High-efficiency HDR in immortalized cell lines by crude rAAV mediated donor template delivery

Abstract

Owing to low efficiency of homology-directed repair (HDR), precise knock-in (KI) of large DNA fragments is a challenge in genome editing. High-efficiency HDR has been reported for primary cells in preclinical gene therapy by combining CRISPR/Cas9 mediated induction of double-strand breaks (DSB) with delivery of a single-stranded DNA HDR-donor-template via highly purified recombinant adeno-associated virus (rAAV). Due in part to the labor and expense of rAAV particle purification, rAAV-mediated HDR-template delivery has been underutilized used to generate large KIs in cultured cell lines. Here, we report application of crude preparations of rAAV to deliver HDR-templates for the KI of large [~]2kb fragments at various genomic loci in several -human as well as mouse cell lines at high efficiency. Our approach should facilitate experiments necessitating KI of large DNA fragments to tag endogenous loci for visualization and/or conditional protein degradation.