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

• Published in: BMC research notes Vol. 10; no. 1; p. 720
• Main Authors: Morsy, Salma G, Tonne, Jason M, Zhu, Yaxi, Lu, Brian, Budzik, Karol, Krempski, James W, Ali, Sherine A, El-Feky, Mohamed A, Ikeda, Yasuhiro
• Format: Journal Article
• Language: English
• Published: England BioMed Central Ltd 08-12-2017; BioMed Central; BMC
• Subjects: Adeno Associated viral vector; Bacterial Proteins; Cell Line; CRISPR-Associated Protein 9; CRISPR-Associated Proteins - genetics; CRISPR-Cas Systems; Dependovirus; Dependoviruses; Disease Susceptibility; DNA sequencing; Endonucleases - genetics; Exome Sequencing; Gene Editing; Genetic aspects; Genetic engineering; Genetic Vectors; Genome editing resistance; Green Fluorescent Proteins; HEK293 Cells; Humans; Off-target; Physiological aspects; Research Note; RNA, Guide, CRISPR-Cas Systems; SaCas9 optimization; Staphylococcus aureus - enzymology; Whole exome sequencing; United States; Egypt; SaCas9 optimization; Genome editing resistance; Off-target; Adeno Associated viral vector; Whole exome sequencing
• ISSN: 1756-0500; 1756-0500
• DOI: 10.1186/s13104-017-3028-4

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.
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.