Divergent susceptibilities to AAV-SaCas9-gRNA vector-mediated genome-editing in a single-cell-derived cell population

7Citations
Citations of this article
28Readers
Mendeley users who have this article in their library.

This article is free to access.

Abstract

Objective: Recombinant adeno-Associated virus (AAV)-based vectors are characterized by their robust and safe transgene delivery. The CRISPR/Cas9 and guide RNA (gRNA) system present a promising genome-editing platform, and a recent development of a shorter Cas9 enzyme from Staphylococcus aureus (SaCas9) allows generation of high titer single AAV vectors which carry both saCas9-and gRNA-expression cassettes. Here, we used two AAV-SaCas9 vectors with distinct GFP-Targeted gRNA sequences and determined the impact of AAV-SaCas9-gRNA vector treatment in a single cell clone carrying a GFP-expression cassette. Results: Our results showed comparable GFP knockout efficiencies (40-50%) upon a single low-dose infection. Three consecutive transductions of 25-fold higher doses of vectors showed 80% GFP knockout efficiency. To analyze the "AAV-SaCas9-resistant cell population", we sorted the residual GFP-positive cells and assessed their permissiveness to super-infection with two AAV-Cas9-GFP vectors. We found the sorted cells were significantly more resistant to the GFP knockout mediated by the same AAV vector, but not by the other GFP-Targeted AAV vector. Our data therefore demonstrate highly efficient genome-editing by the AAV-SaCas9-gRNA vector system. Differential susceptibilities of single cell-derived cells to the AAV-SaCas9-gRNA-mediated genome editing may represent a formidable barrier to achieve 100% genome editing efficiency by this vector system.

Cite

CITATION STYLE

APA

Morsy, S. G., Tonne, J. M., Zhu, Y., Lu, B., Budzik, K., Krempski, J. W., … Ikeda, Y. (2017). Divergent susceptibilities to AAV-SaCas9-gRNA vector-mediated genome-editing in a single-cell-derived cell population. BMC Research Notes, 10(1). https://doi.org/10.1186/s13104-017-3028-4

Register to see more suggestions

Mendeley helps you to discover research relevant for your work.

Already have an account?

Save time finding and organizing research with Mendeley

Sign up for free