Abstract
Purpose :
CRISPR/Cas9-mediated targeting of the human rhodopsin gene (RHO) is being explored as a treatment for dominantly inherited retinitis pigmentosa. CRISPR/Cas9 relies on a guide RNA (gRNA) binding to a target site in the genome via sequence homology. Discrepancies between the gRNA sequence and the target site can reduce CRISPR/Cas9 activity. This study investigates how single base pair mismatches between the gRNA and the target sequence affect the level of gene disruption at two loci in RHO in an in vitro human cell line.
Methods :
RHO was screened for Staphylococcus aureus Cas9 target sites, of which two were selected (locus A and locus B). For each site two gRNAs were designed: a “homologous gRNA” which has 100% homology to the target sequence, and a “mismatch gRNA” which has a one base pair mismatch with the target sequence at nucleotide 19 of the 21-nucleotide gRNA. At locus A the mutation on the mismatch gRNA was G>T (purine to pyrimidine transition); at locus B it was A>G (purine transition). These were cloned into a plasmid expressing both Staphylococcus aureus Cas9 and a single gRNA. All 4 plasmids and a no template control were transfected into HEK293-EGFP cells and the level of RHO disruption was measured by Tracking of Indels by DEcomposition (TIDE) analysis. A two-way ANOVA was conducted comparing the RHO disruption detected with the homologous gRNA, the mismatch gRNA, and the no template control at each locus.
Results :
At both locus A and B, the homologous gRNA created significantly more events of RHO disruption compared to the no template control, with 6.4% disruption at locus A and 4.4% at locus B (p < 0.0001 and p = 0.02, respectively). At locus A the purine to pyrimidine change in one nucleotide reduced activity of the gRNA to the same level as the no template control. At locus B, however, substituting one purine base for another in the gRNA resulted in a RHO disruption level of 4.5%, which was similar to the homologous gRNA sequence.
Conclusions :
The two loci studied in this experiment were successfully targeted to produce Cas9-mediated disruption of RHO using a gRNA with 100% sequence homology to the target. A one base pair mismatch between the target and the gRNA extinguished all detectable CRISPR/Cas9 activity at one site where a purine was substituted for a pyrimidine, but not at the other site where two purine bases were interchanged.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.