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

Salma G. Morsy, Jason M. Tonne, Yaxi Zhu, Brian Lu, Karol Budzik, James W. Krempski, Sherine A. Ali, Mohamed A. El-Feky, Yasuhiro Ikeda

Research output: Contribution to journalArticlepeer-review

2 Scopus citations


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.

Original languageEnglish (US)
Article number720
JournalBMC Research Notes
Issue number1
StatePublished - Dec 8 2017


  • Adeno Associated viral vector
  • Genome editing resistance
  • Off-Target
  • SaCas9 optimization
  • Whole exome sequencing

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)


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