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

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.

Keywords: Adeno Associated viral vector; Genome editing resistance; Off-target; SaCas9 optimization; Whole exome sequencing.

Fig. 1

a Schematic representation of the AAV vector constructs used in this study. GFP-targeted SaCas9 AAV vectors were produced through introduction of 20 homologous nucleotide sequences for the target GFP sequences into the pX601 plasmid. SaCas9 and gRNA are expressed under the control of CMV and U6 promotors, respectively. b GFP expressing HT1080 single cell clones were generated by transduction with lenti-GFP-Puro vector at an MOI 0.0001, followed by puromycin selection and expansion of single cell clones. Fluorescence-activated cell sorting was performed to confirm GFP positive cell populations (middle panels). When GFP#F cells were transduced with AAV-Cas9-GFP1 and AAV-Cas9-GFP2 at MOI 2 × 10⁴, 42.7 and 55.5% GFP knockout efficiency was observed (right panels, n = 2). c Targeted genome editing was verified by sequencing. gRNA-targeted regions were amplified and sequenced. Deletions at the targeted sequences by gRNA#1 and #2 are shown with the original GFP sequences

Fig. 2

a GFP#F cells were transduced with two AAV vectors for three consecutive days at MOI 2 × 10⁴. When treated with AAV-Cas9-GFP1 (left panel), 57.7 ± 0.7, 40.6 ± 0.2 and 35.6 ± 0.1% cells were found GFP-positive, while 37.5 ± 3.3, 25.2 ± 1.1, 27.7 ± 4.5% of cells were found GFP-positive with AAV-Cas9-GFP2 (right panel) vector infection after one, two or three administrations, respectively. Error bars indicate STDEV. *P < 0.05, **P < 0.01, and ***P < 0.001. b We sorted the GFP-positive cell population in the GFP#F cells, which were treated by AAV-Cas9-GFP2 vector at MOI 2 × 10⁴ for three consecutive days. Sorted cells were used for AAV-Cas9-GFP vector superinfection study. Representative FACS images for sorted cells (left panel) and AAV-superinfected cells (right panel) were shown. c Parental GFP#F cells and sorted cells were transduced with AAV-Cas9-GFP2 at MOI of 8 × 10², 4 × 10³, 2 × 10⁴ gc and GFP-positive cell populations were analyzed by FACS (upper panel). Lower panel shows the results of GFP-positive cell populations after super-infection with another GFP-targeting vector, AAV-Cas9-GFP1 vector. The averages of two independent experiments are shown. Error bars represent SEM (*P < 0.05)

Fig. 3

Three off-target deletions identified by exome-sequencing were shown. GFP#F cells were treated by AAV-Cas9-GFP2 vector at high dose (MOI = 2 × 10⁴ gc, × 1), AAV-Cas9-GFP2 vector at low dose four times (MOI = 8 × 10² gc, × 4) or AAV-Cas9-GFP1 vector at high doses three times (MOI = 2 × 10⁴ gc, × 3) to get 70% GFP knockout. Total DNA samples from the three samples, along with DNA sample from uninfected control cells, were analyzed by exome-sequencing to identify off-targeted genome editing sites