A Single "All-in-One" Helper-Dependent Adenovirus to Deliver Donor DNA and CRISPR/Cas9 for Efficient Homology-Directed Repair

Molecular Therapy. Methods & Clinical Development
Donna PalmerPhilip Ng

Abstract

In this study, we developed a single helper-dependent adenovirus (HDAd) to deliver all of the components (donor DNA, CRISPR-associated protein 9 [Cas9], and guide RNA [gRNA]) needed to achieve high-efficiency gene targeting and homology-directed repair in transduced cells. We show that these "all-in-one" HDAds are up to 117-fold more efficient at gene targeting than donor HDAds that do not express CRISPR/Cas9 in human induced pluripotent stem cells (iPSCs). The vast majority (>90%) of targeted recombinants had only one allele targeted, and this was accompanied by high-frequency indel formation in the non-targeted allele at the site of Cas9 cleavage. These indels varied in size and nature, and included large deletions of ∼8 kb. The remaining minority of recombinants had both alleles targeted (so-called bi-allelic targeting). These all-in-one HDAds represent an important platform for accomplishing and expanding the utility of homology-directed repair, especially for difficult-to-transfect cells and for in vivo applications.

References

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Sep 5, 2008·Proceedings of the National Academy of Sciences of the United States of America·Keiichiro SuzukiKohnosuke Mitani
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Dec 5, 2018·Molecular Therapy. Nucleic Acids·Emily XiaJim Hu
May 14, 2019·Molecular Therapy. Methods & Clinical Development·Donna J PalmerPhilip Ng
Jan 1, 2020·Molecular Therapy. Methods & Clinical Development·Donna J PalmerPhilip Ng

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Citations

May 28, 2020·International Journal of Molecular Sciences·Ana RicobarazaRuben Hernandez-Alcoceba
Jun 13, 2020·Cancers·Praveensingh B HajeriMasato Yamamoto
Aug 21, 2020·Human Gene Therapy·Amber Vu, Paul B McCray
Dec 23, 2020·Nanoscale·Li DuanJiang Xia
Aug 18, 2020·Human Gene Therapy·Alejandro Da Silva SanchezJames E Dahlman
Mar 6, 2021·Molecular Therapy. Methods & Clinical Development·Dominik BrücherAndreas Plückthun
May 11, 2021·Frontiers in Pharmacology·J MercierL Guillot
Jun 24, 2021·JHEP Reports : Innovation in Hepatology·Sheila MaestroGloria Gonzalez-Aseguinolaza
Jul 25, 2021·International Journal of Molecular Sciences·Mousa A AlghuthaymiKamel A Abd-Elsalam

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Methods Mentioned

BETA
PCR
genetic modification
PCRs

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