The use of adeno-associated viruses (AAVs) as donors for homology-directed repair (HDR)-mediated genome engineering is limited by safety issues, manufacturing constraints and restricted packaging limits. Non-viral targeted genetic knock-ins rely primarily on double-stranded DNA (dsDNA) and linear single-stranded DNA (lssDNA) donors.
The use of adeno-associated viruses as donors for homology-directed repair -mediated genome engineering is limited by safety issues, manufacturing constraints and restricted packaging limits. Non-viral targeted genetic knock-ins rely primarily on double-stranded DNA and linear single-stranded DNA donors. dsDNA is known to have low efficiency and high cytotoxicity, while lssDNA is challenging for scaled manufacture.
We thank T. Cha for designing and cloning initial phagemid constructs and for guidance and suggestions on cssDNA production. We thank Quintara Biosciences for plasmid construction and DNA/RNA sequencing service for this project; LumiGenics for the in vivo CAR-T functional assays; and MaxCyte for technical support and suggestions for the electroporation experiment. We also thank M. C. Lorence for comments and suggestions on the manuscript. Cartoon illustrations were generated with BioRender.
Springer Nature or its licensor holds exclusive rights to this article under a publishing agreement with the author or other rightsholder; author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
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