Abstract
Prime editors (PEs) mediate genome modification without utilizing double-stranded DNA breaks or exogenous donor DNA as a template. PEs facilitate nucleotide substitutions or local insertions or deletions within the genome based on the template sequence encoded within the prime editing guide RNA (pegRNA). However, the efficacy of prime editing in adult mice has not been established. Here we report an NLS-optimized SpCas9-based prime editor that improves genome editing efficiency in both fluorescent reporter cells and at endogenous loci in cultured cell lines. Using this genome modification system, we could also seed tumor formation through somatic cell editing in the adult mouse. Finally, we successfully utilize dual adeno-associated virus (AAVs) for the delivery of a split-intein prime editor and demonstrate that this system enables the correction of a pathogenic mutation in the mouse liver. Our findings further establish the broad potential of this genome editing technology for the directed installation of sequence modifications in vivo, with important implications for disease modeling and correction.
Competing Interest Statement
UMass has filed a patent application on PE2* and AAT pegRNAs in this work (inventors: PL, SQL, SAW, and WX, patent filed/pending). S.A.W. is a consultant for Chroma Medicine. All remaining authors declare that the research was conducted in the absence of commercial or financial conflict of interest. The authors declare no competing non-financial interests.
Footnotes
Conflict of interest: The authors declare no potential conflicts of interest.
We performed an additional experiment to examine the difference in vivo in nuclear localization for the Liu PE2 construct relative to our PE2* construct (with the increased number of NLSs), since the comparative in vivo data are the important advance for the field within this manuscript. The new immunohistochemistry data in mouse liver are shown in Figure 3 c/d, where in Figure 3c we display a representative image showing the difference in cytoplasmic versus nuclear staining for PE2 versus PE2*, and in Figure 3d we display a calculation of the nuclear ratio of each protein based on the staining. These data show variable nuclear staining for the original PE2 protein, whereas the new PE2* protein is almost completely nuclear consistent with the cell culture confocal imaging in Supplementary Figure 1.
Abbreviation
- CRISPR/Cas9
- clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9
- PEs
- prime editors (PEs)
- pegRNA
- prime editing guide RNA
- PBS
- primer binding site
- RT
- reverse transcriptase
