus, OH 43215, USA., Guo G; Institute for Genomic Medicine, Abigail Wexner Research Institute, Nationwide Children's Hospital, 700 Children's Drive, Columbus, OH 43215, USA., Vetter TA; Center for Gene Therapy, Abigail Wexner Research Institute, Nationwide Children's Hospital, 700 Children's Drive, Columbus, OH 43215, USA.; Department of Pediatrics, The Ohio State University, Columbus, OH 43210, USA., Cormet-Boyaka E; Department of Veterinary Biosciences, The Ohio State University, Columbus, OH 43210, USA., Hester ME; Institute for Genomic Medicine, Abigail Wexner Research Institute, Nationwide Children's Hospital, 700 Children's Drive, Columbus, OH 43215, USA.; Department of Pediatrics, The Ohio State University, Columbus, OH 43210, USA., Vaidyanathan S; Center for Gene Therapy, Abigail Wexner Research Institute, Nationwide Children's Hospital, 700 Children's Drive, Columbus, OH 43215, USA.; Department of Pediatrics, The Ohio State University, Columbus, OH 43210, USA.
Jazyk:	angličtina
Zdroj:	Nucleic acids research [Nucleic Acids Res] 2024 Nov 21. Date of Electronic Publication: 2024 Nov 21.
DOI:	10.1093/nar/gkae1069
Abstrakt:	Single-stranded DNA (ssDNA) templates along with Cas9 have been used for knocking-in exogenous sequences in the genome but suffer from low efficiency. Here, we show that ssDNA with chemical modifications in 12-19% of internal bases, which we denote as enhanced ssDNA (esDNA), improve knock-in (KI) by 2-3-fold compared to end-modified ssDNA in airway basal stem cells (ABCs), CD34 + hematopoietic cells (CD34 + cells), T-cells and endothelial cells. Over 50% of alleles showed KI in three clinically relevant loci (CFTR, HBB and CCR5) in ABCs using esDNA and up to 70% of alleles showed KI in the HBB locus in CD34 + cells in the presence of a DNA-PKcs inhibitor. This level of correction is therapeutically relevant and is comparable to adeno-associated virus-based templates. The esDNA templates did not improve KI in induced pluripotent stem cells (iPSCs). This may be due to the absence of the nuclease TREX1 in iPSCs. Indeed, knocking out TREX1 in other cells improved KI using unmodified ssDNA. esDNA can be used to modify 20-30 bp regions in primary cells for therapeutic applications and biological modeling. The use of this approach for gene length insertions will require new methods to produce long chemically modified ssDNA in scalable quantities. (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)
Databáze:	MEDLINE
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