Abstract
Purpose :
Prime editing is a versatile tool that can introduce point mutations as well as small insertions and deletions into the genome. Nevertheless, low efficiency can be an obstacle for use of prime editing in human induced pluripotent stem cells (hiPSC). This is especially true when the genetic context precludes the use of multiple prime editing guide RNAs (pegRNAs) and other strategies must be employed to achieve the desired edit. Such is the case of the c.25G>A (p.V9M) mutation in the NMNAT1 gene, causative of severe early onset inherited retinal degeneration (IRD).
Methods :
In this study, we efficiently generated an isogenic model of the p.V9M mutation in the NMNAT1 gene, optimizing the prime editing workflow without manipulating key prime editing components. We designed 10 pegRNAs and analyze them to predict off targets and nicking efficiency. The pegRNA, PE and nicking gRNA plasmids were delivered by electroporation into hiPSCs. Electroporated clones were identified with a transient GFP reporter in the nicking gRNA plasmid and analyzed after 48h using fluorescence microscopy and FACS. Single clones were manually selected, expanded, and analyzed with Sanger sequencing to confirm prime editing.
Results :
After an in silico analysis of the genomic region of the c.25G>A NMNAT1 mutation, two pegRNAs were selected for molecular cloning based on the distance to the mutation and sequence length. Initial co-electroporation of PEmax, pegRNAs and nicking gRNA in hiPSCs showed transfection in few clones and low (<1%) editing efficiency. We modified the workflow of hiPSC prime editing, including plasmid concentrations and prime editing components ratios and several conditions of the delivery method and we demonstrated that our optimized workflow enhanced editing efficiency. Fluorescent microscopy and flow cytometry of the electroporated hiPSC showed 60% transfection efficiency. Through NGS we identified 10% correctly edited alleles for the c.25G>A NMNAT1 mutation. After clonal expansion we identified up to 5.38% homozygous clones and 20.43% heterozygous clones.
Conclusions :
These findings show that our optimized workflow enhanced the prime editing efficiency in hiPSC and allowed for the generation of clonal isogenic cell lines with the heritable p.V9M mutation causative of NMNAT1-associated IRD. Our approach has proven to be useful in generating other isogenic models of IRDs.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.